Mechanism of MutS searching for DNA mismatches and signaling repair.
DNA mismatch repair is initiated by the recognition of mismatches by MutS proteins. The mechanism by which MutS searches for and recognizes mismatches and subsequently signals repair remains poorly understood. We used single-molecule analyses of atomic force microscopy images of MutS-DNA complexes, coupled with biochemical assays, to determine the distributions of conformational states, the DNA binding affinities, and the ATPase activities of wild type and two mutants of MutS, with alanine substitutions in the conserved Phe-Xaa-Glu mismatch recognition motif. We find that on homoduplex DNA, the conserved Glu, but not the Phe, facilitates MutS-induced DNA bending, whereas at mismatches, both Phe and Glu promote the formation of an unbent conformation. The data reveal an unusual role for the Phe residue in that it promotes the unbending, not bending, of DNA at mismatch sites. In addition, formation of the specific unbent MutS-DNA conformation at mismatches appears to be required for the inhibition of ATP hydrolysis by MutS that signals initiation of repair. These results provide a structural explanation for the mechanism by which MutS searches for and recognizes mismatches and for the observed phenotypes of mutants with substitutions in the Phe-Xaa-Glu motif.
Department of Chemistry and Curriculum in Applied Sciences and Engineering, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Ingrid
I
Tessmer
Yong
Y
Yang
Jie
J
Zhai
Chungwei
C
Du
Peggy
P
Hsieh
Manju M
MM
Hingorani
Dorothy A
DA
Erie
D000255:Adenosine Triphosphate / Q000737:chemistry; D020816:Amino Acid Motifs; D020137:Base Pair Mismatch; D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D004260:DNA Repair; D006868:Hydrolysis; D018625:Microscopy, Atomic Force; D008954:Models, Biological; D008957:Models, Genetic; D008969:Molecular Sequence Data; D051719:MutS DNA Mismatch-Binding Protein / Q000235:genetics / Q000502:physiology*; D010641:Phenotype; D011487:Protein Conformation; D013824:Thermus / Q000378:metabolism
D000255:Adenosine Triphosphate; D004247:DNA; D051719:MutS DNA Mismatch-Binding Protein