Consequences of 6-thioguanine incorporation into DNA on polymerase, ligase, and endonuclease reactions.
Identifieur interne : 002815 ( PubMed/Checkpoint ); précédent : 002814; suivant : 002816Consequences of 6-thioguanine incorporation into DNA on polymerase, ligase, and endonuclease reactions.
Auteurs : Y H Ling ; J Y Chan ; K L Beattie ; J A NelsonSource :
- Molecular pharmacology [ 0026-895X ] ; 1992.
Descripteurs français
- KwdFr :
- ADN (métabolisme), Autoradiographie, DNA ligases (métabolisme), DNA-directed DNA polymerase (métabolisme), Données de séquences moléculaires, Endonucleases (métabolisme), Matrices (génétique), Spécificité du substrat, Séquence nucléotidique, Tioguanine (métabolisme), Électrophorèse sur gel de polyacrylamide.
- MESH :
English descriptors
- KwdEn :
- MESH :
Abstract
The incorporation of 6-thioguanine (S6G) in place of guanine proceeds readily in DNA synthesis reactions catalyzed by mammalian and bacterial polymerases. This report summarizes the consequences of such incorporation studied to date. S6G was incorporated into one strand of a defined M13mp18 phage sequence in a (+)reaction catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I. After denaturation of the newly synthesized strand (containing S6G) and annealing with a reverse (-) 32P-labeled primer, polymerization catalyzed by the Klenow enzyme as well as by human DNA polymerases alpha, gamma, and delta was slowed considerably, compared with that across the corresponding guanine-containing template. To evaluate S6G-containing DNA as a substrate for DNA ligases, two oligodeoxynucleotides (19- and 20-mers) antisense to a 40-mer were synthesized so that the 40-mer coded for guanine at the 3' terminus of the 19-mer. After annealing of the synthetic oligonucleotides to form a duplex DNA containing a one-nucleotide gap (opposite cytosine in the 40-mer), the 19-mer was extended with 2'-deoxythioguanosine 5'-triphosphate using DNA polymerase, forming a nicked duplex DNA. The abilities of T4 DNA ligase and HeLa and calf thymus DNA ligase I to join the 5'-phosphate with the 3'-S6G-OH were severely inhibited, compared with the 3'-guanine-extended control. This finding suggests that incorporation of S6G at the 3' terminus of Okazaki fragments would inhibit lagging strand DNA synthesis. In other experiments, cleavage of S6G-containing DNA by some but not all restriction endonucleases progressed poorly, compared with the control guanine-containing DNA, independently of the location of S6G at recognition or cleavage sites, as previously observed by Iwaniec et al. [Mol. Pharmacol. 39:299-306 (1991)] with a different spectrum of enzymes. These findings indicate altered DNA-protein interactions due to S6G incorporation. The poor template function of S6G-containing DNA is consistent with the known delayed cytotoxicity and DNA damage previously reported to occur in S6G-treated cells.
PubMed: 1331762
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Consequences of 6-thioguanine incorporation into DNA on polymerase, ligase, and endonuclease reactions.</title><author><name sortKey="Ling, Y H" sort="Ling, Y H" uniqKey="Ling Y" first="Y H" last="Ling">Y H Ling</name><affiliation><nlm:affiliation>Department of Experimental Pediatrics, University of Texas M.D. Anderson Cancer Center, Houston 77030.</nlm:affiliation><wicri:noCountry code="subField">Houston 77030</wicri:noCountry></affiliation></author><author><name sortKey="Chan, J Y" sort="Chan, J Y" uniqKey="Chan J" first="J Y" last="Chan">J Y Chan</name></author><author><name sortKey="Beattie, K L" sort="Beattie, K L" uniqKey="Beattie K" first="K L" last="Beattie">K L Beattie</name></author><author><name sortKey="Nelson, J A" sort="Nelson, J A" uniqKey="Nelson J" first="J A" last="Nelson">J A Nelson</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="1992">1992</date><idno type="RBID">pubmed:1331762</idno><idno type="pmid">1331762</idno><idno type="wicri:Area/PubMed/Corpus">002943</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">002943</idno><idno type="wicri:Area/PubMed/Curation">002943</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Curation">002943</idno><idno type="wicri:Area/PubMed/Checkpoint">002815</idno><idno type="wicri:explorRef" wicri:stream="Checkpoint" wicri:step="PubMed">002815</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Consequences of 6-thioguanine incorporation into DNA on polymerase, ligase, and endonuclease reactions.</title><author><name sortKey="Ling, Y H" sort="Ling, Y H" uniqKey="Ling Y" first="Y H" last="Ling">Y H Ling</name><affiliation><nlm:affiliation>Department of Experimental Pediatrics, University of Texas M.D. Anderson Cancer Center, Houston 77030.</nlm:affiliation><wicri:noCountry code="subField">Houston 77030</wicri:noCountry></affiliation></author><author><name sortKey="Chan, J Y" sort="Chan, J Y" uniqKey="Chan J" first="J Y" last="Chan">J Y Chan</name></author><author><name sortKey="Beattie, K L" sort="Beattie, K L" uniqKey="Beattie K" first="K L" last="Beattie">K L Beattie</name></author><author><name sortKey="Nelson, J A" sort="Nelson, J A" uniqKey="Nelson J" first="J A" last="Nelson">J A Nelson</name></author></analytic><series><title level="j">Molecular pharmacology</title><idno type="ISSN">0026-895X</idno><imprint><date when="1992" type="published">1992</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Autoradiography</term><term>Base Sequence</term><term>DNA (metabolism)</term><term>DNA Ligases (metabolism)</term><term>DNA-Directed DNA Polymerase (metabolism)</term><term>Electrophoresis, Polyacrylamide Gel</term><term>Endonucleases (metabolism)</term><term>Molecular Sequence Data</term><term>Substrate Specificity</term><term>Templates, Genetic</term><term>Thioguanine (metabolism)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ADN (métabolisme)</term><term>Autoradiographie</term><term>DNA ligases (métabolisme)</term><term>DNA-directed DNA polymerase (métabolisme)</term><term>Données de séquences moléculaires</term><term>Endonucleases (métabolisme)</term><term>Matrices (génétique)</term><term>Spécificité du substrat</term><term>Séquence nucléotidique</term><term>Tioguanine (métabolisme)</term><term>Électrophorèse sur gel de polyacrylamide</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>DNA</term><term>DNA Ligases</term><term>DNA-Directed DNA Polymerase</term><term>Endonucleases</term><term>Thioguanine</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>ADN</term><term>DNA ligases</term><term>DNA-directed DNA polymerase</term><term>Endonucleases</term><term>Tioguanine</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Autoradiography</term><term>Base Sequence</term><term>Electrophoresis, Polyacrylamide Gel</term><term>Molecular Sequence Data</term><term>Substrate Specificity</term><term>Templates, Genetic</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Autoradiographie</term><term>Données de séquences moléculaires</term><term>Matrices (génétique)</term><term>Spécificité du substrat</term><term>Séquence nucléotidique</term><term>Électrophorèse sur gel de polyacrylamide</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The incorporation of 6-thioguanine (S6G) in place of guanine proceeds readily in DNA synthesis reactions catalyzed by mammalian and bacterial polymerases. This report summarizes the consequences of such incorporation studied to date. S6G was incorporated into one strand of a defined M13mp18 phage sequence in a (+)reaction catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I. After denaturation of the newly synthesized strand (containing S6G) and annealing with a reverse (-) 32P-labeled primer, polymerization catalyzed by the Klenow enzyme as well as by human DNA polymerases alpha, gamma, and delta was slowed considerably, compared with that across the corresponding guanine-containing template. To evaluate S6G-containing DNA as a substrate for DNA ligases, two oligodeoxynucleotides (19- and 20-mers) antisense to a 40-mer were synthesized so that the 40-mer coded for guanine at the 3' terminus of the 19-mer. After annealing of the synthetic oligonucleotides to form a duplex DNA containing a one-nucleotide gap (opposite cytosine in the 40-mer), the 19-mer was extended with 2'-deoxythioguanosine 5'-triphosphate using DNA polymerase, forming a nicked duplex DNA. The abilities of T4 DNA ligase and HeLa and calf thymus DNA ligase I to join the 5'-phosphate with the 3'-S6G-OH were severely inhibited, compared with the 3'-guanine-extended control. This finding suggests that incorporation of S6G at the 3' terminus of Okazaki fragments would inhibit lagging strand DNA synthesis. In other experiments, cleavage of S6G-containing DNA by some but not all restriction endonucleases progressed poorly, compared with the control guanine-containing DNA, independently of the location of S6G at recognition or cleavage sites, as previously observed by Iwaniec et al. [Mol. Pharmacol. 39:299-306 (1991)] with a different spectrum of enzymes. These findings indicate altered DNA-protein interactions due to S6G incorporation. The poor template function of S6G-containing DNA is consistent with the known delayed cytotoxicity and DNA damage previously reported to occur in S6G-treated cells.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">1331762</PMID><DateCompleted><Year>1992</Year><Month>12</Month><Day>09</Day></DateCompleted><DateRevised><Year>2013</Year><Month>11</Month><Day>21</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">0026-895X</ISSN><JournalIssue CitedMedium="Print"><Volume>42</Volume><Issue>5</Issue><PubDate><Year>1992</Year><Month>Nov</Month></PubDate></JournalIssue><Title>Molecular pharmacology</Title><ISOAbbreviation>Mol. Pharmacol.</ISOAbbreviation></Journal><ArticleTitle>Consequences of 6-thioguanine incorporation into DNA on polymerase, ligase, and endonuclease reactions.</ArticleTitle><Pagination><MedlinePgn>802-7</MedlinePgn></Pagination><Abstract><AbstractText>The incorporation of 6-thioguanine (S6G) in place of guanine proceeds readily in DNA synthesis reactions catalyzed by mammalian and bacterial polymerases. This report summarizes the consequences of such incorporation studied to date. S6G was incorporated into one strand of a defined M13mp18 phage sequence in a (+)reaction catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I. After denaturation of the newly synthesized strand (containing S6G) and annealing with a reverse (-) 32P-labeled primer, polymerization catalyzed by the Klenow enzyme as well as by human DNA polymerases alpha, gamma, and delta was slowed considerably, compared with that across the corresponding guanine-containing template. To evaluate S6G-containing DNA as a substrate for DNA ligases, two oligodeoxynucleotides (19- and 20-mers) antisense to a 40-mer were synthesized so that the 40-mer coded for guanine at the 3' terminus of the 19-mer. After annealing of the synthetic oligonucleotides to form a duplex DNA containing a one-nucleotide gap (opposite cytosine in the 40-mer), the 19-mer was extended with 2'-deoxythioguanosine 5'-triphosphate using DNA polymerase, forming a nicked duplex DNA. The abilities of T4 DNA ligase and HeLa and calf thymus DNA ligase I to join the 5'-phosphate with the 3'-S6G-OH were severely inhibited, compared with the 3'-guanine-extended control. This finding suggests that incorporation of S6G at the 3' terminus of Okazaki fragments would inhibit lagging strand DNA synthesis. In other experiments, cleavage of S6G-containing DNA by some but not all restriction endonucleases progressed poorly, compared with the control guanine-containing DNA, independently of the location of S6G at recognition or cleavage sites, as previously observed by Iwaniec et al. [Mol. Pharmacol. 39:299-306 (1991)] with a different spectrum of enzymes. These findings indicate altered DNA-protein interactions due to S6G incorporation. The poor template function of S6G-containing DNA is consistent with the known delayed cytotoxicity and DNA damage previously reported to occur in S6G-treated cells.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Ling</LastName><ForeName>Y H</ForeName><Initials>YH</Initials><AffiliationInfo><Affiliation>Department of Experimental Pediatrics, University of Texas M.D. Anderson Cancer Center, Houston 77030.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Chan</LastName><ForeName>J Y</ForeName><Initials>JY</Initials></Author><Author ValidYN="Y"><LastName>Beattie</LastName><ForeName>K L</ForeName><Initials>KL</Initials></Author><Author ValidYN="Y"><LastName>Nelson</LastName><ForeName>J A</ForeName><Initials>JA</Initials></Author></AuthorList><Language>eng</Language><GrantList CompleteYN="Y"><Grant><GrantID>CA-28034</GrantID><Acronym>CA</Acronym><Agency>NCI NIH HHS</Agency><Country>United States</Country></Grant><Grant><GrantID>GM-25530</GrantID><Acronym>GM</Acronym><Agency>NIGMS NIH HHS</Agency><Country>United States</Country></Grant></GrantList><PublicationTypeList><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>Mol Pharmacol</MedlineTA><NlmUniqueID>0035623</NlmUniqueID><ISSNLinking>0026-895X</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>9007-49-2</RegistryNumber><NameOfSubstance UI="D004247">DNA</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 2.7.7.7</RegistryNumber><NameOfSubstance UI="D004259">DNA-Directed DNA Polymerase</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 3.1.-</RegistryNumber><NameOfSubstance UI="D004720">Endonucleases</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 6.5.1.-</RegistryNumber><NameOfSubstance UI="D011088">DNA Ligases</NameOfSubstance></Chemical><Chemical><RegistryNumber>FTK8U1GZNX</RegistryNumber><NameOfSubstance UI="D013866">Thioguanine</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D001345" MajorTopicYN="N">Autoradiography</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D001483" MajorTopicYN="N">Base Sequence</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D004247" MajorTopicYN="N">DNA</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D011088" MajorTopicYN="N">DNA Ligases</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D004259" MajorTopicYN="N">DNA-Directed DNA Polymerase</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D004591" MajorTopicYN="N">Electrophoresis, Polyacrylamide Gel</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D004720" MajorTopicYN="N">Endonucleases</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D008969" MajorTopicYN="N">Molecular Sequence Data</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D013379" MajorTopicYN="N">Substrate Specificity</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D013698" MajorTopicYN="N">Templates, Genetic</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D013866" MajorTopicYN="N">Thioguanine</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="pubmed"><Year>1992</Year><Month>11</Month><Day>1</Day></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>1992</Year><Month>11</Month><Day>1</Day><Hour>0</Hour><Minute>1</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>1992</Year><Month>11</Month><Day>1</Day><Hour>0</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">1331762</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list></list><tree><noCountry><name sortKey="Beattie, K L" sort="Beattie, K L" uniqKey="Beattie K" first="K L" last="Beattie">K L Beattie</name><name sortKey="Chan, J Y" sort="Chan, J Y" uniqKey="Chan J" first="J Y" last="Chan">J Y Chan</name><name sortKey="Ling, Y H" sort="Ling, Y H" uniqKey="Ling Y" first="Y H" last="Ling">Y H Ling</name><name sortKey="Nelson, J A" sort="Nelson, J A" uniqKey="Nelson J" first="J A" last="Nelson">J A Nelson</name></noCountry></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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