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/Curation ); 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 [États-Unis] ; Dale W. Mosbaugh [États-Unis]Source :
- 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.
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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 wicri:level="2"><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><country xml:lang="fr">États-Unis</country><placeName><region type="state">Oregon</region></placeName><wicri:cityArea>Departments of Environmental and Molecular Toxicology and Biochemistry and Biophysics and theEnvironmental Health Science Center, Oregon State University, Corvallis</wicri:cityArea></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 wicri:level="2"><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><country xml:lang="fr">États-Unis</country><placeName><region type="state">Oregon</region></placeName><wicri:cityArea>Departments of Environmental and Molecular Toxicology and Biochemistry and Biophysics and theEnvironmental Health Science Center, Oregon State University, Corvallis</wicri:cityArea></affiliation><affiliation wicri:level="1"><mods:affiliation> To whom correspondence should be addressed. Phone: (541) 737-1797. Fax: (541) 737-0497. E-mail: mosbaugd@ucs.orst.edu.</mods:affiliation><country wicri:rule="url">États-Unis</country></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><idno type="wicri:Area/Istex/Curation">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 wicri:level="2"><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><country xml:lang="fr">États-Unis</country><placeName><region type="state">Oregon</region></placeName><wicri:cityArea>Departments of Environmental and Molecular Toxicology and Biochemistry and Biophysics and theEnvironmental Health Science Center, Oregon State University, Corvallis</wicri:cityArea></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 wicri:level="2"><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><country xml:lang="fr">États-Unis</country><placeName><region type="state">Oregon</region></placeName><wicri:cityArea>Departments of Environmental and Molecular Toxicology and Biochemistry and Biophysics and theEnvironmental Health Science Center, Oregon State University, Corvallis</wicri:cityArea></affiliation><affiliation wicri:level="1"><mods:affiliation> To whom correspondence should be addressed. Phone: (541) 737-1797. Fax: (541) 737-0497. E-mail: mosbaugd@ucs.orst.edu.</mods:affiliation><country wicri:rule="url">États-Unis</country></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></record>Pour manipuler ce document sous Unix (Dilib)
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