Incorporation of 5',8-cyclo-2'deoxyadenosines by DNA repair polymerases via base excision repair

Abstract

5',8-cyclo-2-deoxy nucleosides (cdPus) are the smallest tandem purine lesions including 5',8-cyclo-2'-deoxyadenosine (cdA) and 5',8-cyclo-2'-deoxyguanosine (cdG). They can inhibit DNA and RNA polymerases causing mutations, DNA strand breaks, and termination of DNA replication and gene transcription. cdPus can be removed by nucleotide excision repair with low efficiency allowing them to accumulate in the genome. Recent studies suggest that cdPus can be induced in damaged nucleotide pools and incorporated into the genome by DNA polymerases. However, it remains unknown if and how DNA polymerases can incorporate cdPus. In this study, we examined the incorporation of cdAs by human DNA repair polymerases, DNA polymerases β (pol β), and pol η during base excision repair. We then determined the efficiency of cdA incorporation by the polymerases using steady-state kinetics. We found that pol β and pol η incorporated cdAs opposite dT and dC with low efficiency, and incorporated cdAs were readily extended and ligated into duplex DNA. Using molecular docking analysis, we found that the 5',8-covalent bond in cdA disrupted its hydrogen bonding with a template base suggesting that the phosphodiester bond between the 3'-terminus nucleotide and the α-phosphate of cdATP were generated in the absence of hydrogen bonding. The enzyme kinetics analysis further suggests that pol β and pol η increased their substrate binding to facilitate the enzyme catalysis for cdA incorporation. Our study reveals unique mechanisms underlying the accumulation of cdPu lesions in the genome resulting from nucleotide incorporation by repair DNA polymerases.

Keywords: 5′,8-cyclo-2′-deoxyadenosine; Base excision repair, steady-state kinetics of cyclo-deoxypurine incorporation; DNA polymerase β; DNA polymerase η; Incorporation of cyclo-deoxypurine lesions.