Catalytic contributions of key residues in the adenine glycosylase MutY revealed by pH-dependent kinetics and cellular repair assays

Abstract

MutY prevent DNA mutations associated with 8-oxoguanine (OG) by catalyzing the removal of adenines opposite OG. pH dependence of the adenine glycosylase activity establish that Asp 138 of MutY must be deprotonated for maximal activity consistent with its role in stabilizing the oxacarbenium ion transition state in an S(N)1 mechanism. A cellular OG:A repair assay allowed further validation of the critical role of Asp 138. Conservative substitutions of the catalytic residues Asp 138 and Glu 37 resulted in enzymes with a range of activity that were used to correlate the efficiency of adenine excision with overall OG:A repair and suppression of DNA mutations in vivo. The results show that MutY variations that exhibit reduced mismatch affinity result in more dramatic reductions in cellular OG:A repair than those that only compromise adenine excision catalysis.

Figure 1

A. Mutations caused by 8-oxoguanine and repair mediated by MutY/MUTYH and MutM/hOGG1. B. Proposed mechanism for adenine glycosylase activity of MutY based on kinetic isotope effect studies and X-ray crystallography. The residue that participates in activating the water nucleophile remains uncertain, and thus, is not shown in this mechanism.

Figure 2

A. Kinetic scheme used to analyze activity of MutY. B. pH dependence of k_obs for WT, D138E, E37D, and D138C MutY with OG:A-containing DNA determined under single-turnover conditions at 37°C. See also Figure S1 (enzyme stability at pH extremes) and Figure S2 (pH dependence with G:A substrate).

Figure 3

Cellular repair assay to study mutated MutY enzymes. A plasmid vector containing a central OG:A mismatch within a BMTI restriction site is transformed into JM101 muty- E. coli cells expressing a specific MutY enzyme (WT or modified MutY). Restriction fragment and sequence analyses of the resulting plasmids isolated from E. coli indicate the amount of G:C bp at the original lesion site defining the extent of MutY-mediated repair.

Figure 4

A. Bar graph of the percent G:C bp at the lesion site for transformation of the reporter vector into E. coli expressing WT MutY, modified active site mutants, and MutYΔ226-350 determined by restriction fragment analysis. Mean values (95% confidence intervals) are as follows: muty-: 39 ± 2; WT: 100; D138N: 29 ± 2; D138E: 100; D138C: 47 ± 3; E37S: 35 ± 1; E37C: 31 ± 2; E37Q: 30 ± 3; E37D: 68 ± 2; and MutYΔ226-350: 27 ± 1. Averages are from a minimum of 8 separate experiments. B. Representative DNA sequence analysis for different active site mutants and MutYΔ226-350. The “K” in the nucleotide sequence of D138C and E37Q MutY indicates that the sequencing program could not distinguish between the mixes of bases at that position, though the scans clearly show that T dominates over G, consistent with the mutation spectrum of cells deficient in MutY. See also Figure S3 that shows relative expression of mutated MutY enzymes.