DNA replication past bulky carcinogenic DNA adducts: Importance of Escherichia coli DNA polymerase I (KF) sub-domains in translesion DNA synthesis.

摘要

It has previously been shown that the thumb sub-domain of Pol A family polymerases contributes to processivity. Here we assessed the role of this region in AAF lesion bypass utilizing an E.coli exonuclease deficient KF mutant (Delta590-613) in which 24 amino acids at the tip of the thumb have been removed. In two primer-template systems studied, normal sequence and the NarI palindrome, it was found that WT KF and Delta590-613 extension patterns were very similar to one another in absence of adduct. DNA synthesis on AAF adducted templates produced differing results. Both polymerases were unable to perform efficient lesion bypass on normal template; each stalling one nucleotide prior to the adduct site with very little full length product synthesized. Albeit on NarI-AAF, WT KF was able to efficiently bypass the lesion generating a -2 deletion product similar to that seen with E.coli Pol II, while no appreciable incorporation of nucleotide across from or beyond the adduct could be detected for Delta590-613 To elucidate this observation human DNA polymerase eta (Pol eta) extension was conducted using the same primer-template systems. It was found that in either case, Pol eta was able to replicate across AAF efficiently. However, extension on NarI-AAF gave rise to both full length and -2 deletion product which is indicative of the palindrome existing in a state of equilibrium between an unlooped and looped structure. Looped template distortions were further assessed with respect to frameshifting utilizing a primer-template containing a 2-thymine template bulge. These results were correlative with NarI extension studies showing that Delta590-613 was unable to replicate past the template loop structure, while both WT KF and Pol eta were able to synthesize product. These studies suggest that the thumb sub-domain of KF plays and important role in replication past bulged sequences and supports the hypothesis of NarI-AFF undergoing loop formation upon DNA replication. This work also demonstrates that Pol eta preferentially extends from a NarI-AAF straight sequence template, while WT KF efficiently extends from one that is looped; correlative to differences in the active site constraints of Pol A and Pol Y family polymerases.