Oxidized nucleotide insertion by pol β confounds ligation during base excision repair

Abstract

Oxidative stress in cells can lead to accumulation of reactive oxygen species and oxidation of DNA precursors. Oxidized purine nucleotides can be inserted into DNA during replication and repair. The main pathway for correcting oxidized bases in DNA is base excision repair (BER), and in vertebrates DNA polymerase β (pol β) provides gap filling and tailoring functions. Here we report that the DNA ligation step of BER is compromised after pol β insertion of oxidized purine nucleotides into the BER intermediate in vitro. These results suggest the possibility that BER mediated toxic strand breaks are produced in cells under oxidative stress conditions. We observe enhanced cytotoxicity in oxidizing-agent treated pol β expressing mouse fibroblasts, suggesting formation of DNA strand breaks under these treatment conditions. Increased cytotoxicity following MTH1 knockout or treatment with MTH1 inhibitor suggests the oxidation of precursor nucleotides.

Role of DNA polymerase β oxidized nucleotide insertion in DNA ligation failure

“…Furthermore, in a previous study [22], we observed more cytotoxicity and DNA strand breaks in mouse embryonic fibroblast cells expressing pol β than in pol β null cells. Our in vitro findings using cell extracts demonstrated that there is a limited role of DNA end-processing enzymes in the repair of impaired BER intermediates, after pol β oxidized nucleotide insertion and ligation failure.…”

“…In a previous study, we were able to measure pol β nucleotide insertion into a single-nucleotide gapped DNA substrate with template bases dA, dG, dC or dT, and ligation reactions in the same reaction mixture, as a function of time [22]. This biochemical approach enabled us to compare various products of ligation failure (i.e.…”

“…Gel images showing the products for oxidized nucleotide insertion, ligation and ligation failure after 8-oxodGTP insertion against template base C (a), A (b), G (c) and T (d). We previously published the gel image that shows the products for insertion, ligation, and abortive ligation for 8-oxodGMP insertion against template base C [22]. The illustration in Panel a is the result of a repeat experiment.…”

“…The materials and methods used to obtain the experimental results shown here have been previously reported [22]. The reactions were performed under steady-state conditions, where the DNA substrate was in excess relative to the enzymes, i.e.…”

“…In a previous study [22], we demonstrated that oxidized nucleotide insertion by pol β results in ligation failure and impairs BER. This ligation failure after insertion of an oxidized purine nucleotide corresponds to formation of the BER intermediate with blocked 3′-8-oxo-7,8-dihydro-2′-deoxyguanosine (3′-8-oxoG) and 5′-adenylated, (5′-AMP)–containing ends.…”