Substrate discrimination by formamidopyrimidine-DNA glycosylase: a mutational analysis.
Formamidopyrimidine-DNA glycosylase (Fpg) is a primary participant in the repair of 8-oxoguanine, an abundant oxidative DNA lesion. Although the structure of Fpg has been established, amino acid 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 8-oxoguanine accommodated in the binding pocket. Consistent with our computational studies, mutation of Lys-217 selectively reduced the ability of Fpg to excise 8-oxoguanine from DNA. Dihydrouracil, also a substrate for Fpg, served as a nonspecific control. Other residues involved in damage recognition (His-89, Arg-108, and Arg-109) were identified by combined conservation/structure analysis. Arg-108, which forms two hydrogen bonds with cytosine in Fpg-DNA, is a major determinant of opposite-base specificity. Mutation of this residue reduced excision of 8-oxoguanine from thermally unstable mispairs with guanine or thymine, while excision from the stable cytosine and adenine base pairs was less affected. Mutation of His-89 selectively diminished the rate of excision of 8-oxoguanine, whereas mutation of Arg-109 nearly abolished binding of Fpg to damaged DNA. Taken together, these results suggest that His-89 and Arg-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 Arg-108 provides specificity for bases positioned opposite the lesion.
Zaika
Elena I
EI
Laboratory of Chemical Biology, Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794, USA.
Perlow
Rebecca A
RA
Matz
Eileen
E
Broyde
Suse
S
Gilboa
Rotem
R
Grollman
Arthur P
AP
Zharkov
Dmitry O
DO
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology / Q000235:genetics; D001665:Binding Sites; D004249:DNA Damage; D004260:DNA Repair; D043382:DNA-Formamidopyrimidine Glycosylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013816:Thermodynamics
D011994:Recombinant Proteins; D043382:DNA-Formamidopyrimidine Glycosylase