Mutation in the magnesium binding site of hMSH6 disables the hMutSalpha sliding clamp from translocating along DNA.
In human cells, binding of base/base mismatches and small insertion/deletion loops is mediated by hMutSalpha, a heterodimer of hMSH2 and hMSH6. In the presence of ATP and magnesium, hMutSalpha dissociates from the mismatch by following the DNA contour in the form of a sliding clamp. This process is enabled by a conformational change of the heterodimer, which is driven by the binding of ATP and magnesium in the Walker type A and B motifs of the polypeptides, respectively. We show that a purified recombinant hMutSalpha variant, hMutSalpha 6DV, which contains an aspartate to valine substitution in the Walker type B motif of the hMSH6 subunit, fails to undergo the conformational change compatible with translocation. Instead, its direct dissociation from the mismatch-containing DNA substrate in the presence of ATP and magnesium precludes the assembly of a functional mismatch repair complex. The "translocation-prone" conformation of wild type hMutSalpha could be observed solely under conditions that favor hydrolysis of the nucleotide and mismatch repair in vitro. Thus, whereas magnesium could be substituted with manganese, ATP could not be replaced with its slowly or nonhydrolyzable homologues ATP-gammaS or AMPPNP, respectively. The finding that ATP induces different conformational changes in hMutSalpha in the presence and in the absence of magnesium helps explain the functional differences between hMutSalpha variants incapable of binding ATP as compared with those unable to bind the metal ion.
Iaccarino
I
I
Institute of Medical Radiobiology of the University of Zürich and the Paul Scherrer Institute, August Forel Strasse 7, CH-8008 Zürich, Switzerland.
Marra
G
G
Dufner
P
P
Jiricny
J
J
D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D020137:Base Pair Mismatch / Q000502:physiology; D001665:Binding Sites; D020134:Catalytic Domain; D002918:Chymotrypsin / Q000494:pharmacology; D004260:DNA Repair / Q000502:physiology; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D029968:Escherichia coli Proteins; D006801:Humans; D008274:Magnesium / Q000378:metabolism*; D008969:Molecular Sequence Data; D051719:MutS DNA Mismatch-Binding Protein; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation / Q000187:drug effects; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C094443:G-T mismatch-binding protein; D000255:Adenosine Triphosphate; D002918:Chymotrypsin; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli; D008274:Magnesium