Isolation and characterization of the Escherichia coli mutH gene product.
The Escherichia coli mutH gene product has been isolated in near homogeneous form using an in vitro complementation assay for DNA mismatch correction (Lu, A.-L., Clark, S., and Modrich, P. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4639-4643) which is dependent on mutH function. The protein has a subunit Mr of 25,000, and purified preparations contain a Mg2+-dependent endonuclease activity which cleaves 5' to the dG of d(GATC) sequences to generate 5'-phosphoryl and 3'-hydroxyl termini. Symmetrically methylated d(GATC) sites are resistant to the endonuclease, hemimethylated sequences are cleaved on the unmethylated strand, and unmethylated d(GATC) sites are usually subject to scission on only one DNA strand. Although this endonuclease activity is extremely weak (less than 1 scission/h/mutH monomer equivalent) and cleavage at a d(GATC) site does not depend on the presence of a mismatched base pair within the DNA substrate, the activity does not appear to be a contaminant of mutH preparations. d(GATC) endonuclease activity and mutH complementing activity co-purify through multiple column steps without change in relative specific activities, and both activities co-electrophorese under native conditions. These findings suggest that the mutH product functions at the strand discrimination stage of mismatch correction and that this stage of the reaction involves scission of the unmethylated DNA strand.
Welsh
K M
KM
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
Lu
A L
AL
Clark
S
S
Modrich
P
P
D001426:Bacterial Proteins / Q000302:isolation & purification*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000032:analysis; D004720:Endonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D008274:Magnesium / Q000378:metabolism; D008745:Methylation; D008970:Molecular Weight; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004720:Endonucleases; D008274:Magnesium