Adenine Release Is Fast in MutY-catalyzed Hydrolysis of G:A and 8-Oxo-G:A DNA Mismatches

Joe A.B Mccann,Paul J Berti

doi:10.1074/jbc.m212474200

Abstract

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.

Highlights

Taneous strand breakage and are substrates for MutM, a DNA glycosylase/␤-lyase [5] that excises OG from OG:C mismatches
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 MutY1⁄7AP-DNA1⁄7[8-14C]adenine complex, with a rate constant greater than 5 min؊1
The steady state rate constant is much lower, 0.003 minϪ1 with OG:A and 0.02 minϪ1 for G:A, which has been attributed to rate-limiting AP-DNA release [9]

Summary

EXPERIMENTAL PROCEDURES

MutY—MutY was prepared [7] and the active site concentration was determined [6] using an overexpressing Escherichia coli strain supplied by Prof. Chromatographic Resolution—The adenine release rate calculation was based in part on the time needed for high molecular weight species (DNA and protein) to be separated from adenine on the gel filtration column. Primers (20 ␮M) and 20 ␮M [8-14C]dATP (500,000 cpm) were incubated in 230 ␮l of 50 mM Tris1⁄7HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 200 ␮M TTP, 200 ␮M dGTP, 200 ␮M dCTP, and 1150 U M-MLV reverse transcriptase for 13 h at 37 °C and concentrated under vacuum to 60 ␮l, and DNA was precipitated with an equal volume of isopropanol. Substrate DNA (200 nM, 1000 cpm 14C, with 7.5 mM KCl) was equilibrated with reaction buffer (20 mM Tris1⁄7HCl, pH 7.6, 12.5 mM KCl, 10 mM EDTA, 0.1 mg/ml bovine serum albumin) in 45 ␮l. Where [[8-14C]adenine]free,t is the concentration of free [8-14C]adenine at time ϭ t, [[14A]-DNA]0 is the initial substrate DNA concentration, and

Mr are the amounts of

RESULTS

OG:A