Influence of local nucleotide sequence on substitution of 2-aminopurine for adenine during deoxyribonucleic acid synthesis in vitro

Abstract

Three highly purified DNA polymerases, Escherichia coli polymerase I (enzyme A) and the polymerases induced by wild-type T4 phage and by T4 phage mutant L141 (antimutator phenotype), have been examined with respect to their tendency to incorporate the deoxyribonucleotide of 2-aminopurine [(AP)] for deoxyadenylate at specific sites in deoxyribonucleic acid (DNA). Using phi X174 phage DNA as a template and selected phi X174 restriction fragments as specific primers, we synthesized short sequences of phi X174 DNA in vitro by the polymerase of interest, with the 5'-triphosphate of 2-aminopurine deoxyriboside and dATP at equimolar concentration. The relative incorporation of (AP) at the various adenine sites was determined by providing the newly synthesized DNA fragment with a specific terminal radioactive label, subjecting the DNA fragment to thermal depurination as a DNA cleavage reaction highly selective for (AP), and analyzing the resulting radioactive fragments by denaturing gel electrophoresis, autoradiography, and microdensitometry. The L141 polymerase shows very pronounced site-dependent variations in (AP) incorporation. For the wild-type T4 polymerase, the pattern of (AP) incorporation follows the biases seen for the L141 enzyme, although in a less pronounced form. Sequence preferences for (AP) incorporation are least marked for E. coli polymerase I (enzyme A); in several instances, they run counter to the sequence biases observed with the T4 enzymes. For the enzyme showing the most pronounced sequence effects, L141 polymerase, the extent of (AP) incorporation was determined at 57 different sites. No simple principle governing the sequence dependence of (AP) incorporation could be deduced from these results.

ASFV DNA Polymerase X Is Extremely Error-Prone under Diverse Assay Conditions and within Multiple DNA Sequence Contexts

“…(ii) DNA substrate sequence: since DNA sequence context can influence the catalytic properties (including fidelity) of a polymerase (20)(21)(22)(23)(24)(25), Pol X was reassayed using the same substrate employed by Salas. (iii) Pre-steady state vs. steady state: our previous analyses of Pol X were conducted in the pre-steady state (10), while those of Salas and coworkers were done in the steady state (14).…”

Abasic sites from cytosine as termination signals for DNA synthesis

“…These experiments also illustrate the biochemical relationship between 2-amino purine and adenine. 2-Amino purine is recognized as adenine at the exonucleolytic site and incorporated like adenine at the polymerization site although with some differences in detail, as might be expected from the complexity of factors governing its incorporation in different sites (22).…”

NMR studies of DNA oligomers and their interactions with minor groove binding ligands

“…This has potential implications for the observed dependence of single-site mutation rates with nearest-neighboring bases (Pless & Bessman, 1983;Mendelman et al, 1989;Joyce et al, 1992;Mitra et al, 1993). The mutagenicity of AP has been shown to be strongly dependent on the local DNA sequence at the mutation site (Ronen, 1979).…”

“…The mutagenicity of AP has been shown to be strongly dependent on the local DNA sequence at the mutation site (Ronen, 1979). The local DNA sequence, specifically the extent to which the primer-3'-nearest neighbor can stabilize an incoming dNTP substrate at the polymerase active site, has been shown to influence efficiency of AP misinsertion opposite T by DNA polymerase (Pless & Bessman, 1983;Petruska & Goodman, 1985;Bloom et al, 1993).…”