title: Assembly and molecular activities of the MutS tetramer.
abstract: Analytical equilibrium ultracentrifugation indicates that Escherichia coli MutS exists as an equilibrating mixture of dimers and tetramers. The association constant for the dimer-to-tetramer transition is 2.1 x 10(7) M-1, indicating that the protein would consist of both dimers and tetramers at physiological concentrations. The carboxyl terminus of MutS is required for tetramer assembly because a previously described 53-amino acid carboxyl-terminal truncation (MutS800) forms a limiting species of a dimer (Obmolova, G., Ban, C., Hsieh, P., and Yang, W. (2000) Nature 407, 703-710; Lamers, M. H., Perrakis, A., Enzlin, J. H., Winterwerp, H. H., de Wind, N., and Sixma, T. K. (2000) Nature 407, 711-717). MutS800 binds a 20-base pair heteroduplex an order of magnitude more weakly than full-length MutS, and at saturating protein concentrations, the heteroduplex-bound mass observed with MutS800 is only half that observed with the full length protein, indicating that the subunit copy number of heteroduplex-bound MutS is twice that of MutS800. Analytical equilibrium ultracentrifugation using a fluorescein-tagged 20-base pair heteroduplex demonstrated that native MutS forms a tetramer on this single site-sized heteroduplex DNA. Equilibrium fluorescence experiments indicated that dimer-to-tetramer assembly promotes mismatch binding by MutS and that the tetramer can bind only a single heteroduplex molecule, implying nonequivalence of the two dimers within the tetramer. Compared with native MutS, the ability of MutS800 to promote MutL-dependent activation of MutH is substantially reduced.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Keith P
authors.initials: KP
authors.lastname: Bjornson
authors.forename: Leonard J
authors.initials: LJ
authors.lastname: Blackwell
authors.forename: Harvey
authors.initials: H
authors.lastname: Sage
authors.forename: Celia
authors.initials: C
authors.lastname: Baitinger
authors.forename: Dwayne
authors.initials: D
authors.lastname: Allen
authors.forename: Paul
authors.initials: P
authors.lastname: Modrich
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry*; D001426:Bacterial Proteins; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins / Q000737:chemistry; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004706:Endodeoxyribonucleases / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D051719:MutS DNA Mismatch-Binding Protein; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance; D014461:Ultracentrifugation
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; C059682:methyl-directed mismatch repair protein, E coli; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli; D045643:DNA Repair Enzymes