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 o f DNA mutations resulting from the presence of the oxidatively damaged lesion 7,8-dihydro-8-oxo-2'-deoxyguanssine (OG) in DNA by the removal of misincorporated adenine residues in OG:A mispairs. MutY also exhib its adenine glycosylase activity toward adenine in G:A and C:A mismatc hes, although the importance of this activity in vivo has not been est ablished. We have investigated the kinetic properties of MutY's glycos ylase 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 fo r adenine removal by MutY from an OG:A substrate was found to be at le ast 6-fold faster than that from the corresponding G:A substrate. Howe ver, under conditions where [MutY] much less than [DNA], OG:A substrat es 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 f acile, such that complete conversion of the substrate to product can b e achieved under similar conditions. The kinetic results illustrate th at the glycosylase reaction catalyzed by MutY has significant differen ces depending on the characteristics of the substrate. The lingering o f MutY with the product of its reaction with OG:A mispairs may be biol ogically significant to prevent premature removal of OG. Thus, this ap proach is providing insight into factors that may be influencing the r epair of damaged and mismatched DNA in vivo by base-excision repair gl ycosylases.