Kinetic analysis of bypass of abasic site by the catalytic core of yeast DNA polymerase eta

摘要

Abasic sites (Apurinic/apyrimidinic (AP) sites), produced ~50,000 times/cell/day, are very blocking and miscoding. To better understand miscoding mechanisms of abasic site for yeast DNA polymerase Eta, pre-steady-state nucleotide incorporation and LC-MS/MS sequence analysis of extension product were studied using pol Eta_(core) (catalytic core, residues 1-513), which can completely eliminate the potential effects of the C-terminal C_2H_2 motif of pol Eta on dNTP incorporation. The extension beyond the abasic site was very inefficient. Compared with incorporation of dCTP opposite G, the incorporation efficiencies opposite abasic site were greatly reduced according to the order of dGTP > dATP?dCTP and dTTP. Pol Eta_(core) showed no fast burst phase for any incorporation opposite G or abasic site, suggesting that the catalytic step is not faster than the dissociation of polymerase from DNA. LC-MS/MS sequence analysis of extension products showed that 53% products were dGTP misincorporation, 33% were dATP and 14% were -1 frameshift, indicating that Pol Eta_(core) bypasses abasic site by a combined G-rule, A-rule and -1 frameshift deletions. Compared with full-length pol Eta, pol Eta_(core) relatively reduced the efficiency of incorporation of dCTP opposite G, increased the efficiencies of dNTP incorporation opposite abasic site and the exclusive incorporation of dGTP opposite abasic site, but inhibited the extension beyond abasic site, and increased the priority in extension of A: abasic site relative to G: abasic site. This study provides further understanding in the mutation mechanism of abasic sites for yeast DNA polymerase Eta.