Characterization of a DNA mismatch-binding activity in yeast extracts.
An activity present in nuclear extracts of the yeast Saccharomyces cerevisiae binds specifically to oligonucleotides containing DNA mismatches, as judged by a band shift assay. The specificity of this activity for mismatched DNA was confirmed by competition experiments; binding to radiolabeled heteroduplexes was abolished in the presence of excess unlabeled heteroduplex but not when excess unlabeled homoduplex was added. Both T/G and T/- (single base deletion) mispairs were recognized in each of two sequence contexts. Binding was also observed with G/G, G/A, A/C, and T/C mismatches, but recognition of a C/C mispair was very weak. Competition studies with the various mismatches were consistent with the idea that a single activity recognizes all mispairs tested. Extracts from strains mutant in either or both of two putative mismatch recognition functions, MSH2 and MSH3, were also tested. Mismatch-binding activity was present in extracts of msh3- strains but completely absent in msh2- strains. The molecular weight of the major binding protein was estimated by UV cross-linking experiments to be approximately 110 kDa, in good agreement with the size predicted for Msh2 protein (Reenan, R. A. and Kolodner, R. D. (1992) Genetics 132, 963-973).
Miret
J J
JJ
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester 01655.
Milla
M G
MG
Lahue
R S
RS
D001482:Base Composition; D001483:Base Sequence; D001667:Binding, Competitive; D002467:Cell Nucleus / Q000378:metabolism*; D004271:DNA, Fungal / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
D004271:DNA, Fungal; D009692:Nucleic Acid Heteroduplexes; D009838:Oligodeoxyribonucleotides