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

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.

Characterization of a Mutagenic DNA Adduct Formed from 1,2-Dibromoethane by O6-Alkylguanine-DNA Alkyltransferase

“…Both G:C to A:T transitions and G:C to T:A transversions leading to rifampicin resistance were observed in AGT-mediated response to DBE (Table I). The G to T transversions, which represent 20% of the mutations we observed in the rpoB gene are consistent with depurination of the Gua adduct and subsequent processing of the abasic site (46,47). However, in both the rpoB and the lacI genes, the most common mutations were G to A transitions.…”

Altering DNA base excision repair: Use of nuclear and mitochondrial‐targeted N‐methylpurine DNA glycosylase to sensitize astroglia to chemotherapeutic agents

“…Interestingly, the intermediate AP sites and SSB generated in this pathway are more cytotoxic in many cases than the modified base lesions themselves (Chaudhry and Weinfeld, 1997;Lockhart et al, 1982;Loeb and Preston, 1986;Sagher and Strauss, 1983;Schaaper et al, 1983;Whitehouse et al, 2001). MNU was used in the present studies to generate alkylation damage.…”

Amino Acid Templating Mechanisms in Selection of Nucleotides Opposite Abasic Sites by a Family A DNA Polymerase

“…S1) have shown that purines, in particular adenosine, and to a lesser extent guanosine, are most frequently incorporated opposite the lesion. The strong preference for adenosine has been termed "A-rule" (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20).…”