Adenine release is fast in MutY-catalyzed hydrolysis of G:A and 8-Oxo-G:A DNA mismatches.
MutY, a DNA repair enzyme, is unusual in that it binds exceedingly tightly to its products after the chemical steps of catalysis. Until now it was not known whether the product being released in the rate-limiting step was DNA, adenine, or both. MutY hydrolyzes adenine from 8-oxo-G:A (OG:A) base pair mismatches as the first step in the base excision repair pathway, as well as from G:A mismatches. The products are adenine and DNA containing an apurinic (AP) site. Tight product binding may have a physiological role in preventing further damage at the OG:AP site. We developed a rate assay using [8-14C]adenine in OG:A or G:A mismatches that distinguishes between adenine hydrolysis and adenine release. [8-14C]Adenine was released quickly from the MutY.AP-DNA.[8-14C]adenine complex, with a rate constant greater than 5 min-1. This was much faster than the rate-limiting step, at 0.006-0.015 min-1. Gel retardation experiments showed that AP-DNA release was very slow, consistent with it being the rate-limiting step. Thus, the kinetic mechanism involves fast adenine release after hydrolysis followed by rate-limiting AP-DNA release. Adenine appears to be buried deep in the protein.DNA interface, but there is enough flexibility or open space for it to dissociate from the MutY.APDNA.adenine complex. These results have implications for the catalytic mechanism of MutY.
McCann
Joe A B
JA
Department of Biochemistry, McMaster University, Hamilton, Ontario L8S 4M1, Canada.
Berti
Paul J
PJ
D000225:Adenine / Q000737:chemistry / Q000378:metabolism*; D020137:Base Pair Mismatch; D001483:Base Sequence; D002384:Catalysis; D002845:Chromatography; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism; D045647:DNA Glycosylases; D004260:DNA Repair; D004926:Escherichia coli / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D013997:Time Factors
D009841:Oligonucleotides; D004247:DNA; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C061738:mutY adenine glycosylase; D000225:Adenine