The DNA glycosylase MutY prevents G:C to T:A transversion mutations by removing misincorporated adenine across 8-oxo-guanine (OG), an oxidation product of guanine. In this study the role of an active site residue N146 in the adenine excision mechanism has been explored in Geobacillus stearothermophilus (Gs) MutY using site-directed mutagenesis, kinetic studies and X-Ray crystallography. The crystal structure of Gs MutY bound to the transition state analog (1N) was instrumental in identifying hydrogen bond contacts N146 forms with the DNA phosphate backbone and catalytic residue D144. Interestingly, mutation of this residue to a serine corresponds to a MUTYH-Associated Polyposis (MAP) variant that predisposes individuals to colorectal cancer. The catalytic activity of N146S was hindered significantly in comparison to wild-type, and the pH-dependent kinetics showed an increase in the pKa of D144. This result suggests that N146S impacts the efficiency of the reaction by altering certain electrostatic interactions between catalytic residues and the reaction intermediates. In order to understand the role of N146 in the adenine excision mechanism at a molecular level, we used an acid labile substrate analog, purine (P), for biochemical analysis and crystallography. Surprisingly, the crystal structure of N146S with purine shows coordination of a Ca2+ ion in the additional space formed by N146S substitution. Furthermore, the purine base is still intact in the active site despite the ability of N146S Gs MutY to excise P. We believe Ca2+ coordination in the active site prevents the cleavage of P. Indeed, crystallization of N146S with P in the absence of Ca2+ resulted in abasic site product. Our aim is to remove Ca2+ from the active site in crystalline state to initiate and trap different steps of the reaction including intermediates that have been elusive thus far. Finally, the preliminary results of N146S-OG:P crystals soaked in EDTA containing solution showed lowered Ca2+ occupancy in the active site. In conclusion, by monitoring the base excision mechanism of N146S Gs MutY, we hope to gain insights into why this MAP variant has altered activity and suggest potential treatments.

Structural and biochemical insights into adenine excision by N146S Gs MutY

ACS Fall 2021

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