Escherichia coli Double-Strand Uracil-DNA Glycosylase: Involvement in Uracil-Mediated DNA Base Excision Repair and Stimulation of Activity by Endonuclease IV†
Identifieur interne : 001A93 ( Istex/Corpus ); précédent : 001A92; suivant : 001A94Escherichia coli Double-Strand Uracil-DNA Glycosylase: Involvement in Uracil-Mediated DNA Base Excision Repair and Stimulation of Activity by Endonuclease IV†
Auteurs : Jung-Suk Sung ; Dale W. MosbaughSource :
- Biochemistry [ 0006-2960 ] ; 2000.
Abstract
Escherichia coli double-strand uracil-DNA glycosylase (Dug) was purified to apparent homogeneity as both a native and recombinant protein. The molecular weight of recombinant Dug was 18 670, as determined by matrix-assisted laser desorption−ionization mass spectrometry. Dug was active on duplex oligonucleotides (34-mers) that contained site-specific U·G, U·A, ethenoC·G, and ethenoC·A targets; however, activity was not detected on DNA containing a T·G mispair or single-stranded DNA containing either a site-specific uracil or ethenoC residue. One of the distinctive characteristics of Dug was that the purified enzyme excised a near stoichiometric amount of uracil from U·G-containing oligonucleotide substrate. Electrophoretic mobility shift assays revealed that the lack of turnover was the result of strong binding by Dug to the reaction product apyrimidinic-site (AP) DNA. Addition of E. coli endonuclease IV stimulated Dug activity by enhancing the rate and extent of uracil excision by promoting dissociation of Dug from the AP·G-containing 34-mer. Catalytically active endonuclease IV was apparently required to mediate Dug turnover, since the addition of 5 mM EDTA mitigated the effect. Further support for this interpretation came from the observations that Dug preferentially bound 34-mer containing an AP·G target, while binding was not observed on a substrate incised 5‘ to the AP-site. We also investigated whether Dug could initiate a uracil-mediated base excision repair pathway in E. coli NR8052 cell extracts using M13mp2op14 DNA (form I) containing a site-specific U·G mispair. Analysis of reaction products revealed a time dependent appearance of repaired form I DNA; addition of purified Dug to the cell extract stimulated the rate of repair.
Url:
DOI: 10.1021/bi0007066
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<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title>Escherichia coli Double-Strand Uracil-DNA Glycosylase: Involvement in Uracil-Mediated DNA Base Excision Repair and Stimulation of Activity by Endonuclease IV†</title><author><name sortKey="Sung, Jung Suk" sort="Sung, Jung Suk" uniqKey="Sung J" first="Jung-Suk" last="Sung">Jung-Suk Sung</name><affiliation><mods:affiliation>Departments of Environmental and Molecular Toxicology and Biochemistry and Biophysics and theEnvironmental Health Science Center, Oregon State University, Corvallis, Oregon 97731</mods:affiliation></affiliation></author><author><name sortKey="Mosbaugh, Dale W" sort="Mosbaugh, Dale W" uniqKey="Mosbaugh D" first="Dale W." last="Mosbaugh">Dale W. Mosbaugh</name><affiliation><mods:affiliation>Departments of Environmental and Molecular Toxicology and Biochemistry and Biophysics and theEnvironmental Health Science Center, Oregon State University, Corvallis, Oregon 97731</mods:affiliation></affiliation><affiliation><mods:affiliation> To whom correspondence should be addressed. Phone: (541) 737-1797. Fax: (541) 737-0497. E-mail: mosbaugd@ucs.orst.edu.</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:5F6B0C51A0EFE03047B7BA123BD16C54F9101355</idno><date when="2000" year="2000">2000</date><idno type="doi">10.1021/bi0007066</idno><idno type="url">https://api.istex.fr/ark:/67375/TPS-2CZV0JP1-T/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">001A93</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">001A93</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main"><hi rend="italic">Escherichia coli</hi> Double-Strand Uracil-DNA Glycosylase: Involvement in
Uracil-Mediated DNA Base Excision Repair and Stimulation of Activity by
Endonuclease IV<ref type="bib" target="#bi0007066AF2"><hi rend="superscript">†</hi></ref></title><author><name sortKey="Sung, Jung Suk" sort="Sung, Jung Suk" uniqKey="Sung J" first="Jung-Suk" last="Sung">Jung-Suk Sung</name><affiliation><mods:affiliation>Departments of Environmental and Molecular Toxicology and Biochemistry and Biophysics and theEnvironmental Health Science Center, Oregon State University, Corvallis, Oregon 97731</mods:affiliation></affiliation></author><author><name sortKey="Mosbaugh, Dale W" sort="Mosbaugh, Dale W" uniqKey="Mosbaugh D" first="Dale W." last="Mosbaugh">Dale W. Mosbaugh</name><affiliation><mods:affiliation>Departments of Environmental and Molecular Toxicology and Biochemistry and Biophysics and theEnvironmental Health Science Center, Oregon State University, Corvallis, Oregon 97731</mods:affiliation></affiliation><affiliation><mods:affiliation> To whom correspondence should be addressed. Phone: (541) 737-1797. Fax: (541) 737-0497. E-mail: mosbaugd@ucs.orst.edu.</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">Biochemistry</title><title level="j" type="abbrev">Biochemistry</title><idno type="ISSN">0006-2960</idno><idno type="eISSN">1520-4995</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published">2000</date><date type="published">2000</date><biblScope unit="vol">39</biblScope><biblScope unit="issue">33</biblScope><biblScope unit="page" from="10224">10224</biblScope><biblScope unit="page" to="10235">10235</biblScope></imprint><idno type="ISSN">0006-2960</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0006-2960</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract">Escherichia coli double-strand uracil-DNA glycosylase (Dug) was purified to apparent homogeneity as both a native and recombinant protein. The molecular weight of recombinant Dug was 18 670, as determined by matrix-assisted laser desorption−ionization mass spectrometry. Dug was active on duplex oligonucleotides (34-mers) that contained site-specific U·G, U·A, ethenoC·G, and ethenoC·A targets; however, activity was not detected on DNA containing a T·G mispair or single-stranded DNA containing either a site-specific uracil or ethenoC residue. One of the distinctive characteristics of Dug was that the purified enzyme excised a near stoichiometric amount of uracil from U·G-containing oligonucleotide substrate. Electrophoretic mobility shift assays revealed that the lack of turnover was the result of strong binding by Dug to the reaction product apyrimidinic-site (AP) DNA. Addition of E. coli endonuclease IV stimulated Dug activity by enhancing the rate and extent of uracil excision by promoting dissociation of Dug from the AP·G-containing 34-mer. Catalytically active endonuclease IV was apparently required to mediate Dug turnover, since the addition of 5 mM EDTA mitigated the effect. Further support for this interpretation came from the observations that Dug preferentially bound 34-mer containing an AP·G target, while binding was not observed on a substrate incised 5‘ to the AP-site. We also investigated whether Dug could initiate a uracil-mediated base excision repair pathway in E. coli NR8052 cell extracts using M13mp2op14 DNA (form I) containing a site-specific U·G mispair. Analysis of reaction products revealed a time dependent appearance of repaired form I DNA; addition of purified Dug to the cell extract stimulated the rate of repair.</div></front></TEI><istex><corpusName>acs</corpusName><keywords><teeft><json:string>coli</json:string><json:string>endonuclease</json:string><json:string>glycosylase</json:string><json:string>uracil</json:string><json:string>edta</json:string><json:string>excision</json:string><json:string>mosbaugh</json:string><json:string>coli endonuclease</json:string><json:string>rdug</json:string><json:string>htdg</json:string><json:string>recombinant</json:string><json:string>biol</json:string><json:string>chem</json:string><json:string>biochemistry</json:string><json:string>denaturing</json:string><json:string>reaction product</json:string><json:string>jiricny</json:string><json:string>oligonucleotide</json:string><json:string>reaction mixture</json:string><json:string>mispair</json:string><json:string>polyacrylamide</json:string><json:string>glycosylase activity</json:string><json:string>mispairs</json:string><json:string>spectrometry</json:string><json:string>oligonucleotides</json:string><json:string>molecular weight</json:string><json:string>polypeptide</json:string><json:string>dithiothreitol</json:string><json:string>assay</json:string><json:string>duplex</json:string><json:string>guanine</json:string><json:string>agarose</json:string><json:string>binding reaction</json:string><json:string>phosphorimager</json:string><json:string>mispaired</json:string><json:string>ndug</json:string><json:string>amino</json:string><json:string>coli glycosylase</json:string><json:string>mutation</json:string><json:string>various amount</json:string><json:string>incision</json:string><json:string>electrophoretic</json:string><json:string>pmol</json:string><json:string>equal volume</json:string><json:string>electrophoretic mobility shift assay</json:string><json:string>base excision repair</json:string><json:string>denaturing polyacrylamide</json:string><json:string>uracil excision</json:string><json:string>more active</json:string><json:string>bennett</json:string><json:string>recombinant protein</json:string><json:string>substrate specificity</json:string><json:string>free uracil</json:string><json:string>binding reaction mixture</json:string><json:string>apparent homogeneity</json:string><json:string>active fraction</json:string><json:string>gene product</json:string><json:string>nucleotide position</json:string><json:string>standard glycosylase reaction mixture</json:string><json:string>supernatant fraction</json:string><json:string>uracil residue</json:string><json:string>imagequant software</json:string><json:string>bromphenol blue</json:string><json:string>protein band</json:string><json:string>same buffer</json:string><json:string>maldi mass spectrometry</json:string><json:string>amino acid</json:string><json:string>base pair</json:string><json:string>electrophoresis</json:string><json:string>duplex oligonucleotides</json:string><json:string>environmental health science center</json:string><json:string>enzyme activity</json:string><json:string>ammonium sulfate</json:string><json:string>room temperature</json:string><json:string>efficient excision</json:string><json:string>silver staining</json:string><json:string>present study</json:string><json:string>oregon state university</json:string><json:string>base excision</json:string><json:string>protein concentration</json:string><json:string>coli extract</json:string><json:string>coli cell extract</json:string><json:string>nucleic acid</json:string><json:string>apparent molecular weight</json:string><json:string>cell extract</json:string><json:string>catalytic activity</json:string><json:string>stimulatory effect</json:string><json:string>mispaired uracil</json:string><json:string>sonification buffer</json:string><json:string>coli cell</json:string><json:string>enzyme</json:string></teeft></keywords><author><json:item><name>SUNG Jung-Suk</name><affiliations><json:string>Departments of Environmental and Molecular Toxicology and Biochemistry and Biophysics and theEnvironmental Health Science Center, Oregon State University, Corvallis, Oregon 97731</json:string></affiliations></json:item><json:item><name>MOSBAUGH Dale W.</name><affiliations><json:string>Departments of Environmental and Molecular Toxicology and Biochemistry and Biophysics and theEnvironmental Health Science Center, Oregon State University, Corvallis, Oregon 97731</json:string><json:string>To whom correspondence should be addressed. Phone: (541) 737-1797. Fax: (541) 737-0497. E-mail: mosbaugd@ucs.orst.edu.</json:string></affiliations></json:item></author><arkIstex>ark:/67375/TPS-2CZV0JP1-T</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>research-article</json:string></originalGenre><abstract>Escherichia coli double-strand uracil-DNA glycosylase (Dug) was purified to apparent homogeneity as both a native and recombinant protein. The molecular weight of recombinant Dug was 18 670, as determined by matrix-assisted laser desorption−ionization mass spectrometry. Dug was active on duplex oligonucleotides (34-mers) that contained site-specific U·G, U·A, ethenoC·G, and ethenoC·A targets; however, activity was not detected on DNA containing a T·G mispair or single-stranded DNA containing either a site-specific uracil or ethenoC residue. One of the distinctive characteristics of Dug was that the purified enzyme excised a near stoichiometric amount of uracil from U·G-containing oligonucleotide substrate. Electrophoretic mobility shift assays revealed that the lack of turnover was the result of strong binding by Dug to the reaction product apyrimidinic-site (AP) DNA. Addition of E. coli endonuclease IV stimulated Dug activity by enhancing the rate and extent of uracil excision by promoting dissociation of Dug from the AP·G-containing 34-mer. Catalytically active endonuclease IV was apparently required to mediate Dug turnover, since the addition of 5 mM EDTA mitigated the effect. Further support for this interpretation came from the observations that Dug preferentially bound 34-mer containing an AP·G target, while binding was not observed on a substrate incised 5‘ to the AP-site. We also investigated whether Dug could initiate a uracil-mediated base excision repair pathway in E. coli NR8052 cell extracts using M13mp2op14 DNA (form I) containing a site-specific U·G mispair. Analysis of reaction products revealed a time dependent appearance of repaired form I DNA; addition of purified Dug to the cell extract stimulated the rate of repair.</abstract><qualityIndicators><score>10</score><pdfWordCount>10593</pdfWordCount><pdfCharCount>63778</pdfCharCount><pdfVersion>1.2</pdfVersion><pdfPageCount>12</pdfPageCount><pdfPageSize>612 x 792 pts (letter)</pdfPageSize><pdfWordsPerPage>883</pdfWordsPerPage><pdfText>true</pdfText><refBibsNative>true</refBibsNative><abstractWordCount>255</abstractWordCount><abstractCharCount>1766</abstractCharCount><keywordCount>0</keywordCount></qualityIndicators><title>Escherichia coli Double-Strand Uracil-DNA Glycosylase: Involvement in Uracil-Mediated DNA Base Excision Repair and Stimulation of Activity by Endonuclease IV†</title><genre><json:string>research-article</json:string></genre><host><title>Biochemistry</title><language><json:string>unknown</json:string></language><issn><json:string>0006-2960</json:string></issn><eissn><json:string>1520-4995</json:string></eissn><volume>39</volume><issue>33</issue><pages><first>10224</first><last>10235</last></pages><genre><json:string>journal</json:string></genre></host><ark><json:string>ark:/67375/TPS-2CZV0JP1-T</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - biochemistry & molecular biology</json:string></wos><scienceMetrix><json:string>1 - health sciences</json:string><json:string>2 - biomedical research</json:string><json:string>3 - biochemistry & molecular biology</json:string></scienceMetrix><scopus><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string><json:string>3 - Biochemistry</json:string></scopus><inist><json:string>1 - sciences appliquees, technologies et medecines</json:string><json:string>2 - sciences biologiques et medicales</json:string><json:string>3 - sciences biologiques fondamentales et appliquees. psychologie</json:string></inist></categories><publicationDate>2000</publicationDate><copyrightDate>2000</copyrightDate><doi><json:string>10.1021/bi0007066</json:string></doi><id>5F6B0C51A0EFE03047B7BA123BD16C54F9101355</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-2CZV0JP1-T/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-2CZV0JP1-T/bundle.zip</uri></json:item><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-2CZV0JP1-T/fulltext.txt</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/TPS-2CZV0JP1-T/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main"><hi rend="italic">Escherichia coli</hi> Double-Strand Uracil-DNA Glycosylase: Involvement in
Uracil-Mediated DNA Base Excision Repair and Stimulation of Activity by
Endonuclease IV<ref type="bib" target="#bi0007066AF2"><hi rend="superscript">†</hi></ref></title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>American Chemical Society</publisher><availability><licence>Copyright © 2000 American Chemical Society</licence><p>American Chemical Society</p></availability><date type="published">2000</date><date type="Copyright" when="2000">2000</date></publicationStmt><notesStmt><note type="content-type" source="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="publication-type" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note></notesStmt><sourceDesc><biblStruct type="article"><analytic><title level="a" type="main"><hi rend="italic">Escherichia coli</hi> Double-Strand Uracil-DNA Glycosylase: Involvement in
Uracil-Mediated DNA Base Excision Repair and Stimulation of Activity by
Endonuclease IV<ref type="bib" target="#bi0007066AF2"><hi rend="superscript">†</hi></ref></title><author xml:id="author-0000"><persName><surname>Sung</surname><forename type="first">Jung-Suk</forename></persName><affiliation><orgName type="institution">Departments of Environmental and Molecular Toxicology and Biochemistry and Biophysics and the Environmental Health Science Center</orgName><orgName type="institution">Oregon State University</orgName><address><addrLine>Corvallis</addrLine><addrLine>Oregon 97731</addrLine></address></affiliation></author><author xml:id="author-0001" role="corresp"><persName><surname>Mosbaugh</surname><forename type="first">Dale W.</forename></persName><affiliation><orgName type="institution">Departments of Environmental and Molecular Toxicology and Biochemistry and Biophysics and the Environmental Health Science Center</orgName><orgName type="institution">Oregon State University</orgName><address><addrLine>Corvallis</addrLine><addrLine>Oregon 97731</addrLine></address></affiliation><affiliation role="corresp"> To whom correspondence should be addressed. Phone: (541) 737-1797. Fax: (541) 737-0497. E-mail: mosbaugd@ucs.orst.edu.</affiliation></author><idno type="istex">5F6B0C51A0EFE03047B7BA123BD16C54F9101355</idno><idno type="ark">ark:/67375/TPS-2CZV0JP1-T</idno><idno type="DOI">10.1021/bi0007066</idno></analytic><monogr><title level="j" type="main">Biochemistry</title><title level="j" type="abbrev">Biochemistry</title><idno type="acspubs">bi</idno><idno type="coden">bichaw</idno><idno type="pISSN">0006-2960</idno><idno type="eISSN">1520-4995</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published">2000</date><date type="published">2000</date><biblScope unit="vol">39</biblScope><biblScope unit="issue">33</biblScope><biblScope unit="page" from="10224">10224</biblScope><biblScope unit="page" to="10235">10235</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><encodingDesc><schemaRef type="ODD" url="https://xml-schema.delivery.istex.fr/tei-istex.odd"></schemaRef><appInfo><application ident="pub2tei" version="1.0.41" when="2020-04-06"><label>pub2TEI-ISTEX</label><desc>A set of style sheets for converting XML documents encoded in various scientific publisher formats into a common TEI format.
<ref target="http://www.tei-c.org/">We use TEI</ref></desc></application></appInfo></encodingDesc><profileDesc><abstract><p><hi rend="italic">Escherichia coli</hi> double-strand uracil-DNA glycosylase (Dug) was purified to apparent
homogeneity as both a native and recombinant protein. The molecular weight of recombinant Dug was
18 670, as determined by matrix-assisted laser desorption−ionization mass spectrometry. Dug was active
on duplex oligonucleotides (34-mers) that contained site-specific U·G, U·A, ethenoC·G, and ethenoC·A
targets; however, activity was not detected on DNA containing a T·G mispair or single-stranded DNA
containing either a site-specific uracil or ethenoC residue. One of the distinctive characteristics of Dug
was that the purified enzyme excised a near stoichiometric amount of uracil from U·G-containing
oligonucleotide substrate. Electrophoretic mobility shift assays revealed that the lack of turnover was the
result of strong binding by Dug to the reaction product apyrimidinic-site (AP) DNA. Addition of <hi rend="italic">E. coli</hi>
endonuclease IV stimulated Dug activity by enhancing the rate and extent of uracil excision by promoting
dissociation of Dug from the AP·G-containing 34-mer. Catalytically active endonuclease IV was apparently
required to mediate Dug turnover, since the addition of 5 mM EDTA mitigated the effect. Further support
for this interpretation came from the observations that Dug preferentially bound 34-mer containing an
AP·G target, while binding was not observed on a substrate incised 5‘ to the AP-site. We also investigated
whether Dug could initiate a uracil-mediated base excision repair pathway in <hi rend="italic">E. coli</hi> NR8052 cell extracts
using M13mp2op14 DNA (form I) containing a site-specific U·G mispair. Analysis of reaction products
revealed a time dependent appearance of repaired form I DNA; addition of purified Dug to the cell extract
stimulated the rate of repair.
</p></abstract><textClass ana="subject"><keywords scheme="document-type-name"><term>Article</term></keywords></textClass><langUsage><language ident="en"></language></langUsage></profileDesc><revisionDesc><change when="2020-04-06" who="#istex" xml:id="pub2tei">formatting</change></revisionDesc></teiHeader></istex:fulltextTEI></fulltext><metadata><istex:metadataXml wicri:clean="corpus acs not found" wicri:toSee="no header"><istex:xmlDeclaration>version="1.0" encoding="UTF-8"</istex:xmlDeclaration><istex:document><article article-type="research-article" specific-use="acs2jats-1.1.23" dtd-version="1.1d1"><front><journal-meta><journal-id journal-id-type="acspubs">bi</journal-id><journal-id journal-id-type="coden">bichaw</journal-id><journal-title-group><journal-title>Biochemistry</journal-title><abbrev-journal-title>Biochemistry</abbrev-journal-title></journal-title-group><issn pub-type="ppub">0006-2960</issn><issn pub-type="epub">1520-4995</issn><publisher><publisher-name>American Chemical Society</publisher-name></publisher><self-uri>pubs.acs.org/biochemistry</self-uri></journal-meta><article-meta><article-id pub-id-type="doi">10.1021/bi0007066</article-id><article-categories><subj-group subj-group-type="document-type-name"><subject>Article</subject></subj-group></article-categories><title-group><article-title><italic toggle="yes">Escherichia coli</italic> Double-Strand Uracil-DNA Glycosylase: Involvement in
Uracil-Mediated DNA Base Excision Repair and Stimulation of Activity by
Endonuclease IV<xref rid="bi0007066AF2"><sup>†</sup></xref></article-title></title-group><contrib-group><contrib contrib-type="author"><name name-style="western"><surname>Sung</surname><given-names>Jung-Suk</given-names></name></contrib><contrib contrib-type="author" corresp="yes"><name name-style="western"><surname>Mosbaugh</surname><given-names>Dale W.</given-names></name><xref rid="bi0007066AF1">*</xref></contrib><aff>Departments of Environmental and Molecular Toxicology and Biochemistry and Biophysics and the
Environmental Health Science Center, Oregon State University, Corvallis, Oregon 97731
</aff></contrib-group><author-notes><corresp id="bi0007066AF1">
To whom correspondence should be addressed. Phone: (541) 737-1797. Fax: (541) 737-0497. E-mail: mosbaugd@ucs.orst.edu.</corresp></author-notes><pub-date pub-type="epub"><day>29</day><month>07</month><year>2000</year></pub-date><pub-date pub-type="ppub"><day>22</day><month>08</month><year>2000</year></pub-date><volume>39</volume><issue>33</issue><fpage>10224</fpage><lpage>10235</lpage><supplementary-material xlink:href="bi0007066_s.pdf" orientation="portrait" position="float"></supplementary-material><history><date date-type="received"><day>29</day><month>03</month><year>2000</year></date><date date-type="rev-recd"><day>23</day><month>06</month><year>2000</year></date><date date-type="asap"><day>29</day><month>07</month><year>2000</year></date><date date-type="issue-pub"><day>22</day><month>08</month><year>2000</year></date></history><permissions><copyright-statement>Copyright © 2000 American Chemical Society</copyright-statement><copyright-year>2000</copyright-year><copyright-holder>American Chemical Society</copyright-holder></permissions><abstract><p><italic toggle="yes">Escherichia coli</italic> double-strand uracil-DNA glycosylase (Dug) was purified to apparent
homogeneity as both a native and recombinant protein. The molecular weight of recombinant Dug was
18 670, as determined by matrix-assisted laser desorption−ionization mass spectrometry. Dug was active
on duplex oligonucleotides (34-mers) that contained site-specific U·G, U·A, ethenoC·G, and ethenoC·A
targets; however, activity was not detected on DNA containing a T·G mispair or single-stranded DNA
containing either a site-specific uracil or ethenoC residue. One of the distinctive characteristics of Dug
was that the purified enzyme excised a near stoichiometric amount of uracil from U·G-containing
oligonucleotide substrate. Electrophoretic mobility shift assays revealed that the lack of turnover was the
result of strong binding by Dug to the reaction product apyrimidinic-site (AP) DNA. Addition of <italic toggle="yes">E. coli</italic>
endonuclease IV stimulated Dug activity by enhancing the rate and extent of uracil excision by promoting
dissociation of Dug from the AP·G-containing 34-mer. Catalytically active endonuclease IV was apparently
required to mediate Dug turnover, since the addition of 5 mM EDTA mitigated the effect. Further support
for this interpretation came from the observations that Dug preferentially bound 34-mer containing an
AP·G target, while binding was not observed on a substrate incised 5‘ to the AP-site. We also investigated
whether Dug could initiate a uracil-mediated base excision repair pathway in <italic toggle="yes">E. coli</italic> NR8052 cell extracts
using M13mp2op14 DNA (form I) containing a site-specific U·G mispair. Analysis of reaction products
revealed a time dependent appearance of repaired form I DNA; addition of purified Dug to the cell extract
stimulated the rate of repair.
</p></abstract><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>bi0007066</meta-value></custom-meta></custom-meta-group></article-meta><notes id="bi0007066AF2"><label>†</label><p>
This work was supported by National Institutes of Health Grants
GM32823 and ES00210. Technical Report 11651 from the Oregon
Agricultural Experiment Station.</p></notes></front><body><sec id="d7e122"><title></title><p><italic toggle="yes">Escherichia coli</italic> uracil-mediated DNA base excision repair
(BER)<xref rid="bi0007066b00001" ref-type="bibr"></xref> is initiated by uracil-DNA glycosylase which plays
an important role in mutation avoidance (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0007066b00001" ref-type="bibr"></xref>−<xref rid="bi0007066b00002" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi0007066b00003" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi0007066b00004" ref-type="bibr"></xref></named-content></italic>). <italic toggle="yes">E. coli</italic> uracil-DNA glycosylase (Ung) is a monofunctional enzyme encoded by the <italic toggle="yes">ung</italic> gene (<italic toggle="yes"><xref rid="bi0007066b00005" ref-type="bibr"></xref></italic>) which produces a single
polypeptide with a molecular weight of 25 558, as determined
by MALDI mass spectrometry (<italic toggle="yes"><xref rid="bi0007066b00006" ref-type="bibr"></xref></italic>). The enzyme exhibits a
∼2-fold preference for uracil residues located in single-stranded DNA compared to double-stranded DNA containing
U·G mispairs and is more active (2.4-fold) on U·G- rather
than U·A-containing DNA (<italic toggle="yes"><xref rid="bi0007066b00007" ref-type="bibr"></xref></italic>). Catalysis occurs when the
uracil-deoxyribose N1−C1‘ glycosylic bond is destabilized
and subjected to an activated water nucleophilic attack that
results in the release of free uracil and the production of
apyrimidinic-site DNA (<italic toggle="yes"><xref rid="bi0007066b00008" ref-type="bibr"></xref></italic>). Following hydrolysis, Ung dissociates from the AP-site and may implement a processive
search mechanism to locate additional uracil nucleotides (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0007066b00007" ref-type="bibr"></xref>,
<xref rid="bi0007066b00009" ref-type="bibr"></xref></named-content></italic>). With a turnover rate of ∼800 min<sup>-1</sup>, measured on U·A-containing DNA (<italic toggle="yes"><xref rid="bi0007066b00010" ref-type="bibr"></xref></italic>) Ung is considered kinetically efficient
compared to other monofunctional DNA glycosylases (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0007066b00011" ref-type="bibr"></xref>−<xref rid="bi0007066b00012" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi0007066b00013" ref-type="bibr"></xref></named-content></italic>). Ung is inhibited noncompetitively by uracil (<italic toggle="yes">K</italic><sub>i</sub> = 0.12
mM), competitively by AP-sites (<italic toggle="yes">K</italic><sub>i</sub> = 1 μM), and irreversibly by binding to the bacteriophage PBS1 and PBS2 uracil-DNA glycosylase inhibitor (Ugi) protein (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0007066b00010" ref-type="bibr"></xref>, <xref rid="bi0007066b00014" ref-type="bibr"></xref></named-content></italic>). Significant
amino acid sequence homology exists between <italic toggle="yes">E. coli</italic> Ung
and uracil-DNA glycosylases from other biological sources,
such as human, which shares ∼56% identity with Ung (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0007066b00015" ref-type="bibr"></xref>,
<xref rid="bi0007066b00016" ref-type="bibr"></xref></named-content></italic>).
</p><p>Wiebauer and Jiricny (<italic toggle="yes"><xref rid="bi0007066b00017" ref-type="bibr"></xref></italic>) initially detected a novel BER
repair process in HeLa cell extracts that excised aberrant
thymine bases from T·G mispairs. Subsequent experiments
revealed that the thymine-excision activity involved a DNA
glycosylase which was designated mismatch-specific thymine-DNA glycosylase, TDG (<italic toggle="yes"><xref rid="bi0007066b00018" ref-type="bibr"></xref></italic>). This enzyme was purified
from HeLa cell extracts, and identified as a 55 000 molecular
weight polypeptide, based on active analysis following SDS−polyacrylamide gel electrophoresis (<italic toggle="yes"><xref rid="bi0007066b00019" ref-type="bibr"></xref></italic>). The substrate
specificity of HeLa TDG was determined as U·G > T·G ≫ T·C
≫ T·T (<italic toggle="yes"><xref rid="bi0007066b00019" ref-type="bibr"></xref></italic>); however, efficient excision of 3,<italic toggle="yes">N</italic><sup>4</sup>-ethenocytosine residues from ethenoC·G-containing oligonucleotides
was recently demonstrated (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0007066b00020" ref-type="bibr"></xref>, <xref rid="bi0007066b00021" ref-type="bibr"></xref></named-content></italic>). Unlike HeLa uracil-DNA
glycosylase, TDG was reportedly inactive on U·A base pairs
or uracil-containing single-stranded DNA and was not
inhibited by either uracil or Ugi (<italic toggle="yes"><xref rid="bi0007066b00022" ref-type="bibr"></xref></italic>). PCR-mediated cloning
of human TDG cDNA revealed that the gene (hTDG)
contained an open reading frame encoding a 410 amino acid
polypeptide with a deduced molecular weight of ∼46 000
(<italic toggle="yes"><xref rid="bi0007066b00023" ref-type="bibr"></xref></italic>). A databank search disclosed a murine homologue with
88% identity; however, no amino acid sequence motifs
common to other types of DNA glycosylases were identified,
including uracil-DNA glycosylase (<italic toggle="yes"><xref rid="bi0007066b00023" ref-type="bibr"></xref></italic>). In a database search
for proteins with a methyl-CpG-binding domain (MBD),
Hendrich et al. (<italic toggle="yes"><xref rid="bi0007066b00024" ref-type="bibr"></xref></italic>) identified mouse and human full-length
cDNAs encoding a protein that was designated MBD4.
Subsequently, MBD4 was shown to bind preferentially to
m<sup>5</sup>CpG-TpG mismatches and possess DNA glycosylase
activity against U·G and T·G mispairs. However, no
significant amino acid sequence conservation was reported
for MBD4 and TDG (<italic toggle="yes"><xref rid="bi0007066b00024" ref-type="bibr"></xref></italic>).
</p><p>In a study of hTDG N- and C-terminal deletion mutants,
Gallinari and Jiriciny (<italic toggle="yes"><xref rid="bi0007066b00025" ref-type="bibr"></xref></italic>) identified a 248 amino acid core
region of hTDG capable of processing U·G but not T·G
mispairs. This core hTDG region revealed limited homology
with bacterial open reading frames found in <italic toggle="yes">E. coli</italic> and
<italic toggle="yes">Serratia marcescens</italic> (<italic toggle="yes"><xref rid="bi0007066b00025" ref-type="bibr"></xref></italic>). The <italic toggle="yes">E. coli</italic> ORF (169 amino
acids)<xref rid="bi0007066b00002" ref-type="bibr"></xref> shared ∼30% identity with the core hTDG sequence
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0007066b00011" ref-type="bibr"></xref>, <xref rid="bi0007066b00025" ref-type="bibr"></xref></named-content></italic>). When the <italic toggle="yes">E. coli</italic> recombinant protein was expressed
in either reticulocyte lysates or <italic toggle="yes">E. coli</italic> cells, the extracts
efficiently processed U·G but not T·G mispairs or U·A base
pairs (<italic toggle="yes"><xref rid="bi0007066b00025" ref-type="bibr"></xref></italic>). Furthermore, excision of uracil residues from
single-stranded DNA was not observed nor was the DNA
glycosylase activity inhibited by Ugi (<italic toggle="yes"><xref rid="bi0007066b00025" ref-type="bibr"></xref></italic>). Like hTDG but
unlike Ung, this <italic toggle="yes">E. coli</italic> enzyme was shown to efficiently
remove ethenoC residues from duplex DNA (<italic toggle="yes"><xref rid="bi0007066b00021" ref-type="bibr"></xref></italic>). Thus, these
results indicated that the protein encoded by ORF-169 was
distinct from Ung and corresponded to a second class of <italic toggle="yes">E.
coli</italic> uracil-DNA glycosylases. This activity was initially
designated as double-strand-specific uracil-DNA glycosylase
(dsUDG) (<italic toggle="yes"><xref rid="bi0007066b00025" ref-type="bibr"></xref></italic>), but later was referred to as mismatch-specific
uracil-DNA glycosylase (MUG) (<italic toggle="yes"><xref rid="bi0007066b00011" ref-type="bibr"></xref></italic>) and ethenoC-DNA
glycosylase (ethenoCDG) (<italic toggle="yes"><xref rid="bi0007066b00021" ref-type="bibr"></xref></italic>) by other investigators. Based
on characterization presented in this manuscript, and in
compliance with traditional <italic toggle="yes">E. coli</italic> nomenclature, we propose
that dsUDG (double-strand-specific uracil-DNA glycosylase)
be referred to as Dug.
</p><p><italic toggle="yes">E. coli</italic> Dug and Ung lack amino acid sequence homology
(<10% identity); however, these proteins share common
tertiary structural features as described by X-ray crystallography (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0007066b00011" ref-type="bibr"></xref>, <xref rid="bi0007066b00025" ref-type="bibr"></xref></named-content></italic>). Both enzymes utilize a nucleotide-flipping
action to capture the target base in the active site, but use
distinctively different mechanisms for substrate recognition
(<italic toggle="yes"><xref rid="bi0007066b00011" ref-type="bibr"></xref></italic>). Dug purportedly inserts a “polypeptide wedge” into
the DNA double helix at a U·G mispair to facilitate
nucleotide-flipping by a “push” mechanism (<italic toggle="yes"><xref rid="bi0007066b00011" ref-type="bibr"></xref></italic>). The inserted
polypeptide chain (NPSGLSR) is apparently stabilized by
intercalation of Arg146 with the complementary strand base
stack and by three hydrogen bonds formed by Gly143 and
Ser145 that are absolutely specific for the widowed guanine
(<italic toggle="yes"><xref rid="bi0007066b00011" ref-type="bibr"></xref></italic>). In contract, Ung mediates nucleotide-flipping by the
“pinch-push−pull” mechanism that inserts a Leu191 into the
DNA minor groove base stack but overall establishes few
contacts with the complementary DNA strand (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0007066b00016" ref-type="bibr"></xref>, <xref rid="bi0007066b00026" ref-type="bibr"></xref></named-content></italic>). While
X-ray crystallographic studies of Dug protein structure have
offered significant insight into structure/function relationships, the biochemical characteristics of this enzyme, which
differs substantially in size and amino acid sequence from
hTDG, remains to be elucidated.
</p><p>In the present study we have purified to apparent homogeneity a Ugi-insensitive double-strand-specific uracil-DNA
glycosylase from <italic toggle="yes">E. coli</italic> (<italic toggle="yes">ung</italic>) cells that was identified as
Dug. The <italic toggle="yes">dug</italic> gene was cloned, and the gene product was
overproduced, purified, and characterized with respect to
substrate specificity, product binding, and mechanism of
action. We also demonstrated that <italic toggle="yes">E. coli</italic> endonuclease IV
stimulates Dug by instigating catalytic turnover. Furthermore,
we provide the first evidence that Dug initiates a complete
BER reaction using M13mp2 DNA (form I) containing a
site-specific U·G mispair.
</p></sec><sec id="d7e356"><title>Materials and Methods</title><p><italic toggle="yes">Materials.</italic> <italic toggle="yes">E. coli</italic> strains NR8051 [Δ(<italic toggle="yes">pro-lac</italic>) <italic toggle="yes">thi ara</italic>] and
NR8052 [Δ(<italic toggle="yes">pro-lac</italic>) <italic toggle="yes">thi ara trpE9777 ung-1</italic>] were provided
by T. A. Kunkel (NIEHS, National Institutes of Health) and
have been previously described (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0007066b00027" ref-type="bibr"></xref>, <xref rid="bi0007066b00028" ref-type="bibr"></xref></named-content></italic>). <italic toggle="yes">E. coli</italic> JM109
[<italic toggle="yes">recA1</italic> e14(McrA<sup>-</sup>) Δ(<italic toggle="yes">pro-lac</italic>) <italic toggle="yes">thi</italic> <italic toggle="yes">gyrA96</italic> (<italic toggle="yes">NaI<sup>r</sup></italic><sup></sup>) <italic toggle="yes">endoA1</italic><italic toggle="yes">hsdR17</italic> (r<sub>k</sub><sup>-</sup> m<sub>k</sub><sup>+</sup>) <italic toggle="yes">relA1</italic> <italic toggle="yes">supE44</italic>/F‘ <italic toggle="yes">traD36</italic> <italic toggle="yes">lacI<sup>Q</sup></italic><sup></sup> Δ(<italic toggle="yes">lacZ</italic>)<italic toggle="yes">M15</italic> <italic toggle="yes">proA<sup>+</sup></italic><sup></sup><italic toggle="yes">B<sup>+</sup></italic><sup></sup>] was obtained from New England BioLabs.
<italic toggle="yes">E. coli</italic> uracil-DNA glycosylase (fraction V) and Ugi (fraction
IV) were purified as described by Sanderson and Mosbaugh
(<italic toggle="yes"><xref rid="bi0007066b00029" ref-type="bibr"></xref></italic>).
</p><p><italic toggle="yes">E. coli</italic> endonuclease IV (fraction V) was provided by B.
Demple (Harvard University). Restriction endonucleases
(<italic toggle="yes">Eco</italic>RI and <italic toggle="yes">Hin</italic>dIII), T4 polynucleotide kinase, and Vent
DNA polymerase were purchased from New England BioLabs, and T4 DNA ligase from Gibco-BRL. Proteinase K
and creatine phosphokinase (type I, rabbit) were from Sigma,
and ribonuclease A was from Worthington Biochemicals.
</p><p>M13mp2op14 (U·G) DNA (>95% form I) containing a
site-specific U·G mispair<xref rid="bi0007066b00003" ref-type="bibr"></xref> at nucleotide position 79 of the
<italic toggle="yes">lacZ</italic>α gene was prepared as described by Sanderson and
Mosbaugh (<italic toggle="yes"><xref rid="bi0007066b00030" ref-type="bibr"></xref></italic>). Plasmid pKK223−3 was obtained from
Pharmacia Biotech Inc. Oligonucleotides AGCTTGGCTGCAGGTXGACGGATCCCCGGGAATT, where X at nucleotide position 16 corresponds to U (U-34-mer), C (C-34-mer), or T (T-34-mer), and two complementary oligonucleotides (34-mer) containing G or A opposite to X (G-34-mer
and A-34-mer, respectively) were purchased from Midland
Certified Reagent Company. The oligonucleotide containing
3,<italic toggle="yes">N</italic><sup>4</sup>-ethenocytosine (εC-34-mer) at the nucleotide position
X was prepared by Genset. Oligonucleotides CCCAGTCACGTUATTGTAAAACG (U-23-mer), used in the construction of M13mp2op14 (U·G) DNA, and primers
CCAAGCTTTTATCGCCCACGCACTACCAGCGCC (R-33-mer) and
CAGAATTCATGGTTGAGGATATTTTGGCTCCAG (L-33-mer), used in cloning and DNA sequencing experiments, were obtained from Oligos Etc. Oligonucleotides were deblocked, deprotected, purified by denaturing
polyacrylamide gel electrophoresis, and 5‘-end phosphorylated when appropriate using 6000 Ci/mmol [γ-<sup>32</sup>P]ATP
(DuPont-NEN) in place of ATP as previously described (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0007066b00007" ref-type="bibr"></xref>,
<xref rid="bi0007066b00031" ref-type="bibr"></xref></named-content></italic>). Duplex oligonucleotides were prepared by annealing <sup>32</sup>P-labeled U-, T-, or εC-34-mer with the oligonucleotide G-34-mer or A-34-mer (<italic toggle="yes"><xref rid="bi0007066b00007" ref-type="bibr"></xref></italic>). The duplex oligonucleotide [<sup>32</sup>P]AP/G-34-mer was created by reacting [<sup>32</sup>P]U/G-34-mer with
excess <italic toggle="yes">E. coli</italic> uracil-DNA glycosylase to produce a site-specific AP-site; the reaction was quenched by Ugi treatment,
as described by Shroyer et al. (<italic toggle="yes"><xref rid="bi0007066b00008" ref-type="bibr"></xref></italic>). To prepare duplex DNA
(34-mer) that contained an incision on the 5‘-side of the
defined AP-site ([<sup>32</sup>P]*AP/G-34-mer)<xref rid="bi0007066b00004" ref-type="bibr"></xref>, a reaction mixture (50
μL) containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 2
pmol of [<sup>32</sup>P]AP/G-34-mer, and 0.5 units of <italic toggle="yes">E.coli</italic> endonuclease IV (Endo IV) was incubated at 37 °C for 4 h. One
unit of <italic toggle="yes">E. coli</italic> endonuclease IV releases 1 pmol of product/min as described by Levin et al. (<italic toggle="yes"><xref rid="bi0007066b00032" ref-type="bibr"></xref></italic>). Following incision of
>98% of the AP-sites, endonuclease IV was heat-inactivated
at 70 °C for 10 min.
</p><p><italic toggle="yes">Methods.</italic> <italic toggle="yes">Isolation and Cloning of the dug Gene</italic>. A DNA
fragment was obtained from <italic toggle="yes">E. coli</italic> genomic DNA that
contained an ORF (507-bp) encoding the <italic toggle="yes">dug</italic> gene by using
the procedure described by Gallinari and Jiricny (<italic toggle="yes"><xref rid="bi0007066b00025" ref-type="bibr"></xref></italic>) with
some modifications. Briefly, a single colony of <italic toggle="yes">E. coli</italic>
NR8052 (<italic toggle="yes">ung1</italic>) was isolated, diluted into 100 μL of distilled
water and boiled for 10 min to lyse the cells. After
centrifugation for 10 min at 13 000 rpm in a Microspin 24S
(Sorvall) centrifuge at 4 °C, the supernatant fraction was
recovered and a sample (10 μL) was added to a primer
annealing reaction mixture (100 μL) containing 20 mM Tris-HCl (pH 8.8), 2 mM MgSO<sub>4</sub>, 10 mM KCl, 10 mM (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>, 0.1% Triton X-100, and 100 pmol each of R- and L-33-mer oligonucleotide primer. The mixture was heated at 90
°C for 5 min, adjusted to 200 μM each of dATP, dTTP,
dCTP, dGTP, and 2 units of Vent DNA polymerase was
added to the reaction mixture (109 μL). DNA amplification
was then performed for 30 cycles at 94 °C for 1 min, 50 °C
for 1 min, and 72 °C for 3 min. The DNA fragment (523
bp) was isolated using 1.5% agarose gel electrophoresis and
purified by the QIAquick gel extraction protocol (Qiagen).
Following simultaneous restriction endonuclease digestion
with <italic toggle="yes">Eco</italic>RI and <italic toggle="yes">Hin</italic>dIII, the DNA fragment (517-bp) was
isolated, as before, and then inserted into the corresponding
restriction endonuclease cleavage sites of pKK223−3 using
T4 DNA ligase (<italic toggle="yes"><xref rid="bi0007066b00033" ref-type="bibr"></xref></italic>). The resulting construct (pKK-Dug) was
isolated and the nucleotide sequence of the entire <italic toggle="yes">dug</italic> gene
was determined for both DNA strands which verified the
predicted ORF (<italic toggle="yes"><xref rid="bi0007066b00025" ref-type="bibr"></xref></italic>). DNA sequencing was conducted using
an Applied Biosystems model 373A by the Center for Gene
Research and Biotechnology (Oregon State University).
</p><p><italic toggle="yes">Purification of E. coli Double-Strand Uracil-DNA Glycosylase. </italic>Native double-strand uracil-DNA glycosylase was
purified using a purification scheme similar to that initially
described by Lindahl et al. (<italic toggle="yes"><xref rid="bi0007066b00010" ref-type="bibr"></xref></italic>) and modified by Bennett
and Mosbaugh (<italic toggle="yes"><xref rid="bi0007066b00034" ref-type="bibr"></xref></italic>) for purifying <italic toggle="yes">E. coli</italic> Ung. <italic toggle="yes">E. coli</italic> NR8052
cells were grown at 37 °C in 40 L of YT medium (0.5%
yeast extract, 0.8% tryptone, and 0.5% NaCl). After reaching
a density of ∼6.5 × 10<sup>8</sup> cells/mL (1 OD<sub>600</sub> = 8 × 10<sup>8</sup> cells/mL), the cells were harvested by centrifugation at 3500<italic toggle="yes">g</italic> for
15 min in a GSA (Sorvall) rotor, and frozen at −80 °C. Cell
pellets (100 g) were thawed on ice and resuspended in 1 L
of sonification buffer composed of 50 mM Tris-HCl (pH
8.0), 1 mM EDTA, and 0.1 mM dithiothreitol. The cells were
then disrupted by sonification and the lysate was centrifuged
at 20000<italic toggle="yes">g</italic> for 20 min at 4 °C in a SS34 (Sorvall) rotor, as
previously described (<italic toggle="yes"><xref rid="bi0007066b00034" ref-type="bibr"></xref></italic>). Following the addition of 2 ×
10<sup>5</sup> units of Ugi (fraction IV), the cell-free extract was mixed
with an equal volume of 1.6% (w/v) streptomycin sulfate in
sonification buffer. One unit of Ugi inactivates 1 unit of Ung
using reaction conditions as described by Bennett and
Mosbaugh (<italic toggle="yes"><xref rid="bi0007066b00006" ref-type="bibr"></xref></italic>). After gentle stirring for 30 min at 4 °C, the
precipitate was removed by centrifugation at 20000<italic toggle="yes">g</italic> for 20
min at 4 °C, and the supernatant fraction was designated
fraction I. Pulverized ammonium sulfate was slowly added
to fraction I to achieve a final concentration of 50%
(saturation). Precipitated protein was removed by centrifugation at 20000<italic toggle="yes">g</italic> for 20 min at 4 °C. The recovered supernatant
fraction was then adjusted to 80% (saturation) with ammonium sulfate and the precipitate collected as described
above. The pellet was resuspended in 35 mL of UEB buffer
[10 mM Hepes-KOH (pH 7.4), 10 mM 2-mercaptoethanol,
1 mM EDTA, 1 M NaCl and 5% (w/v) glycerol] and dialyzed
extensively against the same buffer; this material constituted
fraction II. Fraction II (68 mL) was loaded onto a Sephadex
G-75 column (6 cm<sup>2</sup> × 88 cm) equilibrated in UEB buffer,
and eluted with the same buffer. Fraction (5 mL) were
collected and assayed for double-strand uracil-DNA glycosylase activity. Active fractions were pooled, concentrated
(∼5-fold) using a Centriprep-10 concentrator (Amicon),
dialyzed against HAB buffer [10 mM potassium phosphate
(pH 7.4), 1 mM dithiothreitol, 200 mM KCl], and designated
fraction III. Fraction III (20 mL) was applied to a hydroxyapatite column (4.9 cm<sup>2</sup> × 8 cm) equilibrated with HAB
buffer. The column was eluted with the same buffer and
fractions (5.2 mL) were collected. To avoid a contaminating
exonuclease activity, only fractions from the leading half of
the activity peak were pooled. These were concentrated (∼6-fold) as before, dialyzed against DAB buffer [30 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol, and 5%
(w/v) glycerol] and corresponded to fraction IV. Fraction
IV (7.5 mL) was loaded onto a single-stranded DNA agarose
column (1.8 cm<sup>2</sup> × 8 cm) equilibrated in DAB buffer. The
column was washed with 50 mL of equilibration buffer, and
a 100 mL linear gradient of 0 to 700 mM NaCl in DAB
buffer was applied. Fractions (3 mL) were collected and
assayed for double-strand uracil-DNA glycosylase activity.
Those fractions containing activity that bound to the resin
and eluted in a symmetrical peak at ∼220 mM NaCl were
pooled and concentrated (∼8-fold) as described above. After
dialysis against DAB buffer, the preparation of native Dug
(nDug) was designated fraction V, and stored at −80 °C.
</p><p><italic toggle="yes">Overproduction and Purification of Recombinant Dug</italic>. <italic toggle="yes">E.
coli</italic> JM109 containing pKK-Dug was grown at 37 °C in 9
L of YT medium supplemented with 0.01% ampicillin. After
reaching a cell density of ∼6.5 × 10<sup>8</sup> cell/mL, 100 mL of
100 mM isopropyl-β-<sc>d</sc>-thiogalactopyranoside was added to
induce <italic toggle="yes">dug</italic> expression and incubation was continued for 4
h at 37 °C. Cells were harvested by centrifugation and lysed
by sonification as described above. Ugi (3 × 10<sup>6</sup> units) was
added to the cell-free extract (400 mL) to inactivate endogenous <italic toggle="yes">E. coli</italic> Ung activity. Recombinant Dug was purified
as described above for the native enzyme with the following
modification: (i) the pellet precipitated by 50−80% ammonium sulfate was resuspended in 20 mL of UEB buffer;
(ii) fraction III was applied to a hydroxyapatite column (19.6
cm<sup>2</sup> × 3.6 cm); (iii) fraction IV was loaded onto a single-stranded DNA agarose column (4.9 cm<sup>2</sup> × 16 cm); and (iv)
fraction V was purified using a DEAE-Sephadex A50 column
(4.9 cm<sup>2</sup> × 6 cm). Active fractions from the DEAE-Sephadex
column were pooled, concentrated (∼2-fold) as described
above, dialyzed against DAB buffer, and designated recombinant Dug (rDug) fraction VI.
</p><p><italic toggle="yes">Enzyme Assays</italic>. Standard uracil-DNA glycosylase reaction
mixtures (10 μL) used for detecting <italic toggle="yes">E. coli</italic> Ung and Dug
contained 25 mM Hepes-KOH (pH 7.9), 0.5 mM EDTA, 1
mM dithiothreitol, 50 mM KCl, 0.01 mM ZnCl<sub>2</sub>, 0.1 mg/mL acetylated BSA, 0.1 pmol of [<sup>32</sup>P]U/G-34-mer, and
various amount of enzyme (2 μL) as indicated in the figure
legends. Where appropriate, enzyme samples were diluted
with Dug buffer. Incubation was conducted at 30 °C for 30
min and the reactions were then terminated by heat treatment
at 70 °C for 3 min. Apyrimidinic sites generated by uracil
removal from [<sup>32</sup>P]U/G-34-mer were quantitatively cleaved
following the addition of 0.1 units of <italic toggle="yes">E. coli</italic> endonuclease
IV, incubation at 37 °C for 30 min, and reaction termination
at 70 °C for 3 min. An equal volume of denaturing sample
buffer was added to the reaction mixtures, which were then
heated at 95 °C for 3 min. Samples (10 μL) were analyzed
by denaturing 12% polyacrylamide/8.3 M urea gel electrophoresis as previously described by Bennett et al. (<italic toggle="yes"><xref rid="bi0007066b00007" ref-type="bibr"></xref></italic>). Gels
were dried under vacuum, autoradiography was performed
using X-OMAT AR film (Eastman Kodak Co.), and quantification of <sup>32</sup>P radioactivity was performed using a PhosphorImager (Molecular Dynamics) and ImageQuant software.
During the purification of <italic toggle="yes">E. coli</italic> Dug, enzyme activity was
measured as described above except (i) reaction mixtures
(100 μL) contained 20 μL of each fraction; (ii) 1000 units
of Ugi was added to each reaction; (iii) 4 pmol of [<sup>32</sup>P]U/G-34-mer was used as substrates; and (iv) incubation
occurred at 30 °C for 16 h. Reactions were terminated with
an equal volume of stop solution (2% SDS, 50 mM EDTA).
Each sample was adjusted to 0.3 mg/mL yeast tRNA and 2
M ammonium acetate, and extracted with phenol:chloroform
(1:1). DNA was then ethanol precipitated, and resuspended
in 20 μL of distilled water. Samples (5 μL) were treated
with <italic toggle="yes">E. coli</italic> endonuclease IV, denaturing polyacrylamide gel
electrophoresis was conducted, and [<sup>32</sup>P]DNA bands were
detected as described above.
</p><p><italic toggle="yes">Polyacrylamide Gel Electrophoresis</italic>. SDS−polyacrylamide slab gel electrophoresis was conducted as described
by Bennett et al. (<italic toggle="yes"><xref rid="bi0007066b00006" ref-type="bibr"></xref></italic>) with some modifications. Briefly,
samples were mixed with an equal volume of denaturation
buffer containing 50 mM Tris-HCl (pH 6.8), 429 mM
2-mercaptoethanol, 30% (w/v) glycerol, 0.04% bromphenol
blue, and 1% SDS. After heating at 100 °C for 3 min,
samples were applied to a discontinuous SDS−polyacrylamide gel (3% stacking gel, 12.5% resolving gel) and
electrophoresis was performed at room temperature and 100
V (stacking gel) and 200 V (resolving gel), until the tracking
dye migrated ∼9 cm. Gels were fixed in 10% acetic acid
and 50% methanol, and protein bands were detected by
staining with 0.05% Coomassie Brilliant Blue G-250 (<italic toggle="yes"><xref rid="bi0007066b00006" ref-type="bibr"></xref></italic>), or
by silver staining with RAPID-Ag−STAIN (ICN Radiochemicals).
</p><p>Activity gel electrophoresis was similarly conducted except
that the denaturation buffer contained 108 mM Tris-HCl (pH
6.8), 2.4 mM EDTA, 244 mM 2-mercaptoethanol, 12.5%
(w/v) glycerol, 1.6% SDS, and 0.06% bromphenol blue. In
addition, 2 mM EDTA was added to both the 3% stacking
gel and 12.5% resolving gel, 50 μg/mL fibrinogen was
included in the resolving gel, and electrophoresis was
performed at 4 °C (<italic toggle="yes"><xref rid="bi0007066b00031" ref-type="bibr"></xref></italic>). Following electrophoresis, the gel
was immersed in 3 gel volumes of SDS-extraction buffer
[10 mM Tris-HCl (pH 7.5), 5 mM mercaptoethanol, and 25%
(v/v) 2-propanol] and agitated for 30 min at room temperature. After repeating the SDS-extraction step, the gel was
sliced horizontally into ∼3.5 mm sections that were crushed
and incubated overnight (16 h) at 4 °C with vigorous shaking
in 500 μL of elution buffer [25 mM Hepes-KOH (pH 7.9),
0.5 mM EDTA, 1 mM dithiothreitol, 50 mM KCl, 0.01 mM
ZnCl<sub>2</sub>, and 0.1 mg/mL acetylated BSA]. Samples (40 μL)
were assayed for uracil-DNA glycosylase activity using
[<sup>32</sup>P]U/G-34-mer as substrate.
</p><p>Denaturing DNA sequencing gels (30 × 40 × 0.08 cm)
containing 12% acrylamide, 0.41% <italic toggle="yes">N</italic>,<italic toggle="yes">N</italic>‘-methylene-bis-acrylamide, 8.3 M urea, and TBE buffer (90 mM Tris-base,
90 mM boric acid, and 2 mM EDTA) were used to analyze
uracil-DNA glycosylase reaction products as previously
described (<italic toggle="yes"><xref rid="bi0007066b00007" ref-type="bibr"></xref></italic>). [<sup>32</sup>P]DNA samples were mixed with an equal
volume of denaturing sample buffer, heated at 95 °C for 3
min, and electrophoresis performed at 1200 V until the
tracking dye migrate 18 cm. Autoradiography was conducted
using X-OMAT AR film, and quantitation of [<sup>32</sup>P]DNA
bands was performed using PhosphorImager (Molecular
Dynamics) and ImageQuant Software.
</p><p><italic toggle="yes">Band Mobility Shift Assays</italic>. Standard DNA binding
reaction mixtures (10 μL) contained 25 mM Hepes-KOH
(pH 7.9), 0.5 mM EDTA, 1 mM dithiothreitol, 50 mM KCl,
0.01 mM ZnCl<sub>2</sub>, 0.1 mM acetylated BSA, 10 nM of various
double-stranded [<sup>32</sup>P]DNA probes, and rDug as indicated.
Competitor oligonucleotide (400 nM C/G-34-mer) was mixed
with duplex [<sup>32</sup>P]34-mer probes on ice prior to adding rDug
as indicated. DNA binding reactions were carried out at 30
°C for 30 min, samples (5 μL) mixed with 1.5 μL of 50%
sucrose, and loaded onto a nondenaturing 6% polyacrylamide
gel (17 × 14 × 0.08 cm). Electrophoresis was conducted at
room temperature and 140 V with TAE buffer [40 mM Tris-acetate and 1 mM EDTA (pH 8.0)] until the bromphenol
blue dye, which was loaded in adjacent lanes, migrated ∼6
cm (<italic toggle="yes"><xref rid="bi0007066b00035" ref-type="bibr"></xref></italic>). The gel was then dried and [<sup>32</sup>P]DNA bands were
detected by autoradiography. The relative amounts of free
and mobility-shifted <sup>32</sup>P-labeled oligonucleotide were measured using a PhosphorImager and ImageQuant software.
</p><p><italic toggle="yes">Base Excision DNA Repair Reactions</italic>. Uracil-mediated
base excision repair reactions were conducted with cell-free
extracts of <italic toggle="yes">E. coli</italic> NR8052 using a procedure similar to that
developed by Sanderson and Mosbaugh (<italic toggle="yes"><xref rid="bi0007066b00030" ref-type="bibr"></xref></italic>). <italic toggle="yes">E. coli</italic> grown
at 37 °C in YT medium to mid-log phase were harvested by
centrifugation, the cell pellet (2.5 g wet weight) was
resuspended in 40 mL of sonification buffer, and cells were
lysed with a Dismembrator (Fisher, model 300) as previously
described (<italic toggle="yes"><xref rid="bi0007066b00034" ref-type="bibr"></xref></italic>). After centrifugation at 20000<italic toggle="yes">g</italic> for 20 min at
4 °C, 14 g of powdered ammonium sulfate was slowly added
to the supernatant fraction (40 mL), and the protein precipitate formed after a 10 min incubation at 0 °C was recovered
by centrifugation. The pellet was resuspended in 10 mL of
R buffer [50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM
dithiothreitol and 10% (w/v) glycerol], dialyzed against the
same buffer, and the cell-free extract protein concentration
(∼12 mg/mL) was determined.
</p><p>Standard BER reaction mixtures contained 100 mM Tris-HCl (pH 7.5), 5 mM MgCl<sub>2</sub>, 1 mM dithiothreitol, 0.1 mM
EDTA, 2 mM ATP, 0.5 mM β-NAD, 20 μM each dATP,
dTTP, dGTP, and dCTP, 5 mM phosphocreatine di-Tris salt,
200 units/ml of phosphocreatine kinase, 10 000 units/mL of
Ugi, 10 μg/mL of M13mp2op14 (U·G) heteroduplex DNA
(form I) and 1 mg/mL of <italic toggle="yes">E. coli</italic> NR8052 cell-free extract.
In some cases, rDug was added to cell-free extracts prior to
incubation at 30 °C for various amounts of time. Samples
(100 μL) were removed, the reaction was terminated on ice,
adjusted to 20 mM EDTA and then heated at 70 °C for 3
min. RNase A was then added to 80 μg/mL and incubated
at 37 °C for 10 min. Following the addition of SDS to 0.5%,
proteinase K was added to 190 μg/mL, and each sample was
incubated for 30 min at 37 °C. The samples were subsequently extracted with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1), ethanol precipitated, washed with
75% ethanol, and resuspended in 20 μL of TE buffer.
</p><p>Repair of the M13mp2op14 DNA recovered from the BER
reactions described above was analyzed as follows. DNA
samples (5 μL) were incubated with 100 units of <italic toggle="yes">E. coli</italic> Ung
for 30 min at 37 °C. One unit of Ung activity is defined as
the amount of enzyme that release 1 nmol of uracil/h under
standard conditions, as previously described (<italic toggle="yes"><xref rid="bi0007066b00006" ref-type="bibr"></xref></italic>). The reaction
was terminated by addition of 1000 units of Ugi, then 1 unit
of <italic toggle="yes">E. coli</italic> endonuclease IV was added and incubation
continued for 30 min at 37 °C. After heating at 70 °C for 3
min, each sample (8 μL) was mixed with 2 μL of agarose
gel sample buffer that contained 0.5% SDS, 50 mM EDTA,
25% (w/v) glycerol and 0.05% bromphenol blue. Samples
(10 μL) were then loaded on to a 0.8% agarose gel (12 ×
13 × 0.5 cm) containing TAE buffer [40 mM Tris-acetate
and 1 mM EDTA (pH 8.0)] and 0.2 μg/mL ethidium
bromide. Electrophoresis was performed in TAE buffer at
100 V until the tracking dye migrated ∼70% of the gel
length. Ethidium-bromide-stained DNA bands were visualized by transillumination (302 nm), and the relative percentage of form I and II DNA was determined using a
concentration series of M13mp2op14 DNA standards (form
I and II) run on the same gel. Ethidium bromide staining
intensity was measured using an Image Store 7500 (Ultra
Violet Products) gel documentation system and ImageQuant
software. After correcting for the ∼0.7-fold reduced staining
intensity of form I DNA relative to form II DNA, the
percentage of form I DNA in each sample was determined.
</p><p><italic toggle="yes">Protein Measurement</italic>. The protein concentration of <italic toggle="yes">E. coli</italic>
crude extracts and partially purified Dug preparations was
determined by the Bradford reaction (<italic toggle="yes"><xref rid="bi0007066b00036" ref-type="bibr"></xref></italic>) using the Bio-Rad
Protein Assay. <italic toggle="yes">E. coli</italic> Ung (fraction V), Dug (fraction V
and VI),<xref rid="bi0007066b00005" ref-type="bibr"></xref> and Ugi (fraction IV) protein concentrations were
determined by absorbance spectroscopy using molar extinction coefficients ε<sub> 280 nm</sub> = 4.2 × 10<sup>4</sup>, 2.4 × 10<sup>4</sup>, and 1.2 ×
10<sup>4</sup> L/mol cm, respectively<xref rid="bi0007066b00006" ref-type="bibr"></xref> (<italic toggle="yes"><xref rid="bi0007066b00006" ref-type="bibr"></xref></italic>).
</p><p><italic toggle="yes">Mass Spectrometry</italic>. MALDI mass spectrometry was
conducted using a custom-built time-of-flight mass spectrometer equipped with a two-stage delayed extraction source
by the Mass Spectrometry Facilities and Service Core Unit
(Environmental Health Science Center, Oregon State University). Approximately 1 μL of purified rDug (fraction VI,
0.16 mg/mL) was mixed with 3 μL of 4-hydroxy-α-cyanocinnamic acid in 0.1% trifluoroacetic acid, 33% acetonitrile.
A droplet (∼0.5 μL) of this analyte/matrix solution was
deposited on a precrystallized matrix sample probe and
allowed to air-dry. Mass spectra were produced by irradiation
of the sample with 30 individual laser pulses and the summed
signals were calibrated using ions from an external calibrant.
</p></sec><sec id="d7e881"><title>Results</title><p><italic toggle="yes">Detection of Ugi-Insensitive Uracil-DNA Glycosylase Activity in E. coli Cell-Free Extracts</italic>. To initiate this investigation
of enzyme activities in <italic toggle="yes">E. coli</italic> cell extracts that catalyze the
removal of uracil from DNA, 5‘-end <sup>32</sup>P-labeled synthetic
duplex oligonucleotides were prepared that contained either
a site-specific U·A base pair or U·G mispair. The 34-mer
DNA substrates (U/A-34-mer and U/G-34-mer) were reacted
with various amounts of <italic toggle="yes">E. coli</italic> NR8051 (<italic toggle="yes">ung<sup>+</sup></italic><sup></sup>) or NR8052
(<italic toggle="yes">ung<sup>-</sup></italic><sup></sup>) cell extracts in the absence or presence of the uracil-DNA glycosylase-specific inhibitor protein (Ugi) as shown
in Figure <xref rid="bi0007066f00001"></xref>. AP-sites generated during the incubation were
cleaved by treatment with exogenous <italic toggle="yes">E. coli</italic> endonuclease
IV, which incised the deoxyribose-phosphate ester bond on
the 5‘-side of the AP-site, resulting in a [<sup>32</sup>P]15-mer product.
Analysis of reaction products by denaturing polyacrylamide
gel electrophoresis revealed that both U/A-34-mer and U/G-34-mer were efficiently processed in the NR8051 cell extract
(Figure <xref rid="bi0007066f00001"></xref>A). When Ugi was included in these reaction
mixtures, uracil-excision activity on the U/A-34-mer was
significantly inhibited; however, a reduced level of activity
was detected on the U/G-34-mer (Figure <xref rid="bi0007066f00001"></xref>B). In the reaction
mixtures containing the NR8052 cell extract, uracil-excision
activity was observed solely on the U/G-34-mer substrate
(Figure <xref rid="bi0007066f00001"></xref>C). Furthermore, the activity was not inhibited by
Ugi (Figure <xref rid="bi0007066f00001"></xref>D). The relative amount of uracil excision
detected for each DNA substrate, U/A-34-mer and U/G-34-mer, was determined and plotted in Figure <xref rid="bi0007066f00001"></xref>, panels E and
F, respectively.
<fig id="bi0007066f00001" position="float" orientation="portrait"><label>1</label><caption><p>Detection of Ugi-insensitive uracil-DNA glycosylase activity in<italic toggle="yes">E. coli </italic>cell-free extracts. Two 5‘-end <sup>32</sup>P-labeled duplex
oligonucleotides ([<sup>32</sup>P]U/A-34-mer and [<sup>32</sup>P]U/G-34-mer) containing
a site-specific uracil residue located at position 16 of the radioactively labeled strand were prepared as described under Materials
and Methods. Reaction mixtures (100 μL) containing 25 mM Hepes-KOH (pH 7.9), 0.5 mM EDTA, 1 mM dithiothreitol, 50 mM KCl,
0.1 mM ZnCl<sub>2</sub>, 0.1 mg/mL acetylated BSA, 0.1 pmol of [<sup>32</sup>P]U/A-
or [<sup>32</sup>P]U/G-34-mer as indicated, and either 0, 0.16, 0.63, 2.5, 10,
or 40 μg of extract protein (lanes 1−6, respectively) from <italic toggle="yes">E. coli</italic>
NR8051 (<italic toggle="yes">ung<sup>+</sup></italic><sup></sup>) cells (A and B) or NR8052 (<italic toggle="yes">ung<sup>-</sup></italic><sup></sup>) cells (C and D)
were incubated with (B and D) or without (A and C) Ugi (1000
units) at 30 °C for 2 h. A control reaction (lane C) was also prepared
that lacked Ugi but contained 400 units of <italic toggle="yes">E. coli</italic> Ung. A similar
reaction (lane I) was conducted following the addition of 1000 units
of Ugi (B and D). After incubation, each reaction was terminated
with stop solution and the DNA was isolated, AP-sites hydrolyzed,
and reaction products resolved by denaturing polyacrylamide as
described under Materials and Methods. <italic toggle="yes">Arrows</italic> indicate the location
on the autoradiogram of unreacted [<sup>32</sup>P]34-mer substrate (S) and
[<sup>32</sup>P]15-mer product (P). The amount of [<sup>32</sup>P]DNA detected in each
band was determined using a PhosphorImager and the percentage
of product formed was calculated by dividing the amount of [<sup>32</sup>P]15-mer by that of [<sup>32</sup>P]15-mer plus [<sup>32</sup>P]34-mer and multiplying
by 100. The percentage of product generated in reactions containing
[<sup>32</sup>P]U/A-34-mer (E) or [<sup>32</sup>P]U/G-34-mer (F) substrate and various
cell-free extracts is shown as <italic toggle="yes">E. coli</italic> NR8052 extract plus Ugi (□),
NR8051 minus Ugi (○), NR8052 plus Ugi (▪), and NR8052 minus
Ugi (·).</p></caption><graphic xlink:href="bi0007066f00001.tif" position="float" orientation="portrait"></graphic></fig></p><p><italic toggle="yes">E. coli</italic> NR8051 cell extract was active on both substrates,
but more active (∼1.6-fold) on the mispaired U/G-34-mer
relative to the paired U/A-34-mer duplex. That the uracil-excision activity of the NR8051 cell extract was significantly
inhibited by Ugi indicated that the enzyme responsible for
the majority of uracil processing on both substrates was most
likely Ung. The extent of Ugi-insensitive uracil-excision on
U/G-34-mer observed in the NR8051 cell extract was
comparable to that observed on U/G-34-mer in the NR8052
cell extract. Since this activity was not inhibited by Ugi and
preferred the mispaired U/G-34-mer, we concluded it could
not be synonymous with Ung.
</p><p><italic toggle="yes">Purification of the Ugi-Insensitive Uracil-DNA Glycosylase Activity.</italic> The Ugi-insensitive uracil-DNA glycosylase
activity was purified from <italic toggle="yes">E. coli</italic> strain NR8052 (<italic toggle="yes">ung<sup>-</sup></italic><sup></sup>) after
Ugi was added to the cell extract to inactivate any residual
Ung activity (see Supporting Information, Figure <xref rid="bi0007066f00001"></xref>). Active
fractions from the final purification step were pooled and
analyzed by SDS−polyacrylamide gel electrophoresis, and
a single protein band was visualized by silver staining (Figure
<xref rid="bi0007066f00002"></xref>A, lane I). Activity gel analysis was carried out to determine
whether the protein band corresponded with the location of
Ugi-insensitive uracil-DNA glycosylase activity (Figure <xref rid="bi0007066f00002"></xref>B).
Using the [<sup>32</sup>P]U/G-34-mer cleavage assay, enzymatic activity was detected which comigrated with the silver-stained
band (Figure <xref rid="bi0007066f00002"></xref>). The apparent molecular weight of the
polypeptide was calculated to be ∼21 000 from both the
location of the silver-stained band and position of activity
gel slice 18 (Figure <xref rid="bi0007066f00002"></xref>C). Thus, the Ugi-insensitive enzyme
was purified to apparent homogeneity and consisted of a
single polypeptide, referred to as Dug.
<fig id="bi0007066f00002" position="float" orientation="portrait"><label>2</label><caption><p>SDS−polyacrylamide gel electrophoresis of native and recombinant Dug preparations. (A) Two sets of samples (40 μL each lane, 4 lanes total) of nDug fraction V (2.2 μg) and rDug fraction VI (6.3 μg) were loaded onto a 12.5% SDS-polyacrylamide gel along with molecular weight standards, and electrophoresis was conducted as described under the Materials and Methods. Following electrophoresis, the gel was cut in half vertically between the sample sets and one-half of the gel containing nDug (lane I) and rDug (lane II) was silver-stained. The direction of migration was from left to right and the location of tracking dye (TD) is indicated by an<italic toggle="yes">arrow</italic>. (B) After electrophoresis, the other half of the gel was
vertically cut between the sample lanes, SDS extracted, and each
lane horizontally sliced into ∼3.5 mm segments. Protein was eluted
and renatured from the gel slices as described in the Materials and
Methods. Samples (40 μL) were assayed for uracil-DNA glycosylase activity, reaction products analyzed on a denaturing 12%
polyacrylamide/8.3 M urea gel, and the autoradiogram corresponding to nDug (fraction V) is shown. The location of the [<sup>32</sup>P]U/G-34-mer substrate (S) and [<sup>32</sup>P]15-mer product (P) are indicated by
<italic toggle="yes">arrows</italic>. (C) A standard curve (log M<sub>r</sub> versus <italic toggle="yes">R<sub>f</sub></italic>) was generated
based on the relative mobility of prestained protein molecular weight
markers (Bio-Rad) for phosphorylase b (<italic toggle="yes">M</italic><sub>r</sub> 111 000), BSA (<italic toggle="yes">M</italic><sub>r</sub>
77 000), ovalbumin (<italic toggle="yes">M</italic><sub>r</sub> 48 200), carbonic anhydrase (<italic toggle="yes">M</italic><sub>r</sub> 33 800),
trypsin inhibitor (<italic toggle="yes">M</italic><sub>r</sub> 28,600), and lysozyme (<italic toggle="yes">M</italic><sub>r</sub> 20 500). The
apparent molecular weight of nDug was determined based on the
Rf for the midpoint of the gel slice (fraction 18, <italic toggle="yes">horizontal line</italic>) as
indicated by the <italic toggle="yes">vertical arrow</italic>.</p></caption><graphic xlink:href="bi0007066f00002.tif" position="float" orientation="portrait"></graphic></fig></p><p><italic toggle="yes">Cloning, Overexpression, and Purification of Recombinant
Dug.</italic> On the basis of the report of Gallinari and Jiricny (<italic toggle="yes"><xref rid="bi0007066b00025" ref-type="bibr"></xref></italic>),
we decided to clone the <italic toggle="yes">E. coli</italic> gene encoded by an open
reading frame of 169 amino acids which was associated with
double-strand-specific uracil-DNA glycosylase activity. The
nucleotide sequence of this ORF was amplified using <italic toggle="yes">E. coli</italic>
NR8052 chromosomal DNA as template and cloned into the
overexpression vector pKK223−3; the resultant construct
was designated pKK-Dug. Attempts to transform <italic toggle="yes">E. coli</italic>
NR8052 (<italic toggle="yes">ung<sup>-</sup></italic><sup></sup>) with pKK-Dug were unsuccessful; however,
transformation of<italic toggle="yes"> E. coli</italic> JM109, an <italic toggle="yes">ung<sup>+</sup></italic><sup></sup> strain, was
successful for overproduction of recombinant protein. To
separate double-strand uracil-DNA glycosylase activity from
the endogenous Ung activity of the host bacterial cells, Ugi
was added to the cell extracts before performing purification.
Recombinant Dug was purified using the procedure described
for the native enzyme with some modifications. DEAE-Sephadex chromatography was included as the final step,
where the activity was detected in the flow-through fractions.
SDS−polyacrylamide gel electrophoresis analysis of the
active fractions revealed a single protein with an apparent
molecular weight of ∼22 000, as visualized by silver staining.
Thus, the purification procedure resulted in the isolation of
an apparently homogeneous protein preparation (see Supporting Information, Figure <xref rid="bi0007066f00002"></xref>).
</p><p><italic toggle="yes">SDS−Polyacrylamide Gel Analysis of Native and Recombinant Double-Strand Uracil-DNA Glycosylase.</italic> To explore
the possibility that the native and recombinant protein might
be the same enzyme, two sets of samples from each
preparation were resolved by SDS−polyacrylamide gel
electrophoresis and protein bands were visualized by silver-staining as shown in Figure <xref rid="bi0007066f00002"></xref>A. Side by side comparison of
their electrophoretic mobility showed that the two protein
bands comigrated. Activity gel analysis established that the
protein band corresponded to the Ugi-insensitive uracil-DNA
glycosylase activity in both the nDug (Figure <xref rid="bi0007066f00002"></xref>) and rDug
(data not shown) preparations. From these observations, we
inferred that the recombinant protein and native Dug were
indistinguishable. Therefore, the recombinant double-strand
uracil-DNA glycosylase preparation was referred to as
recombinant Dug (rDug).
</p><p><italic toggle="yes">Molecular Weight Determination of Dug by MALDI Mass
Spectrometry.</italic> Although the polypeptide molecular weight
estimates (∼21 000) of both the native and recombinant Dug
proteins were close to the predicted molecular weight of the
polypeptide (18 672) encoded by the ORF-169, mass determination by MALDI mass spectrometry was conducted to
more accurately determine the molecular weight and verify
the identity of Dug. The summed spectra produced one major
singly charged peak of molecular weight 18 670 (see
Supporting Information, Figure <xref rid="bi0007066f00003"></xref>). Since the experimentally
determined molecular weight of Dug was in excellent
agreement with that predicted (±0.01%), it appeared that
the protein did not undergo posttranslational modification.
<fig id="bi0007066f00003" position="float" orientation="portrait"><label>3</label><caption><p>DNA substrate specificity of Dug and Ung. Seven sets of standard uracil-DNA glycosylase reaction mixtures containing either single- or double-stranded [<sup>32</sup>P]34-mer oligonucleotide
substrates with U-, U·A-, U·G-, T·G-, εC-, εC·A-, or εC·G-target
residues were prepared as described under Materials and Methods.
After adding 4 and 800 nM rDug (white and black bars, respectively) or 2 pM and 8 nM Ung (striped and stippled bars,
respectively), each reaction (10 μL) was incubated for 30 min at
30 °C. Following the incubation, 5 μL of each reaction was
quenched with 2.5 μL of buffer containing 0.3 M NaOH and 30
mM EDTA. Samples were heated for 30 min at 90 °C to cleave
AP-sites and analyzed on a denaturing 12% polyacrylamide/8.3 M
urea gel as described in Figure <xref rid="bi0007066f00001"></xref>. The percentage of product ([<sup>32</sup>P]15-mer) formed was determined using a PhosphorImager.
</p></caption><graphic xlink:href="bi0007066f00003.tif" position="float" orientation="portrait"></graphic></fig></p><p><italic toggle="yes">Comparison of Dug and Ung Substrate Specificity.</italic> To
compare the relative substrate specificity of <italic toggle="yes">E. coli</italic> Ung and
Dug, 5‘-end <sup>32</sup>P-labeled 34-mer oligonucleotides containing
site-specific U, U·A, U·G, T·G, εC, εC·A, or εC·G residues
were prepared. Each substrate was incubated with Ung or
Dug to catalyze base excision, and the resulting AP-sites
were cleaved by hot-alkali treatment rather than enzymatic
cleavage by <italic toggle="yes">E. coli</italic> endonuclease IV, as single-stranded DNA
is known to be a poor substrate for this enzyme (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0007066b00007" ref-type="bibr"></xref>, <xref rid="bi0007066b00037" ref-type="bibr"></xref></named-content></italic>).
Analysis of reaction products by denaturing polyacrylamide
gel electrophoresis revealed that Dug was more active on
the εC-containing duplex oligonucleotide than on the mispaired U/G-34-mer, and that the excision of εC did not show
strict preference for the opposite strand base (Figure <xref rid="bi0007066f00003"></xref>). In
contrast, Dug was considerably more active on the mispaired
U/G-34-mer than the paired U/A-34-mer oligonucleotide.
However, at a higher concentration of Dug (800 nM) the
excision of uracil from the U/A-34-mer was significantly
increased, whereas Dug activity on single-stranded U- and
εC-34-mer, as well as duplex T/G-34-mer, was not detected.
In contrast, Ung was more active on single-stranded U-34-mer, and about 2-fold more active on the mispaired U/G-34-mer relative to the paired U/A-34-mer duplex. Furthermore, Ung-mediated base-excision was not detected on
substrates containing εC·G or T·G mispairs. When taken
together, these results clearly illustrate a difference in the
substrate specificity of Dug and Ung activities.
</p><p><italic toggle="yes">Effect of Uracil and Ugi on Dug and Ung Activity.</italic>
Inhibition studies were conducted to examine the relative
effect of free uracil and Ugi on Dug and Ung activity.
Standard uracil-DNA glycosylase assays were performed in
the presence of various amounts of free uracil or Ugi, and
the percent of activity relative to the control was determined
(Figure <xref rid="bi0007066f00004"></xref>). Whereas, Ung activity determined on [<sup>32</sup>P]U/G-34-mer was inhibited ∼50% by 1 mM free uracil (Figure
<xref rid="bi0007066f00004"></xref>A), Dug activity was not significantly affected by as much
as 2 mM free uracil. In addition, Ung was inhibited by Ugi,
whereas, the activity of Dug was not affected by a 4-fold
molar excess of Ugi (Figure <xref rid="bi0007066f00004"></xref>B). These results provide
additional evidence that the uracil-excision activity of the
Dug preparation was an intrinsic property of the purified
protein, and not the consequence of <italic toggle="yes">E. coli</italic> Ung contamination.
<fig id="bi0007066f00004" position="float" orientation="portrait"><label>4</label><caption><p>Effect of uracil and Ugi on Dug and Ung activity. Two sets of standard uracil-DNA glycosylase reaction mixtures were prepared containing 10 nM [<sup>32</sup>P]U/G-34-mer and either 5 nM rDug
(○) or 5 pM Ung (·) as described under Materials and Methods.
To each set of reactions various amounts of (A) uracil (0, 0.05,
0.1, 0.25, 0.5, 1, and 2 mM) or (B) Ugi (0, 0.25, 0.5, 2, 8, 16, 32,
and 20 000 pM) were added as indicated above. After incubation
for 30 min at 30 °C, reactions were terminated by heating at 70 °C
for 3 min, then treated with 0.1 units of <italic toggle="yes">E. coli</italic> endonuclease IV.
Reaction products were analyzed on denaturing 12% polyacrylamide/8.3 M urea gels and [<sup>32</sup>P]DNA bands were quantitatively
measured using a PhosphorImager as described in Figure <xref rid="bi0007066f00001"></xref>. Enzyme
activity (%) was determined relative to the control reaction which
lacked uracil or Ugi addition: 100% activity corresponded to 61
and 77 fmol of [<sup>32</sup>P]U/G-34-mer converted to product for Dug and
Ung, respectively.</p></caption><graphic xlink:href="bi0007066f00004.tif" position="float" orientation="portrait"></graphic></fig></p><p><italic toggle="yes">Effect of E. coli Endonuclease IV on Dug Activity.</italic> To
examine the effect of <italic toggle="yes">E. coli</italic> endonuclease IV on the activity
of Dug, the rate of uracil excision was determined for the
[<sup>32</sup>P]U/G-34-mer substrate in the absence or presence of
endonuclease IV (see Supporting Information, Figure <xref rid="bi0007066f00004"></xref>). Dug
activity in standard assays using 2.5 nM Dug exhibited an
initial fast reaction rate (0−1 min), but then the reaction
dramatically slowed and essentially stopped after 1 h. Upon
examination, the concentration of uracil removed (1.7 nM)
after a 1 h incubation was determined to be less than the
concentration of the enzyme added to the reaction. Therefore,
the enzyme did not appear to turn over. In contrast, the
addition of 10 nM endonuclease IV enhanced the reaction
rate of Dug during and after the initial burst phase of the
reaction. Endonuclease IV alone did not exhibit detectable
uracil-DNA glycosylase or incision activity (data not shown).
The maximum extent of uracil excision from the U/G-34-mer was increased ∼5.5-fold relative to the Dug reaction
conducted in the absence of endonuclease IV.
</p><p><italic toggle="yes">Effect of E. coli Endonuclease IV on Dug DNA Binding
and Catalysis.</italic> Electrophoretic mobility shift assays were
conducted to determine whether Dug forms a stable protein−DNA complex. Standard Dug−DNA binding reactions
containing 10 nM [<sup>32</sup>P]U/G-34-mer and various amounts of
Dug were incubated in the absence or presence of 10 nM
endonuclease IV. Following the binding reaction, half of the
sample was analyzed by nondenaturing polyacrylamide gel
electrophoresis (Figure <xref rid="bi0007066f00005"></xref>A). In the absence of endonuclease
IV, the [<sup>32</sup>P]U/G-34-mer probe was mobility shifted in a
concentration-dependent manner. When endonuclease IV was
included in the binding reaction mixture, the extent of Dug−DNA complex formation at equivalent Dug concentrations
became less intense. These results demonstrated that endonuclease IV affected the stability of the Dug−DNA interaction.
<fig id="bi0007066f00005" position="float" orientation="portrait"><label>5</label><caption><p>Effect of<italic toggle="yes">E. coli</italic> endonuclease IV on DNA binding and
catalytic activity of Dug. (A) Electrophoretic mobility shift assay.
Two sets of standard uracil-DNA glycosylase reaction mixtures
containing 0, 0.25, 0.5, 1, 2, 4, and 8 nM rDug (lanes 1−7,
respectively), and 10 nM [<sup>32</sup>P]U/G-34-mer were prepared in the
presence (+) or absence (−) of 10 nM <italic toggle="yes">E. coli</italic> endonuclease IV.
After incubation at 30 °C for 30 min, one-half of each reaction
mixture (5 μL) was mixed with 1.5 μL of 50% sucrose, and samples
(5 μL) were analyzed by nondenaturing polyacrylamide (6%) gel
electrophoresis as described under Materials and Methods. Autoradiography was performed and the location of the free (F) and
bound (B) [<sup>32</sup>P]DNA probe are indicated by <italic toggle="yes">arrows</italic>. (B) To the
remainder of each reaction mixture (5 μL), 2.5 μL of stop solution
containing 0.3 M NaOH and 30 mM EDTA was added, and each
sample was heated at 90 °C for 30 min to cleave the generated
AP-sites. Samples were then analyzed on a denaturing 12%
polyacrylamide/8.3 M urea gel as described in Figure <xref rid="bi0007066f00001"></xref>. Autoradiography was performed and the arrows indicate the location of
the [<sup>32</sup>P]34-mer DNA substrate (S) and reaction products (P)
produced by NaOH treatment alone (−Endo IV) or by a combination of <italic toggle="yes">E.</italic> <italic toggle="yes">coli </italic>endonuclease IV and NaOH treatment (+Endo IV).
(C) The <sup>32</sup>P radioactivity was measured for each band shown in
panels A and B using a PhosphorImager. The amount of [<sup>32</sup>P]34-mer detected (A) in the bound form was calculated for each reaction
mixture conducted in the presence (▪) or absence (□) of endonuclease IV. The amount of cleaved [<sup>32</sup>P]U/G-34-mer (B) was
determined for the reactions performed with (·) and without (○)
endonuclease IV, and plotted as a function of the concentration of
rDug in each reaction mixture.</p></caption><graphic xlink:href="bi0007066f00005.tif" position="float" orientation="portrait"></graphic></fig></p><p>The second aliquot of the binding reaction mixture was
treated with hot-alkali and subjected to denaturing polyacrylamide gel electrophoresis to determine whether uracil-excision had occurred during the binding reaction (Figure
<xref rid="bi0007066f00005"></xref>B). Product analysis of binding reactions carried out in the
absence of endonuclease IV showed a single [<sup>32</sup>P]15-mer
band that increases in intensity with increasing Dug concentration. Analysis of the reactions containing endonuclease
IV revealed the presence of a second <sup>32</sup>P-labeled product
band just above the [<sup>32</sup>P]15-mer generated by hot-alkali
treatment; this relatively slow-migrating band was the
product of cleavage by endonuclease IV. The slow-migrating
band occurred earlier in the concentration series than the
lower band and represents enzyme turnover; the lower band
presumably resulted from Dug-bound AP-site DNA that was
inaccessible to endonuclease IV and was subsequently
cleaved by the hot-alkali treatment. This interpretation was
consistent with an initial β-elimination cleavage reaction
catalyzed by hot alkali on the 3‘-side of the AP-site, followed
by loss of the ring-opened deoxyribose (4-hydroxy-2-pentenal) by a second β-elimination reaction (δ elimination)
to produce a [<sup>32</sup>P]15-mer with a 3‘-terminal phosphate, and
cleavage 5‘ to the AP-site by endonuclease IV, which
generates [<sup>32</sup>P]15-mer with a 3‘-terminal hydroxyl (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0007066b00019" ref-type="bibr"></xref>, <xref rid="bi0007066b00038" ref-type="bibr"></xref></named-content></italic>).
Quantification of the DNA binding results revealed that, in
the absence of endonuclease IV, the amount of Dug-bound
DNA formed was equal to the amount of AP-site-containing
DNA (product DNA) (Figure <xref rid="bi0007066f00005"></xref>C). Moreover, the amount of
oligonucleotide cleaved was approximately equal to the
amount of enzyme in the reaction, suggesting that Dug bound
tightly to the DNA following uracil excision. In the presence
of endonuclease IV, the amount of bound DNA was
significantly reduced, but the rate of uracil excision was
enhanced (Figure <xref rid="bi0007066f00005"></xref>C). These results imply that endonuclease
IV stimulated the dissociation of Dug from product DNA
enabling the enzyme to turnover and participate in further
catalytic events.
</p><p><italic toggle="yes">Effect of EDTA on Endonuclease IV-Mediated Dissociation
of the Dug/AP-Site−DNA Complex.</italic> To determine if the
catalytic activity of endonuclease IV was responsible for
stimulating Dug activity, electrophoretic mobility shift assays
were conducted to examine the endonuclease IV-mediated
dissociation of Dug from AP-site-DNA in the absence or
presence of 5 mM EDTA. Previously, Levin et al. (<italic toggle="yes"><xref rid="bi0007066b00039" ref-type="bibr"></xref></italic>)
demonstrated that EDTA was a potent inhibitor of endonuclease IV activity. Binding reactions containing 20 nM
[<sup>32</sup>P]U/G-34-mer and an equal concentration of Dug resulted
in the mobility shift of more than 90% of the [<sup>32</sup>P]U/G-34-mer DNA probe, both in the presence and absence of EDTA
(Figure <xref rid="bi0007066f00006"></xref>, lane 1, left and right). This indicated that the DNA-binding of Dug was insensitive to EDTA. In the absence of
EDTA (Figure <xref rid="bi0007066f00006"></xref>, left), increasing amounts of endonuclease
IV in the DNA binding reaction resulted in a linear decline
in the amount of bound <sup>32</sup>P-labeled probe, confirming the
results obtained in Figure <xref rid="bi0007066f00005"></xref>. However, in the presence of
EDTA (Figure <xref rid="bi0007066f00006"></xref>, right), the stimulatory effect of endonuclease IV on the dissociation of Dug from its product DNA
was significantly blocked. This result implied that incision
of the AP-site by endonuclease IV was required to promote
dissociation of Dug from its product DNA.
<fig id="bi0007066f00006" position="float" orientation="portrait"><label>6</label><caption><p>Effect of EDTA on<italic toggle="yes">E. coli</italic> endonuclease IV and Dug
binding to DNA. DNA binding reaction mixtures (10 μL) containing
20 nM [<sup>32</sup>P]U/G-34-mer, 20 nM rDug, and various amounts of <italic toggle="yes">E.
coli</italic> endonuclease IV (0, 2, 5, 10, 20, and 40 nM: lanes 1−6,
respectively) were prepared with (+) and without (−) 5 mM EDTA.
After incubation at 30 °C for 30 min, samples (5 μL) were analyzed
by nondenaturing 6% polyacrylamide gel electrophoresis as described under Materials and Methods. Autoradiography was
performed and the location of the free (F) and bound (B) forms of
[<sup>32</sup>P]DNA are indicated by <italic toggle="yes">arrows</italic>. The amount of the [<sup>32</sup>P]DNA
probe detected in each band was determined using a PhosphorImager as described under Materials and Methods.</p></caption><graphic xlink:href="bi0007066f00006.tif" position="float" orientation="portrait"></graphic></fig></p><p><italic toggle="yes">Ability of Dug To Bind Duplex DNA Containing an Incised
AP-Site.</italic> Since endonuclease IV allowed the release of Dug
from its product DNA, we postulated that Dug would not
efficiently bind DNA containing an incision on the 5‘-side
of an AP-site. To test this proposition, incised AP-site duplex
DNA (*AP/G-34-mer) was prepared. Three <sup>32</sup>P-labeled DNA
duplexes (U/G-, AP/G-, and *AP/G-34-mer) were incubated
with various amounts of Dug and electrophoretic mobility
shift assays were performed. Analysis of the binding reactions showed that Dug bound preferentially to AP/G-34-mer
relative to U/G-34-mer, and that Dug was not observed to
bind to the *AP/G-34-mer containing an incision on the 5‘-side of the AP-site (Figure <xref rid="bi0007066f00007"></xref>). These findings supported the
hypothesis that the endonuclease IV-mediated stimulation
of Dug activity was the result of Dug dissociation from
incised AP-sites.
<fig id="bi0007066f00007" position="float" orientation="portrait"><label>7</label><caption><p>Ability of Dug to bind duplex DNA containing an incised AP-site. DNA binding reaction mixtures (10 μL) were prepared containing 0, 2.5, 5, 10, and 20 nM rDug (lanes 1−5, respectively), 10 nM of [<sup>32</sup>P]U/G-, [<sup>32</sup>P]AP/G-, or [<sup>32</sup>P]*AP/G-34-mer (pretreated
with <italic toggle="yes">E. coli </italic>endonuclease IV) as indicated, and 400 nM C/G-34-mer competitor oligonucleotide. After incubation at 30 °C for 30
min, samples (5 μL) were analyzed using nondenaturing 6%
polyacrylamide gel electrophoresis as described under Materials
and Methods. The location of the free (F) and bound (B) oligonucleotide probes are indicated by <italic toggle="yes">arrows</italic> on the autoradiogram.</p></caption><graphic xlink:href="bi0007066f00007.tif" position="float" orientation="portrait"></graphic></fig></p><p><italic toggle="yes">Uracil-Mediated Base Excision Repair Initiated by Dug
in E. coli Cell Extracts</italic>. To ascertain whether Dug may
participate in uracil-mediated base excision repair in <italic toggle="yes">E. coli</italic>,
we conducted BER assays using M13mp2op14 DNA (form
I) containing a single site-specific U·G mispair. To eliminate
Ung-initiated base excision repair, BER assays were performed using <italic toggle="yes">E. coli</italic> NR8052 (<italic toggle="yes">ung<sup>-</sup></italic><sup></sup>) cell extract in the
presence of excess Ugi. Form I DNA was incubated with
cell-free extracts for various times, DNA isolated, and then
treated with excess of exogenous Ung and endonuclease IV
in order to convert unrepaired molecules to form II DNA.
Analysis of reaction products by agarose gel electrophoresis
revealed the time dependent appearance of repaired form I
DNA (Figure <xref rid="bi0007066f00008"></xref>A, lanes 1−7, left). To investigate whether
Dug was indeed involved in the repair reaction, we supplemented the BER reaction with purified rDug. Under this
condition, the amount of Ung/Endo IV-resistant form I DNA
formed was significantly increased (Figure <xref rid="bi0007066f00008"></xref>A, lanes 1−7,
right). Quantification of the reaction products revealed that
the rate of uracil-DNA repair was enhanced and the extent
of repaired form I DNA was increased by ∼2.5-fold after a
60 min reaction (Figure <xref rid="bi0007066f00008"></xref>B). These results demonstrated that
Dug participated in the <italic toggle="yes">E. coli</italic> base excision repair reaction,
by carrying out uracil-excision in the first step of the
pathway.
<fig id="bi0007066f00008" position="float" orientation="portrait"><label>8</label><caption><p>Uracil-mediated base excision DNA repair in<italic toggle="yes">E. coli</italic>
(<italic toggle="yes">ung<sup>-</sup></italic><sup></sup>) cell-free extracts. (A) Two base excision repair reaction
mixtures (800 μL) containing 10 μg/mL of M13mp2op14 (U·G)
DNA and 1 mg/mL of <italic toggle="yes">E. coli</italic> NR8052 cell extract protein were
prepared either with (+) or without (−) 80 pmol of exogenous rDug
(fraction VI). Reactions were incubated at 30 °C, samples (100
μL) removed after 0, 5, 10, 20, 30, 40, and 60 min (lanes 1−7,
respectively), 25 μL of 0.1 M EDTA was added, and the samples
heated at 70 °C for 3 min to terminate the BER reaction. DNA
was isolated, treated with <italic toggle="yes">E. coli</italic> uracil-DNA glycosylase and
endonuclease IV, and then analyzed by 0.8% agarose gel electrophoresis as described under Materials and Methods. Mock-treated
M13mp2op14 (U·G) DNA (1 μg) without cell-free extract and Ung/Endo IV treatment, and a sample containing 1 μg of a 1-kb DNA
ladder (Gibco-BRL) were used as reference standards (lanes C and
M, respectively). The <italic toggle="yes">arrows</italic> indicate the location of form I and II
DNA bands detected by ethidium bromide stain. (B) DNA bands
detected by ethidium bromide staining (A) were quantitatively
measured using a transilluminator and digital camera imaging
system as described in Materials and Methods. The amount of form
I and II DNA was measured relative to corresponding DNA
standards (6.3−100 ng). The percentage of form I DNA detected
in samples with (·) and without (○) rDug addition was calculated
by dividing the amount of form I (ng) by that of form I plus form
II DNA and multiplying by 100.</p></caption><graphic xlink:href="bi0007066f00008.tif" position="float" orientation="portrait"></graphic></fig></p></sec><sec id="d7e1398"><title>Discussion</title><p>We have purified to apparent homogeneity a Ugi-insensitive double-strand specific uracil-DNA glycosylase from <italic toggle="yes">E.
coli</italic> <italic toggle="yes">ung-1</italic> cell extracts. Several observations led to the
conclusion that Dug was distinct from the <italic toggle="yes">E. coli</italic> Ung: (1)
Dug activity was identified and purified in the presence of
Ugi, a potent and irreversible inhibitor of Ung (<italic toggle="yes">14</italic>, <italic toggle="yes">34</italic>, <italic toggle="yes">40</italic>),
(2) the molecular weight of Dug, as estimated by size
exclusion chromatography (∼18 000) and SDS−polyacrylamide gel electrophoresis (∼21 000) was lower than that
obtained for Ung (∼25 000), (3) Dug was inactive on the
single-stranded uracil-containing substrates preferred by Ung
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0007066b00007" ref-type="bibr"></xref>, <xref rid="bi0007066b00041" ref-type="bibr"></xref></named-content></italic>), and (4) the turnover number of Dug, compared to
Ung, was extremely low (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0007066b00010" ref-type="bibr"></xref>, <xref rid="bi0007066b00014" ref-type="bibr"></xref></named-content></italic>). Since many properties of
nDug were similar to the <italic toggle="yes">E. coli</italic> ORF-169 gene product first
referred to as dsUDG (<italic toggle="yes"><xref rid="bi0007066b00025" ref-type="bibr"></xref></italic>), we cloned the gene and purified
the overproduced recombinant enzyme, and found it to be
indistinguishable from Dug. Further, we showed by mass
spectrometry analysis that the molecular weight of purified
Dug (18 670) was in excellent agreement with the deduced
molecular weight of the amino acid sequence, 18 672. In
this study we have demonstrated that Dug acts on U·A-containing DNA, and has a relatively broad substrate
specificity that was restricted to duplex DNA substrate;
therefore, the designations MUG (mismatch-specific uracil-DNA glycosylase) and εCDG (ethenocytosine-DNA glycosylase) appeared to be somewhat limiting. Hence, in accordance with the three-letter system of bacterial nomenclature,
we advocate that dsUDG be referred to as Dug, double-strand
specific uracil-DNA glycosylase.
</p><p>One of the distinctive characteristics of Dug was that the
purified enzyme appeared incapable of excising more than
a stoichiometric amount of uracil residues from DNA.
Formally, the observed lack of turnover is open to several
interpretations: (1) Dug may function as a “suicide enzyme”
that is inactivated after a single cleavage reaction, such as
<italic toggle="yes">O</italic><sup>6</sup>-alkylguanine methyltransferase (<italic toggle="yes"><xref rid="bi0007066b00042" ref-type="bibr"></xref></italic>) or type I restriction
endonucleases (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0007066b00043" ref-type="bibr"></xref>, <xref rid="bi0007066b00044" ref-type="bibr"></xref></named-content></italic>); (2) Dug may bind to the AP-site
containing DNA reaction product with sufficient affinity that
it is subsequently restricted from engaging new substrate;
and (3) a cofactor(s) essential for efficient Dug function may
be absent in the preparation or reaction mix. DNA-binding
experiments involving band shift assays demonstrated that
the low turnover of Dug was the result of strong binding to
the reaction product AP·G-DNA. Furthermore, cleavage
analysis of Dug−DNA binding reactions indicated that the
vast majority of U·G-containing DNA in complex with Dug
was converted into AP·G-containing DNA. We reasoned that
since AP sites in <italic toggle="yes">E. coli</italic>, generated either spontaneously or
through the action of DNA glycosylases, are subjected to a
variety of AP endonuclease activity, the catalytic activity of
Dug might be modulated by an AP-endonuclease. This was
indeed the case as Dug activity was significantly stimulated
(∼5.5-fold) by the addition of an excess (4-fold) of endonuclease IV.
</p><p>Stimulation of Dug activity was apparently promoted by
enzyme dissociation from AP·G-containing DNA, since we
observed less Dug−DNA complex in the presence versus
the absence of endonuclease IV. Endonuclease IV is a class
II AP-endonuclease that cleaves 5‘ to an AP-site and whose
activity is inhibited by EDTA (<italic toggle="yes"><xref rid="bi0007066b00039" ref-type="bibr"></xref></italic>). Interestingly, in the
reactions including both Dug and endonuclease IV, a new
reaction product was observed (Figure <xref rid="bi0007066f00005"></xref>), one that presumably contained a 3‘-terminal hydroxyl rather than the
3‘-terminal phosphate created by alkali cleavage of Dug-generated AP-sites (<italic toggle="yes"><xref rid="bi0007066b00019" ref-type="bibr"></xref></italic>). Further, the increase in Dug activity
stimulated by endonuclease IV corresponded to an increase
in the amount of 3‘-terminal hydroxyl product observed.
Catalytically active endonuclease IV was required for
stimulation, since Dug/Endo IV reactions conducted in the
presence of 5 mM EDTA showed no detectable stimulation.
Therefore, it appeared that Dug did not bind product DNA
nicked by endonuclease IV; this was demonstrated directly
in Figure <xref rid="bi0007066f00007"></xref>. These results suggested that it was the interaction
of endonuclease IV with DNA, rather than a Dug:Endo IV
protein interaction, that promoted displacement of Dug from
AP·G-containing DNA. To our knowledge, this study is the
first to report on the interaction of a uracil-DNA glycosylase
with a 5‘-incised AP-site.
</p><p>Waters et al. (<italic toggle="yes"><xref rid="bi0007066b00013" ref-type="bibr"></xref></italic>) investigated the effect of human AP-endonuclease 1 (HAP1) on the catalytic efficiency of human
thymine-DNA glycosylase. Like Dug, purified hTDG was
characterized by a very low turnover rate (∼0.4 min<sup>-1</sup>) and
a high affinity (half-life ∼10 h) for its reaction product, AP·G
DNA (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0007066b00011" ref-type="bibr"></xref>, <xref rid="bi0007066b00045" ref-type="bibr"></xref></named-content></italic>). The presence of a 10-fold excess of endonuclease IV in hTDG reactions with T·G-containing DNA
was not observed to affect the rate of thymine excision. In
contrast, the addition of HAP1 was found to stimulate
thymine removal in a concentration-dependent manner,
although the stimulatory effect of an equimolar amount of
HAP1 was modest, approximately 0.5-fold in a 5 h reaction
(<italic toggle="yes"><xref rid="bi0007066b00013" ref-type="bibr"></xref></italic>). Interestingly, HAP1-mediated stimulation of hTDG
activity was much greater on U·C-containing DNA (<italic toggle="yes"><xref rid="bi0007066b00013" ref-type="bibr"></xref></italic>). This
finding is consonant with the observation that dissociation
of hTDG, in the presence of 2 mM Mg<sup>2+</sup>, is more rapid from
DNA containing an AP-site opposite a C or <sup>SMe</sup>G nucleotide
than from AP·G-DNA (<italic toggle="yes"><xref rid="bi0007066b00013" ref-type="bibr"></xref></italic>). Following the cloning of the
hTDG cDNA and the purification of the recombinant protein,
N- and C-terminal deletion analysis revealed that hTDG
contained a 249 amino acid catalytic “core” capable of
processing U·G but not T·G mispairs (<italic toggle="yes"><xref rid="bi0007066b00025" ref-type="bibr"></xref></italic>). It would be of
interest to ascertain whether the hTDG catalytic core enzyme
is stimulated by an AP-endonuclease, or whether the N-terminal portion of hTDG, which bears no sequence similarity to the N-terminus of Dug, is required. Determination of
whether the stimulatory effect of HAP1 on hTDG catalytic
efficiency is the result of a specific HAP1:hTDG protein
interaction, a HAP1:DNA interaction, or some other mechanism must await further experimentation.
</p><p>In an effort to clarify the role of Dug in DNA repair,
Lutsenko and Bhagwat (<italic toggle="yes"><xref rid="bi0007066b00046" ref-type="bibr"></xref></italic>) constructed <italic toggle="yes">E. coli</italic> strains mutant
in either or both <italic toggle="yes">ung</italic> and <italic toggle="yes">dug</italic>. Using a kanamycin reversion
assay specific for a C to T mutation at the second C in the
Dcm recognition sequence CCAGG located in the <italic toggle="yes">kan</italic> gene,
no significant change in the frequency of kanamycin reversion was detected for the <italic toggle="yes">E. coli</italic> <italic toggle="yes">ung-1</italic> <italic toggle="yes">dug </italic>double mutant
relative to the isogenic <italic toggle="yes">dug<sup>+</sup></italic><sup></sup> strain (<italic toggle="yes"><xref rid="bi0007066b00046" ref-type="bibr"></xref></italic>). In contrast, the
reversion frequency of the <italic toggle="yes">E. coli</italic> <italic toggle="yes">ung<sup> </sup></italic><sup></sup>mutant strain was
elevated approximately 8−10-fold compared to wild-type
(<italic toggle="yes">ung<sup>+</sup></italic><sup></sup>) strain (<italic toggle="yes"><xref rid="bi0007066b00046" ref-type="bibr"></xref></italic>). Whether the DNA sequence context of
the kanamycin reversion target might modulate any potential
anti-mutator effect of wild-type <italic toggle="yes">dug</italic> in this genetic assay is
not clear. In this regard, we have presented evidence in this
study that extracts of<italic toggle="yes"> E. coli</italic> <italic toggle="yes">ung-1</italic> cells are proficient in
carrying out complete uracil-mediated BER of a U·G mispair,
and that the addition of purified Dug protein accelerates this
process. These observations formally support the argument
that Dug can participate in DNA repair, and that further
elucidation of the role of Dug in BER is required.
</p><p>Using a rifampicin-resistance forward mutation assay,
Lutsenko and Bhagwat (<italic toggle="yes"><xref rid="bi0007066b00046" ref-type="bibr"></xref></italic>) did observe a small <italic toggle="yes">dug</italic>-dependent effect (∼2.7-fold) on mutation frequency. Interestingly, efficient excision of εC from εC·G mispairs was
reported for <italic toggle="yes">E. coli</italic> extracts of <italic toggle="yes">dug<sup>+</sup></italic><sup></sup>, but not <italic toggle="yes">dug<sup>-</sup></italic><sup></sup>, strains
(<italic toggle="yes"><xref rid="bi0007066b00046" ref-type="bibr"></xref></italic>). In the present study, we observed efficient excision of
εC from εC·A as well as εC·G, mispairs. Taken together,
these data further strengthen the hypothesis of Lutsenko and
Bhagwat (<italic toggle="yes"><xref rid="bi0007066b00046" ref-type="bibr"></xref></italic>) and Saparbaev and Laval (<italic toggle="yes"><xref rid="bi0007066b00021" ref-type="bibr"></xref></italic>), that <italic toggle="yes">dug</italic> may
encode the principal εC excision activity in <italic toggle="yes">E. coli</italic> cells.
</p><p>In interpreting the double-strand uracil-DNA glycosylase
X-ray crystal structure, Barrett et al. (<italic toggle="yes"><xref rid="bi0007066b00011" ref-type="bibr"></xref></italic>) suggested that the
protein, lacking the specialized binding pocket of uracil-DNA
glycosylase, achieved substantial specificity from interactions
with the complementary strand. According to this interpretation, the substrate preference of Dug for U·G and T·G is
due to an association with the guanine opposite U that is
mediated by three hydrogen bonds absolutely specific for
guanine (<italic toggle="yes"><xref rid="bi0007066b00011" ref-type="bibr"></xref></italic>). Thus, the carbonyl of Gly 143 forms strong
hydrogen bonding interactions with the exocyclic 2-amino
and endocyclic N1 groups of guanine, and the carbonyl of
Ser 145 interacts similarly with the guanine exocyclic
2-amino group. These amino acids reside on a motif
(NPSGLSR) that forms a “wedge” that penetrates the base
stack of the DNA from the minor groove (<italic toggle="yes"><xref rid="bi0007066b00011" ref-type="bibr"></xref></italic>). Although
this hypothesis is useful to explain the preference of Dug
for U·G- over U·A-containing DNA, it is not entirely
consistent with the efficient catalytic activity observed on
εC·A in this study, nor with the very low activity against
T·G-containing DNA observed by Saparbaev and Laval (<italic toggle="yes"><xref rid="bi0007066b00021" ref-type="bibr"></xref></italic>).
In this regard, it is notable that, following the processing of
a εC·A mispair, Dug was not observed to bind to the resultant
AP·A-DNA in a electrophoretic mobility shift assay (data
not shown). It is possible that the interaction of the enzyme
with the guanine opposite the mispaired uracil or ethenocytosine may occur after nucleotide flipping. In the Dug crystal
structure, the amino acids implicated in the interaction with
the opposite guanine are Gly-143 and Ser-145, which are
found in the “wedge” motif (NPSGLSR) (<italic toggle="yes"><xref rid="bi0007066b00011" ref-type="bibr"></xref></italic>). However,
while these amino acids are not conserved in the related
hTDG motif (MPSSSAR), nevertheless hTDG shows strong
preference for U·G- and T·G-containing DNA over U·A (and
T·A) base pairs. These amino acid differences may explain
in part the differential activity of hTDG and Dug toward
U·G- and εC·G-containing substrates reported by Saparbaev
and Laval (<italic toggle="yes"><xref rid="bi0007066b00021" ref-type="bibr"></xref></italic>). As we have demonstrated, Dug loses affinity
for the “widowed” guanine once the AP-site is 5‘-incised
by endonuclease IV. The structural basis for this loss of
interaction and whether a similar result would be obtained
for a 3‘-incised AP-site are not known at this time.
</p><p>We have demonstrated the involvement of Dug in initiating
uracil-mediated base excision repair in <italic toggle="yes">E. coli </italic>using an
M13mp2 form I DNA containing site-specific U·G mispair.
An examination of the BER reaction in <italic toggle="yes">E. coli </italic>deficient in
Ung activity revealed that ∼20% of the mispaired uracil was
repaired after 60 min of incubation. To ensure that we were
examining Ung-independent uracil base excision repair, an
excess of Ugi was included in the reaction mixture to
inactivate any residual Ung activity. The repair of mispaired
uracil appeared to be mediated by Dug because the extent
of repair was enhanced to ∼55% when the BER reaction
was supplemented with purified Dug. This observation
provides the first line of evidence that uracil-excision repair
in <italic toggle="yes">E. coli</italic> can be instigated by the <italic toggle="yes">dug</italic> gene product in vivo.
These results stand in contrast to those recently reported by
Sandigursky et al. (<italic toggle="yes"><xref rid="bi0007066b00047" ref-type="bibr"></xref></italic>). Previously, repair of a site-specific
U·G mispair in a closed circular plasmid was not detected
in extracts of an <italic toggle="yes">E. coli </italic>strain deficient in Ung activity. On
this basis, it was concluded that Ung was absolutely required
for repair of U·G-containing DNA (<italic toggle="yes"><xref rid="bi0007066b00047" ref-type="bibr"></xref></italic>). In this study, we
show that extracts of <italic toggle="yes">E. coli ung-1</italic> contain a Ugi-resistant
uracil-excision activity attributable to the <italic toggle="yes">dug</italic> gene product
and capable of initiating repair of a U·G mispair. In an earlier
report, this laboratory found that in extracts of a human
glioblastoma cell line, a small but detectable amount (∼7%)
of uracil-excision repair appeared to be insensitive to Ugi,
suggesting that a back-up repair pathway existed, and that
hTDG activity may be involved in the repair of a U·T mispair
(<italic toggle="yes"><xref rid="bi0007066b00030" ref-type="bibr"></xref></italic>).
</p><p>Many DNA glycosylases have overlapping substrate
specificities and may provide back-up functions for each
other (<italic toggle="yes"><xref rid="bi0007066b00048" ref-type="bibr"></xref></italic>). It has been suggested that Dug may act as a back-up or alternative to Ung in the repair of U·G mispairs arising
through spontaneous hydrolytic deamination of cytosine (<italic toggle="yes"><xref rid="bi0007066b00025" ref-type="bibr"></xref></italic>).
In the present study, we have shown that Dug can initiate
uracil-mediated base excision DNA repair in <italic toggle="yes">E. coli</italic>.
However, the <italic toggle="yes">dug</italic> gene product does not appear to play a
strong role in preventing G·C to A·T transition mutations.
Since Dug has been shown to exhibit a strong preference
for εC residues in double-stranded DNA (<italic toggle="yes"><xref rid="bi0007066b00021" ref-type="bibr"></xref></italic>), one might
wonder whether an <italic toggle="yes">E. coli</italic> strain defective in <italic toggle="yes">dug</italic> would
exhibit an elevated mutation rate for G·C to T·A transversions. An understanding of the biological role of <italic toggle="yes">E. coli</italic> Dug
and that of its human homolog, TDG, must await further
experimentation.
</p></sec></body><back><ack><title>Acknowledgments</title><p>We wish to thank Lilo Barofsky for performing mass
spectrometry through the Environmental Health Sciences
Center, Mass Spectrometry Core Unit, the Center for Gene
Research and Biotechnology for conducting the DNA
sequence analysis, and Dr. Samuel Bennett for insightful
discussions and critical reading of the manuscript.
</p></ack><notes notes-type="si"><sec id="d7e1717"><title><ext-link xlink:href="/doi/suppl/10.1021%2Fbi0007066">Supporting Information Available</ext-link></title><p>Figure <xref rid="bi0007066f00001"></xref> presents column purification profiles for Sephadex G-75, hydroxyapatite, and single-stranded DNA agarose
chromatography of native double-strand uracil-DNA glycosylase isolated from <italic toggle="yes">E. coli</italic> NR8052 cells. SDS−polyacrylamide gel electrophoresis analysis of recombinant Dug during
the purification procedure is shown in Figure <xref rid="bi0007066f00002"></xref>. Molecular
weight determination of rDug by matrix-assisted laser
desorption−ionization mass spectrometry is provided in
Figure <xref rid="bi0007066f00003"></xref>. The effect of <italic toggle="yes">E. coli</italic> endonuclease IV on the rate
and extent of the Dug mediated uracil excision reaction is
demonstrated in Figure <xref rid="bi0007066f00004"></xref>. This material is available free of
charge via the Internet at <uri xlink:href="http://pubs.acs.org">http://pubs.acs.org</uri>.
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J.</source><year>1997</year><volume>325</volume><fpage>1</fpage><lpage>16</lpage></element-citation></ref><ref id="bi0007066n00001"><mixed-citation><comment>Abbreviations: BER, base excision repair; Ung, <italic toggle="yes">E. coli</italic> uracil-DNA glycosylase; UDG, human and herpes simplex virus uracil-DNA glycosylase; Ugi, PBS1 and 2 uracil-DNA glycosylase inhibitor protein; dsUDG and Dug, <italic toggle="yes">E. coli</italic> double-strand-specific uracil-DNA glycosylase; TDG, human mismatch-specific thymine-DNA glycosylase; MUG, <italic toggle="yes">E. coli</italic> mismatch-specific uracil-DNA glycosylase; εC, 3,<italic toggle="yes">N</italic><sup>4</sup>-ethenocytosine; εCDG, <italic toggle="yes">E. coli </italic>εC-DNA glycosylase; Endo IV, <italic toggle="yes">E. coli</italic> endonuclease IV; BSA, bovine serum albumin; AP, apurinic/apyrimidinic; <sup>*</sup>AP, incision on the 5‘-side of AP-site; MALDI, matrix-assisted laser desorption−ionization; ORF, open reading frame; PCR, polymerase chain reaction; EDTA, ethylenediaminetetraacetic acid; SDS, sodium dodecyl sulfate.</comment></mixed-citation></ref><ref id="bi0007066n00002"><mixed-citation><comment>ORF-169 is located between nucleotide positions 3,212,608 and 3,213,115 on the <italic toggle="yes">E. coli</italic> chromosome.</comment></mixed-citation></ref><ref id="bi0007066n00003"><mixed-citation><comment>Base pairs and mispairs are described by listing the base in the repaired strand first and then the base in the template strand.</comment></mixed-citation></ref><ref id="bi0007066n00004"><mixed-citation><comment>*AP denotes an apyrimidinic-site cleaved on the 5‘-side to produce 3‘-hydroxyl nucleotide and deoxyribose 5-phosphate termini.</comment></mixed-citation></ref><ref id="bi0007066n00005"><mixed-citation><comment>Fractions V and VI correspond to the final purification step for nDug and rDug, respectively.</comment></mixed-citation></ref><ref id="bi0007066n00006"><mixed-citation><comment>Molar extinction coefficient ε<sub>280nm</sub> for Dug was calculated by the DNASTAR Protean computer program version 3.11.</comment></mixed-citation></ref></ref-list></back></article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo><title>Escherichia coli Double-Strand Uracil-DNA Glycosylase: Involvement in Uracil-Mediated DNA Base Excision Repair and Stimulation of Activity by Endonuclease IV†</title></titleInfo><titleInfo contentType="CDATA"><title>Escherichia coli Double-Strand Uracil-DNA Glycosylase: Involvement in Uracil-Mediated DNA Base Excision Repair and Stimulation of Activity by Endonuclease IV†</title></titleInfo><name type="personal"><namePart type="family">SUNG</namePart><namePart type="given">Jung-Suk</namePart><affiliation>Departments of Environmental and Molecular Toxicology and Biochemistry and Biophysics and theEnvironmental Health Science Center, Oregon State University, Corvallis, Oregon 97731</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal" displayLabel="corresp"><namePart type="family">MOSBAUGH</namePart><namePart type="given">Dale W.</namePart><affiliation>Departments of Environmental and Molecular Toxicology and Biochemistry and Biophysics and theEnvironmental Health Science Center, Oregon State University, Corvallis, Oregon 97731</affiliation><affiliation> To whom correspondence should be addressed. Phone: (541) 737-1797. Fax: (541) 737-0497. E-mail: mosbaugd@ucs.orst.edu.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="research-article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>American Chemical Society</publisher><dateCreated encoding="w3cdtf">2000-07-29</dateCreated><dateIssued encoding="w3cdtf">2000-08-22</dateIssued><copyrightDate encoding="w3cdtf">2000</copyrightDate></originInfo><note type="footnote" ID="bi0007066AF2"> This work was supported by National Institutes of Health Grants GM32823 and ES00210. Technical Report 11651 from the Oregon Agricultural Experiment Station.</note><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract>Escherichia coli double-strand uracil-DNA glycosylase (Dug) was purified to apparent homogeneity as both a native and recombinant protein. The molecular weight of recombinant Dug was 18 670, as determined by matrix-assisted laser desorption−ionization mass spectrometry. Dug was active on duplex oligonucleotides (34-mers) that contained site-specific U·G, U·A, ethenoC·G, and ethenoC·A targets; however, activity was not detected on DNA containing a T·G mispair or single-stranded DNA containing either a site-specific uracil or ethenoC residue. One of the distinctive characteristics of Dug was that the purified enzyme excised a near stoichiometric amount of uracil from U·G-containing oligonucleotide substrate. Electrophoretic mobility shift assays revealed that the lack of turnover was the result of strong binding by Dug to the reaction product apyrimidinic-site (AP) DNA. Addition of E. coli endonuclease IV stimulated Dug activity by enhancing the rate and extent of uracil excision by promoting dissociation of Dug from the AP·G-containing 34-mer. Catalytically active endonuclease IV was apparently required to mediate Dug turnover, since the addition of 5 mM EDTA mitigated the effect. Further support for this interpretation came from the observations that Dug preferentially bound 34-mer containing an AP·G target, while binding was not observed on a substrate incised 5‘ to the AP-site. We also investigated whether Dug could initiate a uracil-mediated base excision repair pathway in E. coli NR8052 cell extracts using M13mp2op14 DNA (form I) containing a site-specific U·G mispair. Analysis of reaction products revealed a time dependent appearance of repaired form I DNA; addition of purified Dug to the cell extract stimulated the rate of repair.</abstract><note type="footnote" ID="bi0007066AF2"> This work was supported by National Institutes of Health Grants GM32823 and ES00210. 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J. 1997 325 1 16</note><part><date>1997</date><detail type="volume"><caption>vol.</caption><number>325</number></detail><extent unit="pages"><start>1</start><end>16</end></extent></part></relatedItem><relatedItem type="references" ID="bi0007066n00001" displayLabel="bibbi0007066n00001"><titleInfo><title>Abbreviations: BER, base excision repair; Ung,E. coliuracil-DNA glycosylase; UDG, human and herpes simplex virus uracil-DNA glycosylase; Ugi, PBS1 and 2 uracil-DNA glycosylase inhibitor protein; dsUDG and Dug,E. colidouble-strand-specific uracil-DNA glycosylase; TDG, human mismatch-specific thymine-DNA glycosylase; MUG,E. colimismatch-specific uracil-DNA glycosylase; εC, 3,N4-ethenocytosine; εCDG,E. coliεC-DNA glycosylase; Endo IV,E. coliendonuclease IV; BSA, bovine serum albumin; AP, apurinic/apyrimidinic;*AP, incision on the 5‘-side of AP-site; MALDI, matrix-assisted laser desorption−ionization; ORF, open reading frame; PCR, polymerase chain reaction; EDTA, ethylenediaminetetraacetic acid; SDS, sodium dodecyl sulfate.</title></titleInfo><note type="content-in-line">Abbreviations: BER, base excision repair; Ung, E. coli uracil-DNA glycosylase; UDG, human and herpes simplex virus uracil-DNA glycosylase; Ugi, PBS1 and 2 uracil-DNA glycosylase inhibitor protein; dsUDG and Dug, E. coli double-strand-specific uracil-DNA glycosylase; TDG, human mismatch-specific thymine-DNA glycosylase; MUG, E. coli mismatch-specific uracil-DNA glycosylase; εC, 3,N4-ethenocytosine; εCDG, E. coli εC-DNA glycosylase; Endo IV, E. coli endonuclease IV; BSA, bovine serum albumin; AP, apurinic/apyrimidinic; *AP, incision on the 5‘-side of AP-site; MALDI, matrix-assisted laser desorption−ionization; ORF, open reading frame; PCR, polymerase chain reaction; EDTA, ethylenediaminetetraacetic acid; SDS, sodium dodecyl sulfate.</note></relatedItem><relatedItem type="references" ID="bi0007066n00002" displayLabel="bibbi0007066n00002"><titleInfo><title>ORF-169 is located between nucleotide positions 3,212,608 and 3,213,115 on theE. colichromosome.</title></titleInfo><note type="content-in-line">ORF-169 is located between nucleotide positions 3,212,608 and 3,213,115 on the E. coli chromosome.</note></relatedItem><relatedItem type="references" ID="bi0007066n00003" displayLabel="bibbi0007066n00003"><titleInfo><title>Base pairs and mispairs are described by listing the base in the repaired strand first and then the base in the template strand.</title></titleInfo><note type="content-in-line">Base pairs and mispairs are described by listing the base in the repaired strand first and then the base in the template strand.</note></relatedItem><relatedItem type="references" ID="bi0007066n00004" displayLabel="bibbi0007066n00004"><titleInfo><title>*AP denotes an apyrimidinic-site cleaved on the 5‘-side to produce 3‘-hydroxyl nucleotide and deoxyribose 5-phosphate termini.</title></titleInfo><note type="content-in-line">*AP denotes an apyrimidinic-site cleaved on the 5‘-side to produce 3‘-hydroxyl nucleotide and deoxyribose 5-phosphate termini.</note></relatedItem><relatedItem type="references" ID="bi0007066n00005" displayLabel="bibbi0007066n00005"><titleInfo><title>Fractions V and VI correspond to the final purification step for nDug and rDug, respectively.</title></titleInfo><note type="content-in-line">Fractions V and VI correspond to the final purification step for nDug and rDug, respectively.</note></relatedItem><relatedItem type="references" ID="bi0007066n00006" displayLabel="bibbi0007066n00006"><titleInfo><title>Molar extinction coefficient ε280nmfor Dug was calculated by the DNASTAR Protean computer program version 3.11.</title></titleInfo><note type="content-in-line">Molar extinction coefficient ε280nm for Dug was calculated by the DNASTAR Protean computer program version 3.11.</note></relatedItem><identifier type="istex">5F6B0C51A0EFE03047B7BA123BD16C54F9101355</identifier><identifier type="ark">ark:/67375/TPS-2CZV0JP1-T</identifier><identifier type="DOI">10.1021/bi0007066</identifier><accessCondition type="use and reproduction" contentType="restricted">Copyright © 2000 American Chemical Society</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-X5HBJWF8-J">acs</recordContentSource><recordOrigin>Converted from (version 1.2.10) to MODS version 3.6.</recordOrigin><recordCreationDate encoding="w3cdtf">2020-04-10</recordCreationDate></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-2CZV0JP1-T/record.json</uri></json:item></metadata><annexes><json:item><extension>tiff</extension><original>true</original><mimetype>image/tiff</mimetype><uri>https://api.istex.fr/document/5F6B0C51A0EFE03047B7BA123BD16C54F9101355/annexes/tiff</uri></json:item></annexes><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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