title: The role of nucleotide cofactor binding in cooperativity and specificity of MutS recognition.
abstract: Mismatch repair (MMR) is essential for eliminating biosynthetic errors generated during replication or genetic recombination in virtually all organisms. The critical first step in Escherichia coli MMR is the specific recognition and binding of MutS to a heteroduplex, containing either a mismatch or an insertion/deletion loop of up to four nucleotides. All known MutS homologs recognize a similar broad spectrum of substrates. Binding and hydrolysis of nucleotide cofactors by the MutS-heteroduplex complex are required for downstream MMR activity, although the exact role of the nucleotide cofactors is less clear. Here, we showed that MutS bound to a 30-bp heteroduplex containing an unpaired T with a binding affinity approximately 400-fold stronger than to a 30-bp homoduplex, a much higher specificity than previously reported. The binding of nucleotide cofactors decreased both MutS specific and nonspecific binding affinity, with the latter marked by a larger drop, further increasing MutS specificity by approximately 3-fold. Kinetic studies showed that the difference in MutS K(d) for various heteroduplexes was attributable to the difference in intrinsic dissociation rate of a particular MutS-heteroduplex complex. Furthermore, the kinetic association event of MutS binding to heteroduplexes was marked by positive cooperativity. Our studies showed that the positive cooperativity in MutS binding was modulated by the binding of nucleotide cofactors. The binding of nucleotide cofactors transformed E. coli MutS tetramers, the functional unit in E. coli MMR, from a cooperative to a noncooperative binding form. Finally, we found that E. coli MutS bound to single-strand DNA with significant affinity, which could have important implication for strand discrimination in eukaryotic MMR mechanism.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, 260 Whitney Avenue, P.O. Box 208114, New Haven, CT 06520-8114, USA.
authors.forename: Shar-yin N
authors.initials: SN
authors.lastname: Huang
authors.forename: Donald M
authors.initials: DM
authors.lastname: Crothers
mesh_terms: D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D001483:Base Sequence; D001667:Binding, Competitive / Q000187:drug effects; D003067:Coenzymes / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D051719:MutS DNA Mismatch-Binding Protein / Q000737:chemistry / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D009692:Nucleic Acid Heteroduplexes; D009711:Nucleotides / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature; D017186:Titrimetry
chemical_list: D003067:Coenzymes; D050505:Mutant Proteins; D009692:Nucleic Acid Heteroduplexes; D009711:Nucleotides; D000266:Adenylyl Imidodiphosphate; D051719:MutS DNA Mismatch-Binding Protein
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