Consequences of 6-thioguanine incorporation into DNA on polymerase, ligase, and endonuclease reactions.
Identifieur interne : 004733 ( Main/Exploration ); précédent : 004732; suivant : 004734Consequences 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
Affiliations:
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Le document en format XML
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<sourceDesc><biblStruct><analytic><title xml:lang="en">Consequences of 6-thioguanine incorporation into DNA on polymerase, ligase, and endonuclease reactions.</title>
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<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>
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<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>
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<term>DNA Ligases</term>
<term>DNA-Directed DNA Polymerase</term>
<term>Endonucleases</term>
<term>Thioguanine</term>
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<keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>ADN</term>
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<term>DNA-directed DNA polymerase</term>
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<term>Données de séquences moléculaires</term>
<term>Matrices (génétique)</term>
<term>Spécificité du substrat</term>
<term>Séquence nucléotidique</term>
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<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>
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<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>
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