In Vitro Effects of a C4‘-Oxidized Abasic Site on DNA Polymerases

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Abstract
Oxidative damage to DNA produces abasic sites resulting from the formal hydrolysis of the nucleotides' glycosidic bonds, along with a variety of oxidized abasic sites. The C4‘-oxidized abasic site (C4-AP) is produced by several DNA-damaging agents. This lesion accounts for ∼40% of the DNA damage produced by bleomycin. The effect of a C4‘-oxidized abasic site incorporated at a defined site in a template was examined on Klenow fragments with and without 3‘ → 5‘ exonuclease activity. Both enzymes preferentially incorporated dA > dG ≫ dC, T opposite C4-AP. Neither enzyme is able to extend the primer past the lesion. Experiments with regular AP sites in an otherwise identical template indicate that Klenow does not differentiate between these two disparate abasic sites. Extension of the primer by alternative polymerases pol II, pol II exo-, pol IV, and pol V was examined. Pol II exo- was most efficient. Qualitative translesion synthesis experiments showed that pol II exo- preferentially incorporates T opposite C4-AP, followed in order by dG, dA, and dC. Thymidine incorporation opposite C4‘-AP is distinct from the pol II exonuclease interaction with a regular AP site in an otherwise identical template. These in vitro experiments suggest that bypass polymerases may play a crucial role in survival of cells in which C4-AP is produced, and unlike a typical AP site, the C4-AP lesion may not follow the “A-rule”. The interaction between bypass polymerases and a C4-AP lesion could explain the high levels of G:C → T:A transversions in cells treated with bleomycin.