UV-DDB, a key protein in human global nucleotide excision repair (NER), binds avidly to abasic sites and 8-oxo-guanine (8-oxoG), suggesting a non-canonical role in base excision repair (BER). We investigated whether UV-DDB can stimulate BER for these two common forms of DNA damage, 8-oxoG and abasic sites, which are repaired by 8-oxoguanine glycosylase (OGG1) and apurinic/apyrimidinic endonuclease (APE1), respectively. UV-DDB increased both OGG1 and APE1 strand cleavage and stimulated subsequent DNA polymerase β gap-filling activity by 30-fold. Single molecule real-time imaging revealed that UV-DDB forms transient complexes with OGG1 or APE1, facilitating their dissociation from DNA. Furthermore, UV-DDB moved to sites of 8-oxoG repair in cells and UV-DDB depletion sensitized cells to oxidative DNA damage. We propose that UV-DDB is a general sensor of DNA damage in both NER and BER pathways, facilitating damage recognition in the context of chromatin.

Base excision repair glycosylases locate and remove damaged bases in DNA with remarkable specificity. The MutY glycosylases, unusual for their excision of undamaged adenines mispaired to the oxidized base 8-oxoguanine (OG), must recognize both bases of the mispair in order to prevent promutagenic activity. Moreover, MutY must effectively find OG:A mismatches within the context of highly abundant and structurally similar T:A base pairs. Very little is known about the factors that initiate MutY’s interaction with the substrate when it first encounters an intrahelical OG:A mispair, or about the order of recognition checkpoints. Here, we used structure–activity relationships (SAR) to investigate the features that influence the in vitro measured parameters of mismatch affinity and adenine base excision efficiency by E. coli MutY. We also evaluated the impacts of the same substrate alterations on MutY-mediated repair in a cellular context. Our results show that MutY relies strongly on the presence of the OG base and recognizes multiple structural features at different stages of recognition and catalysis to ensure that only inappropriately mispaired adenines are excised. Notably, some OG modifications resulted in more dramatic reductions in cellular repair than in the in vitro kinetic parameters, indicating their importance for initial recognition events needed to locate the mismatch within DNA. Indeed, the initial encounter of MutY with its target base pair may rely on specific interactions with the 2-amino group of OG in the major groove, a feature that distinguishes OG:A from T:A base pairs. These results furthermore suggest that inefficient substrate location in human MutY homologue variants may prove predictive for the early onset colorectal cancer phenotype known as MUTYH-Associated Polyposis, or MAP.

MutY glycosylase excises adenines misincorporated opposite the oxidatively damaged lesion, 8-oxo-7,8dihydroguanine (OG), to initiate base excision repair and prevent G to T transversion mutations. Successful repair requires MutY recognition of the OG:A mispair amidst highly abundant and structurally similar undamaged DNA base pairs. Herein we use a combination of in vitro and bacterial cell repair assays with single-molecule fluorescence microscopy to demonstrate that both a Cterminal domain histidine residue and the 2-amino group of OG base are critical for MutY detection of OG:A sites. These studies are the first to directly link deficiencies in MutY lesion detection with incomplete cellular repair. These results suggest that defects in lesion detection of human MutY (MUTYH) variants may prove predictive of early-onset colorectal cancer known an MUTYHassociated polyposis. Furthermore, unveiling these specific molecular determinants for repair makes it possible to envision new MUTYH-specific cancer therapies.

The oxidative base damage, 8-oxo-7,8-dihydroguanine (8-oxoG) is a highly mutagenic lesion because replicative DNA polymerases insert adenine (A) opposite 8-oxoG. In mammalian cells, the removal of A incorporated across from 8-oxoG is mediated by the glycosylase MUTYH during base excision repair (BER). After A excision, MUTYH binds avidly to the abasic site and is thus product inhibited. We have previously reported that UV-DDB plays a non-canonical role in BER during the removal of 8-oxoG by 8-oxoG glycosylase, OGG1 and presented preliminary data that UV-DDB can also increase MUTYH activity. In this present study we examine the mechanism of how UV-DDB stimulates MUTYH. Bulk kinetic assays show that UV-DDB can stimulate the turnover rate of MUTYH excision of A across from 8-oxoG by 4–5-fold. Electrophoretic mobility shift assays and atomic force microscopy suggest transient complex formation between MUTYH and UV-DDB, which displaces MUTYH from abasic sites. Using single molecule fluorescence analysis of MUTYH bound to abasic sites, we show that UV-DDB interacts directly with MUTYH and increases the mobility and dissociation rate of MUTYH. UV-DDB decreases MUTYH half-life on abasic sites in DNA from 8800 to 590 seconds. Together these data suggest that UV-DDB facilitates productive turnover of MUTYH at abasic sites during 8-oxoG:A repair.

The DNA glycosylase MutY prevents deleterious mutations resulting from guanine oxidation by recognition and removal of adenine (A) misincorporated opposite 8-oxo-7,8-dihydroguanine (OG). Correct identification of OG:A is crucial to prevent improper and detrimental MutY-mediatedadenine excision from G:A or T:A base pairs. Here we present a structure–activity relationship (SAR) study using analogues of A to probe the basis for OG:A specificity of MutY. We correlate observed in vitro MutY activity on A analogue substrates with their experimental and calculated acidities to provide mechanistic insight into the factors influencing MutY base excision efficiency. These data show that H-bonding and electrostatic interactions of the base within the MutY active site modulate the lability of the N-glycosidic bond. A analogues that were not excised from duplex DNA as efficiently as predicted by calculations provided insight into other required structural features, such as steric fit and H-bonding within the active site for proper alignment with MutY catalytic residues. We also determined MutY-mediated repair of A analogues paired with OG within the context of a DNA plasmid in bacteria. Remarkably, the magnitudes of decreased in vitro MutY excision rates with different A analogue duplexes do not correlate with the impact on overall MutY-mediated repair. The feature that most strongly correlated with facile cellular repair was the ability of the A analogues to H-bond with the Hoogsteen face of OG. Notably, base pairing of A with OG uniquely positions the 2-amino group of OG in the major groove and provides a means to indirectly select only these inappropriately placed adenines for excision. This highlights the importance of OG lesion detection for efficient MutY-mediated cellular repair. The A analogue SARs also highlight the types of modifications tolerated by MutY and will guide the development of specific probes and inhibitors of MutY.