Insertion of nucleotides opposite apurinic apyrimidinic sites in deoxyribonucleic acid during in vitro synthesis: uniqueness of adenine nucleotides

M13 DNA containing 20-30 apurinic/apyrimidinic (AP) sites per intact circular molecule was prepared by growing phage on an ung- dut- Escherichia coli mutant and treating the DNA with uracil N-glycosylase. AP sites obstruct in vitro DNA synthesis catalyzed by E. coli pol I. The position at which term...

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Veröffentlicht in:Biochemistry (Easton) 1983-09, Vol.22 (19), p.4518-4526
Hauptverfasser: Sagher, Daphna, Strauss, Bernard
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container_title Biochemistry (Easton)
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creator Sagher, Daphna
Strauss, Bernard
description M13 DNA containing 20-30 apurinic/apyrimidinic (AP) sites per intact circular molecule was prepared by growing phage on an ung- dut- Escherichia coli mutant and treating the DNA with uracil N-glycosylase. AP sites obstruct in vitro DNA synthesis catalyzed by E. coli pol I. The position at which termination of synthesis occurs was determined for four enzymes. T4 DNA polymerase terminates one nucleotide before putative AP sites. DNA pol I, AMV reverse transcriptase, and DNA polymerase alpha terminate synthesis either before or at the site of an AP lesion depending on the particular sequence. We determined the identity of the nucleotide inserted opposite an AP site by synthesizing up to the lesion in a first-stage reaction using T4 DNA polymerase and then determining elongation in a second stage. Purines are inserted opposite AP sites more readily than pyrimidines, and dATP is more efficient than dGTP in promoting such elongation. The DNA-dependent conversion of dNTP to dNMP was determined in mixtures of all four dNTP's by using AP DNA. The production of dAMP from dATP occurs most readily. We conclude that there is an inherent specificity for the incorporation of adenine nucleotides opposite AP sites in this in vitro system. Insofar as the model system reflects in vivo mutational events, our data suggest that depurination should produce transversions and depyrimidination should produce transitions.
doi_str_mv 10.1021/bi00288a026
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AP sites obstruct in vitro DNA synthesis catalyzed by E. coli pol I. The position at which termination of synthesis occurs was determined for four enzymes. T4 DNA polymerase terminates one nucleotide before putative AP sites. DNA pol I, AMV reverse transcriptase, and DNA polymerase alpha terminate synthesis either before or at the site of an AP lesion depending on the particular sequence. We determined the identity of the nucleotide inserted opposite an AP site by synthesizing up to the lesion in a first-stage reaction using T4 DNA polymerase and then determining elongation in a second stage. Purines are inserted opposite AP sites more readily than pyrimidines, and dATP is more efficient than dGTP in promoting such elongation. The DNA-dependent conversion of dNTP to dNMP was determined in mixtures of all four dNTP's by using AP DNA. The production of dAMP from dATP occurs most readily. 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Psychology ; Humans ; Kinetics ; Magnesium - pharmacology ; Manganese - pharmacology ; Molecular and cellular biology ; Molecular genetics ; Mutagenesis. 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AP sites obstruct in vitro DNA synthesis catalyzed by E. coli pol I. The position at which termination of synthesis occurs was determined for four enzymes. T4 DNA polymerase terminates one nucleotide before putative AP sites. DNA pol I, AMV reverse transcriptase, and DNA polymerase alpha terminate synthesis either before or at the site of an AP lesion depending on the particular sequence. We determined the identity of the nucleotide inserted opposite an AP site by synthesizing up to the lesion in a first-stage reaction using T4 DNA polymerase and then determining elongation in a second stage. Purines are inserted opposite AP sites more readily than pyrimidines, and dATP is more efficient than dGTP in promoting such elongation. The DNA-dependent conversion of dNTP to dNMP was determined in mixtures of all four dNTP's by using AP DNA. The production of dAMP from dATP occurs most readily. We conclude that there is an inherent specificity for the incorporation of adenine nucleotides opposite AP sites in this in vitro system. Insofar as the model system reflects in vivo mutational events, our data suggest that depurination should produce transversions and depyrimidination should produce transitions.</description><subject>Apurinic Acid - genetics</subject><subject>Biological and medical sciences</subject><subject>Burkitt Lymphoma</subject><subject>Cell Line</subject><subject>Coliphages - genetics</subject><subject>DNA Glycosylases</subject><subject>DNA Polymerase I - metabolism</subject><subject>DNA Replication - drug effects</subject><subject>DNA, Viral - genetics</subject><subject>DNA-Directed DNA Polymerase - metabolism</subject><subject>Escherichia coli - enzymology</subject><subject>Escherichia coli - genetics</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Humans</subject><subject>Kinetics</subject><subject>Magnesium - pharmacology</subject><subject>Manganese - pharmacology</subject><subject>Molecular and cellular biology</subject><subject>Molecular genetics</subject><subject>Mutagenesis. 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Psychology</topic><topic>Humans</topic><topic>Kinetics</topic><topic>Magnesium - pharmacology</topic><topic>Manganese - pharmacology</topic><topic>Molecular and cellular biology</topic><topic>Molecular genetics</topic><topic>Mutagenesis. Repair</topic><topic>N-Glycosyl Hydrolases - metabolism</topic><topic>phage M13</topic><topic>Polynucleotides - genetics</topic><topic>Templates, Genetic</topic><topic>Uracil-DNA Glycosidase</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Sagher, Daphna</creatorcontrib><creatorcontrib>Strauss, Bernard</creatorcontrib><collection>Istex</collection><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Biochemistry (Easton)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Sagher, Daphna</au><au>Strauss, Bernard</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Insertion of nucleotides opposite apurinic apyrimidinic sites in deoxyribonucleic acid during in vitro synthesis: uniqueness of adenine nucleotides</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>1983-09-13</date><risdate>1983</risdate><volume>22</volume><issue>19</issue><spage>4518</spage><epage>4526</epage><pages>4518-4526</pages><issn>0006-2960</issn><eissn>1520-4995</eissn><abstract>M13 DNA containing 20-30 apurinic/apyrimidinic (AP) sites per intact circular molecule was prepared by growing phage on an ung- dut- Escherichia coli mutant and treating the DNA with uracil N-glycosylase. AP sites obstruct in vitro DNA synthesis catalyzed by E. coli pol I. The position at which termination of synthesis occurs was determined for four enzymes. T4 DNA polymerase terminates one nucleotide before putative AP sites. DNA pol I, AMV reverse transcriptase, and DNA polymerase alpha terminate synthesis either before or at the site of an AP lesion depending on the particular sequence. We determined the identity of the nucleotide inserted opposite an AP site by synthesizing up to the lesion in a first-stage reaction using T4 DNA polymerase and then determining elongation in a second stage. Purines are inserted opposite AP sites more readily than pyrimidines, and dATP is more efficient than dGTP in promoting such elongation. The DNA-dependent conversion of dNTP to dNMP was determined in mixtures of all four dNTP's by using AP DNA. The production of dAMP from dATP occurs most readily. We conclude that there is an inherent specificity for the incorporation of adenine nucleotides opposite AP sites in this in vitro system. Insofar as the model system reflects in vivo mutational events, our data suggest that depurination should produce transversions and depyrimidination should produce transitions.</abstract><cop>Washington, DC</cop><pub>American Chemical Society</pub><pmid>6354260</pmid><doi>10.1021/bi00288a026</doi><tpages>9</tpages></addata></record>
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source MEDLINE; American Chemical Society (ACS) Journals
subjects Apurinic Acid - genetics
Biological and medical sciences
Burkitt Lymphoma
Cell Line
Coliphages - genetics
DNA Glycosylases
DNA Polymerase I - metabolism
DNA Replication - drug effects
DNA, Viral - genetics
DNA-Directed DNA Polymerase - metabolism
Escherichia coli - enzymology
Escherichia coli - genetics
Fundamental and applied biological sciences. Psychology
Humans
Kinetics
Magnesium - pharmacology
Manganese - pharmacology
Molecular and cellular biology
Molecular genetics
Mutagenesis. Repair
N-Glycosyl Hydrolases - metabolism
phage M13
Polynucleotides - genetics
Templates, Genetic
Uracil-DNA Glycosidase
title Insertion of nucleotides opposite apurinic apyrimidinic sites in deoxyribonucleic acid during in vitro synthesis: uniqueness of adenine nucleotides
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