Flipping duplex DNA inside out: a double base-flipping reaction mechanism by Escherichia coli MutY adenine glycosylase.
The Escherichia coli MutY adenine glycosylase plays a critical role in repairing mismatches in DNA between adenine and the oxidatively damaged guanine base 8-oxoguanine. Crystallographic studies of the catalytic core domain of MutY show that the scissile adenine is extruded from the DNA helix to be bound in the active site of the enzyme (Guan, Y., Manuel, R. C., Arvai, A. S., Parikh, S. S., Mol, C. D., Miller, J. H., Lloyd, S., and Tainer, J. A. (1998) Nat. Struct. Biol. 5, 1058-1064). However, the structural and mechanistic bases for the recognition of the 8-oxoguanine remain poorly understood. In experiments using a single-stranded 8-bromoguanine-containing synthetic oligodeoxyribonucleotide alone and in a duplex construct mismatched to an adenine, we observed UV cross-linking between MutY and the 8-bromoguanine probe. We further observed enhanced cross-linking in the single strand experiments, suggesting that neither the duplex context nor the mismatch with adenine is required for recognition of the 8-oxoguanine moiety. Stopped-flow fluorescence studies using 2-aminopurine-containing oligodeoxyribonucleotides further revealed the sequential extrusion of the 8-oxoguanine at 108 s(-1) followed by the adenine at 16 s(-1). A protein isomerization step following base flipping at 1.9 s(-1) was also observed and is postulated to provide additional stabilization of the extruded adenine thereby facilitating its capture by the active site for excision.
Bernards
Andrew S
AS
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, USA.
Miller
Jamie K
JK
Bao
Kogan K
KK
Wong
Isaac
I
D045647:DNA Glycosylases; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D006147:Guanine / Q000378:metabolism; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D014466:Ultraviolet Rays
D004269:DNA, Bacterial; D006147:Guanine; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C061738:mutY adenine glycosylase