Single-Turnover and Pre-Steady-State Kinetics of the Reaction of the Adenine Glycosylase MutY with Mismatch-Containing DNA Substrates

Abstract

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.

Unnatural substrates reveal the importance of 8-oxoguanine for in vivo mismatch repair by MutY

“…2c). 17 Of note, the rate constant k 2 was determined under conditions of singleturnover, where [DNA] < [MutY] (Fig. 2).…”

“…We have previously examined the features of mismatched substrates that are required for efficient lesion recognition and adenine excision by MutY using pre-steady state and singleturnover kinetics. [17][18][19] Pre-steady state experiments revealed that MutY has a high affinity for the product such that release of the DNA product is rate-limiting. 17,19,20 Moreover, the identity of the base opposite A greatly affects both the rate of product release as well as the intrinsic rate of adenine removal determined under single-turnover conditions.…”

“…Duplex 2 is the duplex sequence that we have used extensively in analyzing the glycosylase activity of MutY. 17 In the series of experiments described herein, the glycosylase activity was also assayed using two NaCl concentrations, 30 mM and 150 mM, in the reaction buffer. The glycosylase activity of MutY is monitored by measuring the extent of strand scission that occurs at the abasic site produced by the base removal activity of MutY upon quenching the reaction with NaOH (Fig.…”

“…This biphasic profile is due to slow release of MutY from the DNA product. 17,20 Based on this behavior the same minimal kinetic scheme and approach to determine the relevant rate constants as we have described previously was used (Fig. 2c).…”

DNA repair glycosylases with a [4Fe–4S] cluster: A redox cofactor for DNA-mediated charge transport?

“…MutY, structurally similar to Endo III [18][19][20][21], is another BER glycosylase that contains a [4Fe-4S] cluster [20]. However, MutY instead removes adenine from 8-oxo-guanine:adenine mispairs [22][23][24][25][26][27][28][29][30][31][32][33][34].…”