Contrasting effects of mutating active site residues, Aspartic acid 64 and Histidine 187 of <i>Escherichia coli </i>uracil-DNA glycosylase on uracil excision and interaction with an inhibitor protein

Abstract

Uracil, a promutagenic base, arises in DNA by spontaneous deamination of cytosine or by the malfunctioning of DNA polymerases. To maintain the genomic integrity, cells possess a highly conserved base excision repair enzyme. uracil –DNA glycosylase (UDG). UDGs have a notably high turnover number and strict specificity for uracil in DNA. UDGs are inhibited

by a small proteinaceous inhibitor. Ugi, which acts as a transition state substrate mimic. Crystal structure studies have identified the residues crucial in catalysis, and in their interaction with Ugi. Here, we report on the &nbsp;mutational analyses of &nbsp;D64(D64H and D64N) and H187 (H187C, H187L and H187R) in the active site pocket of <i>Escherichia coli </i>UDG. The mutants

<span style="font-size:14.0pt;font-family:" times="" new="" roman";mso-fareast-font-family:="" "times="" roman";mso-ansi-language:en-us;mso-fareast-language:en-us;="" mso-bidi-language:ar-sa"="">were compromised in uracil excision by ~ 200-25,000 fold when compared to the native protein. In contrast. our analysis of the <i>in vivo </i>formed UDG-Ugi complexes on urea gels shows that D64 and H187 contribute minimally to the interaction of the two proteins. Thus, our findings prov de further evidence to the primary functi on of D64 and H187 in catalysis.</span>