Substrate discrimination by XXXX -DNA glycosylase: a mutational analysis.

@entity1 @entity25469 @entity3884 @entity535 @entity44930 @entity534 @entity2774 @entity5536 @entity5538 @entity6244
@entity44930 -DNA glycosylase (Fpg) is a primary @entity1 in the repair of @entity25469 , an abundant oxidative DNA lesion. Although the structure of Fpg has been established, @entity535 residues that define damage recognition have not been identified. We have combined molecular dynamics and bioinformatics approaches to address this issue. Site-specific mutagenesis coupled with enzyme kinetics was used to test our predictions. On the basis of molecular dynamics simulations, Lys-217 was predicted to interact with the O8 of extrahelical @entity25469 accommodated in the binding pocket. Consistent with our computational studies, mutation of Lys-217 selectively reduced the ability of Fpg to excise @entity25469 from DNA. Dihydrouracil, also a substrate for Fpg, served as a nonspecific control. Other residues involved in damage recognition (His-89, @entity534 -108, and @entity534 -109) were identified by combined conservation/structure analysis. @entity534 -108, which forms two @entity3884 bonds with @entity5538 in Fpg-DNA, is a major determinant of opposite-base specificity. Mutation of this residue reduced excision of @entity25469 from thermally unstable mispairs with @entity2774 or @entity6244 , while excision from the stable @entity5538 and @entity5536 base pairs was less affected. Mutation of His-89 selectively diminished the rate of excision of @entity25469 , whereas mutation of @entity534 -109 nearly abolished binding of Fpg to damaged DNA. Taken together, these results suggest that His-89 and @entity534 -109 form part of a reading head, a structural feature used by the enzyme to scan DNA for damage. His-89 and Lys-217 help determine the specificity of Fpg in recognizing the oxidatively damaged base, while @entity534 -108 provides specificity for bases positioned opposite the lesion.
