The damaged base 8-oxoguanine (OG) is particularly sinister due to its mimicry of thymine that evades detection during replication and results in incorrect insertion of A to form OG:A mismatches. The MutY glycosylase is a unique [4Fe-4S]2+-cofactor containing enzyme that prevents mutations by excising adenine from OG:A mismatches to initiate base excision repair (BER). MutY has captured the spotlight due to a direct correlation between inherited defects in the human MutY homologue (MUTYH) and colorectal cancer, referred to as MUTYH-associated polyposis (MAP). Our research laboratory played a key role in the discovery of MAP by providing functional analysis of the two most common variants in MUTYH revealing a hampered ability to recognize OG. We have also used a combination of synthesis of modified substrates, enzymology and X-ray crystallography to reveal features associated with damage recognition and adenine excision by MutY and MUTYH. Notably, using modified substrates and modified enzymes in in vitro and cellular assays we have revealed the key features of OG:A base pairs that MUTYH uses to “find” substrate and ignore structurally similar T:A base pairs, and these insights help illuminate the most deleterious types of MAP variants. We have also synthesized several azaribose transition state analog containing DNA duplexes and determined crystal structures that provide insight into the adenine excision mechanism catalyzed by MutY. Together with measurements of product stereochemistry, we have proposed a revised mechanism for MutY that involves two nucleophilic displacement steps akin to the mechanisms accepted for “retaining” O-glycosidases. This new-for-MutY yet familiar mechanism may also be operative in related BER glycosylases and provides a critical framework for analysis of MAP variants. Our structural and mechanistic insights have also revealed new ways to selectively target MUTYH and other BER glycosylases. Notably, along this line, we have prepared a “clickable” TS mimic that provides a unique strategy to prepare high affinity specific inhibitors of MUTYH, and related glycosylases.

Dare to repair: From DNA chemistry to cancer

2019 Fall National Meeting

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