N-terminal domains of human DNA polymerase lambda promote primer realignment during translesion DNA synthesis.

ABSTRACT: The X-family DNA polymerases ? (Pol?) and ? (Pol?) possess similar 5'-2-deoxyribose-5-phosphate lyase (dRPase) and polymerase domains. Besides these domains, Pol? also possesses a BRCA1 C-terminal (BRCT) domain and a proline-rich domain at its N terminus. However, it is unclear how these non-enzymatic domains contribute to the unique biological functions of Pol?. Here, we used primer extension assays and a newly developed high-throughput short oligonucleotide sequencing assay (HT-SOSA) to compare the efficiency of lesion bypass and fidelity of human Pol?, Pol? and two N-terminal deletion constructs of Pol? during the bypass of either an abasic site or an 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) lesion. We demonstrate that the BRCT domain of Pol? enhances the efficiency of abasic site bypass by approximately 1.6-fold. In contrast, deletion of the N-terminal domains of Pol? did not affect the efficiency of 8-oxodG bypass relative to nucleotide incorporations opposite undamaged dG. HT-SOSA analysis demonstrated that Pol? and Pol? preferentially generated -1 or -2 frameshift mutations when bypassing an abasic site and the single or double base deletion frequency was highly sequence dependent. Interestingly, the BRCT and proline-rich domains of Pol? cooperatively promoted the generation of -2 frameshift mutations when the abasic site was situated within a sequence context that was susceptible to homology-driven primer realignment. Furthermore, both N-terminal domains of Pol? increased the generation of -1 frameshift mutations during 8-oxodG bypass and influenced the frequency of substitution mutations produced by Pol? opposite the 8-oxodG lesion. Overall, our data support a model wherein the BRCT and proline-rich domains of Pol? act cooperatively to promote primer/template realignment between DNA strands of limited sequence homology. This function of the N-terminal domains may facilitate the role of Pol? as a gap-filling polymerase within the non-homologous end joining pathway.

SUBMITTER: Taggart DJ 

PROVIDER: S-EPMC4175094 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature