The oxidation of guanine (G) to 8-oxoguanine (OG) is a common type of DNA damage. While OG can canonically base-pair with a cytosine (C), upon replication it will mispair with an adenine (A), resulting in the formation of a T:A point mutation. The DNA glycosylase MutY is able to specifically recognize the OG:A mispair and remove the A, despite the absence of any significant helix distortion from OG:A. Thus, the ability of MutY to find the OG:A mispair and distinguish it from undamaged DNA is of interest. Previous work has shown that the N-2 exocyclic amino group of OG is required for recognition and crystal structures have shown that when MutY is bound to its OG:A substrate, two histidine residues (positions 292 and 296 in EcMutY) are located in relatively close proximity to this crucial amino group. We are interested in probing the potential role of these residues by modifying them to alanine (H292A) to test the overall importance of histidine’s presence, and to lysine (H296K) to test for the effects of addition of a positively charged residue. The variants were studied by measuring overall conversion to G:C in bacterial cells using a cell assay in which the vector for expression of the variant is transformed into muty- E. coli. The variants were also isolated and purified for use in in vitro kinetics and binding experiments.

Structure-activity relationship analysis of MutY variants for OG:A recognition

2019 Spring National Meeting

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