MutY DNA glycosylase: base release and intermediate complex formation.
MutY protein, a DNA glycosylase found in Escherichia coli, recognizes dA:dG, dA:8-oxodG, and dA:dC mismatches in duplex DNA, excising the adenine moiety. We have investigated the mechanism of action of MutY, addressing several points of disagreement raised by previous studies of this enzyme. MutY forms a covalent intermediate with its DNA substrate but does not catalyze strand cleavage. The covalent intermediate has a half-life of approximately 2.6 h, 2 orders of magnitude greater than the half-life of Schiff bases formed when E. coli formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease III react with their respective substrates. The covalent complex between MutY and its DNA substrate involves Lys-142; however, the position of this residue in the presumptive active site differs from that of catalytic residues involved in Schiff base formation associated with endonuclease III and related DNA glycosylases/AP lyases. MutY converts DNA duplexes containing the dA:8-oxodG mispair to a product containing an abasic site; heat-induced cleavage of this product may account for the several reports in the literature that ascribe AP lyase activity to MutY. The MutY-DNA intermediate complex is highly stable and hinders access by Fpg to DNA, thereby avoiding a double-strand break. Cross-linking of MutY to DNA may play an important role in the regulation of base excision repair.
Zharkov
D O
DO
Department of Pharmacological Sciences, State University of New York at Stony Brook 11794l-8651, USA.
Grollman
A P
AP
D000080242:8-Hydroxy-2'-Deoxyguanosine; D001665:Binding Sites; D001894:Borohydrides / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D045647:DNA Glycosylases; D004260:DNA Repair; D043382:DNA-Formamidopyrimidine Glycosylase; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D006358:Hot Temperature; D006868:Hydrolysis; D008958:Models, Molecular; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D001894:Borohydrides; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; C025364:sodium borohydride; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C061738:mutY adenine glycosylase; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli; D003849:Deoxyguanosine