Abstract
Escherichia coli MutY is an adenine DNA glycosylase active on DNA substrates containing A/G, A/8-oxoG, or A/C mismatches and also has a weak guanine glycosylase activity on G/8-oxoG-containing DNA. The N-terminal domain of MutY, residues 1-226, has been shown to retain catalytic activity. Substrate binding, glycosylase, and Schiff base intermediate formation activities of the truncated and intact MutY were compared. MutY has high binding affinity with 8-oxoG when mispaired with A, G, T, C, or inosine. The truncated protein has more than 18-fold lower affinities for binding various 8-oxoG-containing mismatches when compared with intact MutY. MutY catalytic activity toward A/8-oxoG-containing DNA is much faster than that on A/G-containing DNA whereas deletion of the C-terminal domain reduces its catalytic preference for A/8-oxoG-DNA over A/G-DNA. MutY exerts more inhibition on the catalytic activity of MutM (Fpg) protein than does truncated MutY. The tight binding of MutY with GO mispaired with T, G, and apurinic/apyrimidinic sites may be involved in the regulation of MutM activity. An E. coli mutY strain that produces an N-terminal 249-residue truncated MutY confers a mutator phenotype. These findings strongly suggest that the C-terminal domain of MutY determines the 8-oxoG specificity and is crucial for mutation avoidance by oxidative damage.
Highlights
The short-patch MutY repair pathway of Escherichia coli repairs A/GO and A/G to C/GO and C:G, respectively, and corrects A/C to G:C at a much lower rate [1,2,3,4,5,6,7]
We further investigated the role of the C-terminal domain of MutY
MutM protein was purified to more than 95% homogeneity from E. coli JM109 cells harboring the overproduction plasmid by ammonium sulfate precipitation, phosphocellulose, hydroxylapatite, and heparin chromatographies similar to the method used with MutY enzyme [7]
Summary
E. coli Strains—E. coli PR8 (SuϪ lacZ X74 galU galK Smr), PR70 (like PR8 but micA68::Tn10Kan), and PR68 (like PR8 but micA68::mini Tn10Kan mutL218::Tn10) were obtained from M. Expression and Purification of MutM—The E. coli mutM gene was polymerase chain reaction amplified from the pFPG60 plasmid [7, 48] using primers Chang (5Ј-GCCGGAATTCGGAGATGCTATGCCTGAATT-3Ј) and Chang (5Ј-GCCGGAATTCTTACTTCTGGCACTGCCGAC-3Ј) Both primers contain an EcoRI site at the 5Ј end of the coding sequence or after the stop codon. MutM protein was purified to more than 95% homogeneity from E. coli JM109 cells harboring the overproduction plasmid by ammonium sulfate precipitation, phosphocellulose, hydroxylapatite, and heparin chromatographies similar to the method used with MutY enzyme [7]. The MutY binding reaction mixture contains 20 mM Tris-HCl, pH 7.6, 80 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 2.9% glycerol, 20 ng of poly(dI-dC), and 1.8 fmol of labeled DNA in a total volume of 10 l. The ratio of RifR cells to total cells was the mutation frequency
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