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Dare to Repair: From DNA chemistry, metals and mechanism to cancer
Add to Calendar 2023-03-14T18:30:00 2023-03-14T19:30:00 UTC Dare to Repair: From DNA chemistry, metals and mechanism to cancer 301A Chemistry Building
Start DateTue, Mar 14, 2023
2:30 PM
End DateTue, Mar 14, 2023
3:30 PM
Presented By
Sheila S. David - UC Davis
Event Series: Chemistry Department Colloquium Seminar Series Spring 2023
Sheila David

Sheila S. David - UC Davis


The informational content of DNA is eroded by chemical modifications of the DNA nucleobases.  The common G oxidation, 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/MUTYH glycosylases are unique [4Fe-4S]2+-cofactor containingenzyme 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 insight into the functional properties of two most common variants in MUTYH.

We have used a combination of synthesis of modified substrates, enzymology, X-ray crystallography and cellular assays to reveal features associated with damage recognition and adenine excision by MUTYH and MUTYH variants. Notably, using modified substrates andenzymes 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.  This multipronged approach has also been useful in delineating the impact of MAP variants that are localized near the Fe-S cluster and Zn cofactors.  X-ray structures of MutY bound to an azaribose transition state analog containing DNA duplexes, along with measurements of product stereochemistry, have led us to propose a revised mechanism for MutY that involves formation of a covalent intermediate with a nucleophilic Asp residue, followed by hydrolysis of the acetal intermediate to give an abasic site product. A nearby Asn residue hydrogen-bonds to the catalytic Asp, and is also found mutated to Ser in a cancer associated MUTYH variant.  By capturing structural snapshots of N146S MutY bound to DNA containing a substrate, a transition state analog and enzyme-catalyzed abasic site product, we have gleaned detailed insight into the base excision mechanism.  These results are consistent with the use by MutY of a mechanism akin to that of“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.  In addition, this mechanistic insight provides a unique strategy for developing new inhibitors and chemical biology probes for MUTYH.