Reactive oxygen species (ROS), generated both endogenously and in response to exogenous stress, induce point mutations by mis-replication of oxidized bases and other lesions in the genome. Repair of these lesions via base excision repair (BER) pathway maintains genomic fidelity. Regulation of the BER pathway for mutagenic oxidized bases, initiated by NEIL1 and other DNA glycosylases at the chromatin level remains unexplored. Whether single nucleotide (SN)-BER of a damaged base requires histone deposition or nucleosome remodeling is unknown, unlike nucleosome reassembly which is shown to be required for other DNA repair processes. Here we show that chromatin assembly factor (CAF)-1 subunit A (CHAF1A), the p150 subunit of the histone H3/H4 chaperone, and its partner anti-silencing function protein 1A (ASF1A), which we identified in human NEIL1 immunoprecipitation complex, transiently dissociate from chromatin bound NEIL1 complex in G1 cells after induction of oxidative base damage. CHAF1A inhibits NEIL1 initiated repair in vitro Subsequent restoration of the chaperone-BER complex in cell, presumably after completion of repair, suggests that histone chaperones sequester the repair complex for oxidized bases in non-replicating chromatin, and allow repair when oxidized bases are induced in the genome.

Homologous recombination/end joining (HR/HEJ)-deficient cancers with BRCA mutations utilize alternative DNA double-strand break repair pathways, particularly alternative non-homologous end joining or microhomology-mediated end joining (alt-EJ/MMEJ) during S and G2 cell cycle phases. Depletion of alt-EJ factors, including XRCC1, PARP1 and POLQ, is synthetically lethal with BRCA2 deficiency; yet, XRCC1 roles in HR-deficient cancers and replication stress are enigmatic. Here, we show that after replication stress, XRCC1 forms an active repair complex with POLQ and MRE11 that supports alt-EJ activity in vitro. BRCA2 limits XRCC1 recruitment and repair complex formation to suppress alt-EJ at stalled forks. Without BRCA2 fork protection, XRCC1 enables cells to complete DNA replication at the expense of increased genome instability by promoting MRE11-dependent fork resection and restart. High XRCC1 and MRE11 gene expression negatively impacts Kaplan-Meier survival curves and hazard ratios for HR-deficient breast cancer patients in The Cancer Genome Atlas. The additive effects of depleting both BRCA2 and XRCC1 indicate distinct pathways for replication restart. Our collective data show that XRCC1-mediated processing contributes to replication fork degradation, replication restart and chromosome aberrations in BRCA2-deficient cells, uncovering new roles of XRCC1 and microhomology-mediated repair mechanisms in HR-deficient cancers, with implications for chemotherapeutic strategies targeting POLQ and PARP activities.

XRCC1 plays a key role in the repair of DNA base damage and single-strand breaks. Although it has no known enzymatic activity, XRCC1 interacts with multiple DNA repair proteins and is a subunit of distinct DNA repair protein complexes. Here we used the yeast two-hybrid genetic assay to identify mutant versions of XRCC1 that are selectively defective in interacting with a single protein partner. One XRCC1 mutant, A482T, that was defective in binding to polynucleotide kinase phosphatase (PNKP) not only retained the ability to interact with partner proteins that bind to different regions of XRCC1 but also with aprataxin and aprataxin-like factor whose binding sites overlap with that of PNKP. Disruption of the interaction between PNKP and XRCC1 did not impact their initial recruitment to localized DNA damage sites but dramatically reduced their retention there. Furthermore, the interaction between PNKP and the DNA ligase IIIα-XRCC1 complex significantly increased the efficiency of reconstituted repair reactions and was required for complementation of the DNA damage sensitivity to DNA alkylation agents of xrcc1 mutant cells. Together our results reveal novel roles for the interaction between PNKP and XRCC1 in the retention of XRCC1 at DNA damage sites and in DNA alkylation damage repair.

Microhomology-mediated end joining (MMEJ), an error-prone pathway for DNA double-strand break (DSB) repair, is implicated in genomic rearrangement and oncogenic transformation; however, its contribution to repair of radiation-induced DSBs has not been characterized. We used recircularization of a linearized plasmid with 3΄-P-blocked termini, mimicking those at X-ray-induced strand breaks, to recapitulate DSB repair via MMEJ or nonhomologous end-joining (NHEJ). Sequence analysis of the circularized plasmids allowed measurement of relative activity of MMEJ versus NHEJ. While we predictably observed NHEJ to be the predominant pathway for DSB repair in our assay, MMEJ was significantly enhanced in preirradiated cells, independent of their radiation-induced arrest in the G2/M phase. MMEJ activation was dependent on XRCC1 phosphorylation by casein kinase 2 (CK2), enhancing XRCC1's interaction with the end resection enzymes MRE11 and CtIP. Both endonuclease and exonuclease activities of MRE11 were required for MMEJ, as has been observed for homology-directed DSB repair (HDR). Furthermore, the XRCC1 co-immunoprecipitate complex (IP) displayed MMEJ activity in vitro, which was significantly elevated after irradiation. Our studies thus suggest that radiation-mediated enhancement of MMEJ in cells surviving radiation therapy may contribute to their radioresistance and could be therapeutically targeted.

Human genome stability requires efficient repair of oxidized bases, which is initiated via damage recognition and excision by NEIL1 and other base excision repair (BER) pathway DNA glycosylases (DGs). However, the biological mechanisms underlying detection of damaged bases among the million-fold excess of undamaged bases remain enigmatic. Indeed, mutation rates vary greatly within individual genomes, and lesion recognition by purified DGs in the chromatin context is inefficient. Employing super-resolution microscopy and co-immunoprecipitation assays, we find that acetylated NEIL1 (AcNEIL1), but not its non-acetylated form, is predominantly localized in the nucleus in association with epigenetic marks of uncondensed chromatin. Furthermore, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) revealed non-random AcNEIL1 binding near transcription start sites of weakly transcribed genes and along highly transcribed chromatin domains. Bioinformatic analyses revealed a striking correspondence between AcNEIL1 occupancy along the genome and mutation rates, with AcNEIL1-occupied sites exhibiting fewer mutations compared to AcNEIL1-free domains, both in cancer genomes and in population variation. Intriguingly, from the evolutionarily conserved unstructured domain that targets NEIL1 to open chromatin, its damage surveillance of highly oxidation-susceptible sites to preserve essential gene function and to limit instability and cancer likely originated ∼500 million years ago during the buildup of free atmospheric oxygen.