Structure–Activity Relationships Reveal Key Features of 8-Oxoguanine: A Mismatch Detection by the MutY Glycosylase

Abstract

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.

Single molecule glycosylase studies with engineered 8-oxoguanine DNA damage sites show functional defects of a MUTYH polyposis variant

“…Structural studies show that B. stearothermophilus MutY makes contact with this face of the damaged base through a histidine residue that lies on a short C-terminal loop (41), and it has been suggested that this loop might be in proximity of the major groove in an undamaged DNA substrate (75). A study of E. coli MutY function on various substrate analogs found that the 2-amino group of 8-oxoG is critical for early recognition of an 8-oxoG:A damage site (84). Therefore, it is likely that MUTYH rapidly slides along DNA until the C-terminal domain encounters the 2-amino group of 8-oxoG, whereupon the enzyme stops, repositions, and further interrogates the damage site using the wedge residue.…”

Translesion synthesis by AMV, HIV, and MMLVreverse transcriptases using RNA templates containing inosine, guanosine, and their 8-oxo-7,8-dihydropurine derivatives

“…Interestingly, inosine was found to be a possible candidate in this regard [29]. Other examples highlighting their use as tools to probe for biochemical mechanisms include, where 8-oxoinosine and other C8-subsituted purines have been employed to understand the base excision of 8-oxoG by the MutY glycosylases [30]; or in the replication of DNA with E. coli DNA polymerase I [31].…”

The Role of DNA Repair in Maintaining Mitochondrial DNA Stability

“…Mismatched bases of mtDNA damage originate from DNA replication errors, by either incorporation of wrong nucleotides or nucleotides mainly in oxidized bases [31]. Single base mismatches are identified by Y-box binding protein 1 (YB1) [32] and repaired by the mismatch repair system (MMR) [33]. DNA strand breaks includes single-strand (SSBs) [34] and double-strand breaks (DSBs) [35].…”