Base excision repair (BER) glycosylases initiate DNA repair by cleaving the glycosidic bond of a damaged base, such as 8-oxo-7,8-dihydroguanine (OG). In contrast, the atypical BER glycosylase MutY removes undamaged but miscoding adenines opposite OG lesions, in a secondary BER pathway that prevents G->T mutations. Consistent with this failsafe role, the human homolog (MUTYH) is known to be a tumor suppressor in gastrointestinal cells. Because cellular targeting of DNA damage can have profound implications for both the development and the treatment of cancer, we are investigating the way that MutY glycosylases locate individual OG:A mispairs among undamaged cellular DNA, using substrate base analogs and enzyme variants. Substrate analogs in this work contain relatively subtle modifications of the Watson-Crick or Hoogsteen faces of OG and A; the resulting changes in in vitro binding and catalysis by MutY illustrate important substrate recognition interactions. We also apply these substrate analogs to a cellular assay of MutY-mediated BER, to assess how observed changes in vitro might be muted or amplified in the cellular context. Similarly, we compare the kinetics and overall cellular repair of OG:A mispairs by binding-deficient variants versus wild type MutY. These studies aim to shed light on how MutY's efficiency influences mutagenesis and cell survival under conditions of oxidative stress, and on how we might modulate these repair pathways to our benefit in the battle against cancer. 

Structure-activity relationships in substrate recognition by base excision repair glycosylase MutY

247th National Meeting (2014)

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