Single-turnover and pre-steady-state kinetics of the reaction of the adenine glycosylase MutY with mismatch-containing DNA substrates.
The DNA repair enzyme MutY plays an important role in the prevention of DNA mutations resulting from the presence of the oxidatively damaged lesion 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) in DNA by the removal of misincorporated adenine residues in OG:A mispairs. MutY also exhibits adenine glycosylase activity toward adenine in G:A and C:A mismatches, although the importance of this activity in vivo has not been established. We have investigated the kinetic properties of MutY's glycosylase activity with OG:A and G:A containing DNA duplexes. Our results indicate that MutY's processing of these two substrates is distinctly different. By using single-turnover experiments, the intrinsic rate for adenine removal by MutY from an OG:A substrate was found to be at least 6-fold faster than that from the corresponding G:A substrate. However, under conditions where [MutY] << [DNA], OG:A substrates are not quantitatively converted to product due to the inefficient turnover resulting from slow product release. In contrast, with a G:A substrate MutY's dissociation from the corresponding product is more facile, such that complete conversion of the substrate to product can be achieved under similar conditions. The kinetic results illustrate that the glycosylase reaction catalyzed by MutY has significant differences depending on the characteristics of the substrate. The lingering of MutY with the product of its reaction with OG:A mispairs may be biologically significant to prevent premature removal of OG. Thus, this approach is providing insight into factors that may be influencing the repair of damaged and mismatched DNA in vivo by base-excision repair glycosylases.
Porello
S L
SL
Department of Chemistry, University of Utah, Salt Lake City 84112, USA.
Leyes
A E
AE
David
S S
SS
D000225:Adenine / Q000378:metabolism; D020137:Base Pair Mismatch; D020029:Base Pairing; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D045647:DNA Glycosylases; D004260:DNA Repair; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006147:Guanine / Q000378:metabolism; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000378:metabolism*; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D017186:Titrimetry
D009841:Oligonucleotides; C077119:4,8-dihydro-4-hydroxy-8-oxo-2'-deoxyguanosine; D006147:Guanine; D004247:DNA; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C061738:mutY adenine glycosylase; D003849:Deoxyguanosine; D000225:Adenine