In vitro bypass of malondialdehyde-deoxyguanosine adducts: differential base selection during extension by the Klenow fragment of DNA polymerase I is the critical determinant of replication outcome.

Abstract

The major malondialdehyde-derived adduct in DNA is 3-(2'-deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10(3H)-one (M(1)dG). M(1)dG undergoes hydrolytic ring opening in duplex DNA to 9-(2'-deoxy-beta-D-erythro-pentofuranosyl)-N(2)-(3-oxo-1-propenyl)guanine (N(2)OPdG). Template-primers were constructed containing M(1)dG or N(2)OPdG in a (CpG)(4) repeat sequence and replicated with the Klenow fragment of DNA polymerase I (Kf). Incorporation opposite the lesion and replication beyond the adduct sites by Kf was reduced compared to unadducted controls. The amount of bypass to full-length products was significantly greater with the acyclic adduct, N(2)OPdG, than with the cyclic adduct, M(1)dG. Sequence analysis indicated that the fully extended primers contained dC opposite both adducts when replication was conducted with Kf exo(+). In contrast, with Kf exo(-), primers extended past M(1)dG contained T opposite the adduct, but primers extended past N(2)OPdG contained dC opposite the adduct. Single nucleotide incorporation experiments indicated that Kf exo(-) incorporates all four nucleotides opposite M(1)dG or N(2)OPdG. Kf exo(+) removed dA, dG, and T opposite M(1)dG and N(2)OPdG but was much less active when dC was opposite the adduct. NMR studies on duplex DNA indicated that N(2)OPdG hydrogen bonds with dC in the complementary strand. The fact that base pairing can occur for the acyclic adduct may explain why N(2)OPdG is less blocking than M(1)dG. These results support in vivo findings that the ring-closed adduct, M(1)dG, is more mutagenic than the ring-opened adduct, N(2)OPdG. They also provide a detailed picture of in vitro replication in which the outcome is determined primarily by the selectivity of template-primer extension beyond rather than insertion opposite the adducts.