Structural and functional analysis of the MutS C-terminal tetramerization domain.
The Escherichia coli DNA mismatch repair (MMR) protein MutS is essential for the correction of DNA replication errors. In vitro, MutS exists in a dimer/tetramer equilibrium that is converted into a monomer/dimer equilibrium upon deletion of the C-terminal 53 amino acids. In vivo and in vitro data have shown that this C-terminal domain (CTD, residues 801-853) is critical for tetramerization and the function of MutS in MMR and anti-recombination. We report the expression, purification and analysis of the E.coli MutS-CTD. Secondary structure prediction and circular dichroism suggest that the CTD is folded, with an alpha-helical content of 30%. Based on sedimentation equilibrium and velocity analyses, MutS-CTD forms a tetramer of asymmetric shape. A single point mutation (D835R) abolishes tetramerization but not dimerization of both MutS-CTD and full-length MutS. Interestingly, the in vivo and in vitro MMR activity of MutS(CF/D835R) is diminished to a similar extent as a truncated MutS variant (MutS800, residues 1-800), which lacks the CTD. Moreover, the dimer-forming MutS(CF/D835R) has comparable DNA binding affinity with the tetramer-forming MutS, but is impaired in mismatch-dependent activation of MutH. Our data support the hypothesis that tetramerization of MutS is important but not essential for MutS function in MMR.
Manelyte
Laura
L
Institut für Biochemie, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany.
Urbanke
Claus
C
Giron-Monzon
Luis
L
Friedhoff
Peter
P
D000595:Amino Acid Sequence; D004247:DNA / Q000378:metabolism; D053843:DNA Mismatch Repair; D019281:Dimerization; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D051719:MutS DNA Mismatch-Binding Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
D029968:Escherichia coli Proteins; D004247:DNA; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli