Escherichia coli double-strand uracil-DNA glycosylase: involvement in uracil-mediated DNA base excision repair and stimulation of activity by endonuclease IV.
Identifieur interne : 002579 ( PubMed/Corpus ); précédent : 002578; suivant : 002580Escherichia coli double-strand uracil-DNA glycosylase: involvement in uracil-mediated DNA base excision repair and stimulation of activity by endonuclease IV.
Auteurs : J S Sung ; D W MosbaughSource :
- Biochemistry [ 0006-2960 ] ; 2000.
English descriptors
- KwdEn :
- Base Pair Mismatch, Carbon-Oxygen Lyases (metabolism), Cloning, Molecular, DNA Repair, DNA-(Apurinic or Apyrimidinic Site) Lyase, DNA-Binding Proteins (genetics), DNA-Binding Proteins (isolation & purification), DNA-Binding Proteins (metabolism), Deoxyribonuclease IV (Phage T4-Induced), Escherichia coli (enzymology), Escherichia coli (genetics), Escherichia coli Proteins, Genes, Bacterial, Molecular Weight, N-Glycosyl Hydrolases (genetics), N-Glycosyl Hydrolases (isolation & purification), N-Glycosyl Hydrolases (metabolism), Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Substrate Specificity.
- MESH :
- chemical , genetics : DNA-Binding Proteins, N-Glycosyl Hydrolases.
- chemical , isolation & purification : DNA-Binding Proteins, N-Glycosyl Hydrolases.
- chemical , metabolism : Carbon-Oxygen Lyases, DNA-Binding Proteins, N-Glycosyl Hydrolases.
- enzymology : Escherichia coli.
- genetics : Escherichia coli.
- Base Pair Mismatch, Cloning, Molecular, DNA Repair, DNA-(Apurinic or Apyrimidinic Site) Lyase, Deoxyribonuclease IV (Phage T4-Induced), Escherichia coli Proteins, Genes, Bacterial, Molecular Weight, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Substrate Specificity.
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.
DOI: 10.1021/bi0007066
PubMed: 10956012
Links to Exploration step
pubmed:10956012Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">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, J S" sort="Sung, J S" uniqKey="Sung J" first="J S" last="Sung">J S Sung</name><affiliation><nlm:affiliation>Departments of Environmental and Molecular Toxicology and Biochemistry and Biophysics and the Environmental Health Science Center, Oregon State University, Corvallis 97731, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Mosbaugh, D W" sort="Mosbaugh, D W" uniqKey="Mosbaugh D" first="D W" last="Mosbaugh">D W Mosbaugh</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2000">2000</date><idno type="RBID">pubmed:10956012</idno><idno type="pmid">10956012</idno><idno type="doi">10.1021/bi0007066</idno><idno type="wicri:Area/PubMed/Corpus">002579</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">002579</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">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, J S" sort="Sung, J S" uniqKey="Sung J" first="J S" last="Sung">J S Sung</name><affiliation><nlm:affiliation>Departments of Environmental and Molecular Toxicology and Biochemistry and Biophysics and the Environmental Health Science Center, Oregon State University, Corvallis 97731, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Mosbaugh, D W" sort="Mosbaugh, D W" uniqKey="Mosbaugh D" first="D W" last="Mosbaugh">D W Mosbaugh</name></author></analytic><series><title level="j">Biochemistry</title><idno type="ISSN">0006-2960</idno><imprint><date when="2000" type="published">2000</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Base Pair Mismatch</term><term>Carbon-Oxygen Lyases (metabolism)</term><term>Cloning, Molecular</term><term>DNA Repair</term><term>DNA-(Apurinic or Apyrimidinic Site) Lyase</term><term>DNA-Binding Proteins (genetics)</term><term>DNA-Binding Proteins (isolation & purification)</term><term>DNA-Binding Proteins (metabolism)</term><term>Deoxyribonuclease IV (Phage T4-Induced)</term><term>Escherichia coli (enzymology)</term><term>Escherichia coli (genetics)</term><term>Escherichia coli Proteins</term><term>Genes, Bacterial</term><term>Molecular Weight</term><term>N-Glycosyl Hydrolases (genetics)</term><term>N-Glycosyl Hydrolases (isolation & purification)</term><term>N-Glycosyl Hydrolases (metabolism)</term><term>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization</term><term>Substrate Specificity</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>DNA-Binding Proteins</term><term>N-Glycosyl Hydrolases</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>DNA-Binding Proteins</term><term>N-Glycosyl Hydrolases</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Carbon-Oxygen Lyases</term><term>DNA-Binding Proteins</term><term>N-Glycosyl Hydrolases</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>Escherichia coli</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Escherichia coli</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Base Pair Mismatch</term><term>Cloning, Molecular</term><term>DNA Repair</term><term>DNA-(Apurinic or Apyrimidinic Site) Lyase</term><term>Deoxyribonuclease IV (Phage T4-Induced)</term><term>Escherichia coli Proteins</term><term>Genes, Bacterial</term><term>Molecular Weight</term><term>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization</term><term>Substrate Specificity</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">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><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">10956012</PMID><DateCompleted><Year>2000</Year><Month>09</Month><Day>12</Day></DateCompleted><DateRevised><Year>2019</Year><Month>06</Month><Day>13</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">0006-2960</ISSN><JournalIssue CitedMedium="Print"><Volume>39</Volume><Issue>33</Issue><PubDate><Year>2000</Year><Month>Aug</Month><Day>22</Day></PubDate></JournalIssue><Title>Biochemistry</Title><ISOAbbreviation>Biochemistry</ISOAbbreviation></Journal><ArticleTitle>Escherichia coli double-strand uracil-DNA glycosylase: involvement in uracil-mediated DNA base excision repair and stimulation of activity by endonuclease IV.</ArticleTitle><Pagination><MedlinePgn>10224-35</MedlinePgn></Pagination><Abstract><AbstractText>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.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Sung</LastName><ForeName>J S</ForeName><Initials>JS</Initials><AffiliationInfo><Affiliation>Departments of Environmental and Molecular Toxicology and Biochemistry and Biophysics and the Environmental Health Science Center, Oregon State University, Corvallis 97731, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Mosbaugh</LastName><ForeName>D W</ForeName><Initials>DW</Initials></Author></AuthorList><Language>eng</Language><GrantList CompleteYN="Y"><Grant><GrantID>ES00210</GrantID><Acronym>ES</Acronym><Agency>NIEHS NIH HHS</Agency><Country>United States</Country></Grant><Grant><GrantID>GM32823</GrantID><Acronym>GM</Acronym><Agency>NIGMS NIH HHS</Agency><Country>United States</Country></Grant></GrantList><PublicationTypeList><PublicationType UI="D003160">Comparative Study</PublicationType><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013487">Research Support, U.S. Gov't, P.H.S.</PublicationType></PublicationTypeList></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Biochemistry</MedlineTA><NlmUniqueID>0370623</NlmUniqueID><ISSNLinking>0006-2960</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D004268">DNA-Binding Proteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D029968">Escherichia coli Proteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 3.1.21.2</RegistryNumber><NameOfSubstance UI="D043223">Deoxyribonuclease IV (Phage T4-Induced)</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 3.1.21.2</RegistryNumber><NameOfSubstance UI="C468972">endonuclease IV, E coli</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 3.2.2.-</RegistryNumber><NameOfSubstance UI="D009699">N-Glycosyl Hydrolases</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 3.2.2.-</RegistryNumber><NameOfSubstance UI="C412878">double-strand uracil-DNA glycosylase, E coli</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 4.2.-</RegistryNumber><NameOfSubstance UI="D019757">Carbon-Oxygen Lyases</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 4.2.99.18</RegistryNumber><NameOfSubstance UI="D043603">DNA-(Apurinic or Apyrimidinic Site) Lyase</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D020137" MajorTopicYN="N">Base Pair Mismatch</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D019757" MajorTopicYN="N">Carbon-Oxygen Lyases</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D003001" MajorTopicYN="N">Cloning, Molecular</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D004260" MajorTopicYN="Y">DNA Repair</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D043603" MajorTopicYN="N">DNA-(Apurinic or Apyrimidinic Site) Lyase</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D004268" MajorTopicYN="N">DNA-Binding Proteins</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName><QualifierName UI="Q000302" MajorTopicYN="N">isolation & purification</QualifierName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D043223" MajorTopicYN="N">Deoxyribonuclease IV (Phage T4-Induced)</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D004926" MajorTopicYN="N">Escherichia coli</DescriptorName><QualifierName UI="Q000201" MajorTopicYN="Y">enzymology</QualifierName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D029968" MajorTopicYN="Y">Escherichia coli Proteins</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D005798" MajorTopicYN="N">Genes, Bacterial</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D008970" MajorTopicYN="N">Molecular Weight</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D009699" MajorTopicYN="N">N-Glycosyl Hydrolases</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName><QualifierName UI="Q000302" MajorTopicYN="N">isolation & purification</QualifierName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D019032" MajorTopicYN="N">Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D013379" MajorTopicYN="N">Substrate 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