An 8-oxo-guanine repair pathway coordinated by MUTYH glycosylase and DNA polymerase lambda

Abstract

Reactive oxygen species (ROS) interact with DNA, frequently generating highly mutagenic 7,8-dihydro-8-oxoguanine (8-oxo-G) lesions. Replicative DNA polymerases (pols) often misincorporate adenine opposite 8-oxo-G. The subsequent repair mechanism allowing the removal of adenine and formation of C:8-oxo-G base pair is essential to prevent C:G to A:T transversion mutations. Here, we show by immunofluorescence experiments, in cells exposed to ROS, the involvement of MutY glycosylase homologue (MUTYH) and DNA pol lambda in the repair of A:8-oxo-G mispairs. We observe specific recruitment of MUTYH, DNA pol lambda, proliferating cell nuclear antigen (PCNA), flap endonuclease 1 (FEN1) and DNA ligases I and III from human cell extracts to A:8-oxo-G DNA, but not to undamaged DNA. Using purified human proteins and a DNA template, we reconstitute the full pathway for the faithful repair of A:8-oxo-G mispairs involving MUTYH, DNA pol lambda, FEN1, and DNA ligase I. These results reveal a cellular response pathway to ROS, important to sustain genomic stability and modulate carcinogenesis.

Fig. 1.

Recruitment of BER proteins to the sites of oxidative DNA damage. The experiments were done under the conditions as specified in Materials and Methods. (A and B) Fluorescent microscope images of 355-nm laser-irradiated HeLa cells synchronized in G1/S phase and stained with antibodies against 8-oxo-G and MUTYH (A) or DNA pol λ (B). Colocalization was observed in 50% of the analyzed cells. (C) Immunoblot analysis of MUTYH and DNA pol λ protein level, 5 h upon recovery, in HeLa whole cell extract (WCE) treated with and without H₂O₂. (D and E) Protein recruitment assay using A:8-oxo-G (Left) or C:G (Right) biotinylated hairpin substrate and HeLa WCE in the absence (D) or presence (E) of Mg²⁺ for the times indicated (*, the higher molecular weight band corresponds to MUTYH localized in mitochondria). Only proteins that bind to hairpin substrate can be cross-linked and visualized by immunoblotting.

Fig. 2.

DNA polymerase λ physically interacts with MUTYH and preferentially incorporates the correct dCTP opposite an 8-oxo-G after inaccurate replication by DNA polymerase δ. The experiments were done under the conditions as specified in Materials and Methods. (A) MUTYH titration in the presence of the indicated amounts of DNA pol δ, APE1, dGTP, dTTP and dATP (lanes 1–6) or dCTP (lanes 7–12). (B) Interaction of MUTYH and DNA pol λ using a GST pull-down assay. Incubation of APE1, DNA pols δ, and λ with GST-MUTYH (lanes 1–3) and GST (lanes 4–6). Lanes 7–9: input purified recombinant APE1, DNA pols δ, and λ, respectively. (C) dGTP, dTTP, and dATP (lanes 1–6) or dCTP (lanes 7–12) incorporation by DNA pol λ with MUTYH and APE1. (D) Summary of DNA pol λ activity in the presence of dATP (●) or dCTP (■) from three different experiments as the one documented in C; error bars, ± SD values.

Fig. 3.

RP-A and PCNA stimulate accurate incorporation by DNA polymerase λ and this product is specifically processed by FEN1 and DNA ligase I. The experiments were done under the conditions specified in Materials and Methods. (A and B) Incorporation of dGTP, dTTP, and dATP (white bars) or dCTP (black bars) by DNA pol λ in the presence of RP-A (A) or PCNA (B). Error bars, ± SD values of three independent experiments. (C) Products of the DNA pol λ repair synthesis containing A:8-oxo-G mispair (white bars) or C:8-oxo-G base pair (black bars) ligated by DNA ligase III in the presence of MUTYH and APE1. Error bars, ± SD values of three independent experiments. (D) dGTP, dTTP, and dATP (lanes 1–6) or dCTP (lanes 7–12) incorporation by DNA pol λ with MUTYH, APE1, and FEN1. (E) Products of the DNA pol λ repair synthesis containing A:8-oxo-G mispair (white bars) or C:8-oxo-G base pair (black bars) ligated by DNA ligase I in the presence of MUTYH, APE1, and FEN1. Error bars, ± SD values of three independent experiments.

Fig. 4.

Model for the MUTYH initiated long patch BER of 8-oxo-G, after misincorporation by the replication machinery. 1) DNA replication over an 8-oxo-G by DNA pol δ. 2) recognition of an A:8-oxo-G mispair by MUTYH, removal of the A and formation of an AP site (designated as B). 3) recruitment of APE1 mediated by MUTYH/PCNA and generation of 5′-P, 3′-OH gapped intermediate. 4) protection of the 1-nt gap by RP-A and PCNA mediated recruitment of DNA pol λ, with accurate gap filling (dCTP incorporation). 5) PCNA mediated recruitment of FEN1 and removal of 1-nt flap. 6) ligation of the nick by recruited DNA ligase I and further faithful OGG1 initiated DNA pol β mediated SP-BER of C:8-oxo-G product. Alternatively an inaccurate loop is initiated: (A) DNA pol λ catalyzes inaccurate gap filling; (B) recruitment of DNA ligase III/XRCC1-mediated by PCNA and ligation of the nick; (C) recognition of A:8-oxo-G mispair by MUTYH, removal of A and generation of AP site (designated as B); and (D) recruitment of APE1 mediated by MUTYH/PCNA, generation of 5′-P, 3′-OH gapped intermediate. This creates an opportunity for DNA pol λ to catalyze accurate LP-BER. For further details, see text.