hMSH2 and hMSH6 play distinct roles in mismatch binding and contribute differently to the ATPase activity of hMutSalpha.
In extracts of human cells, base-base mismatches and small insertion/deletion loops are bound primarily by hMutSalpha, a heterodimer of hMSH2 and hMSH6 (also known as GTBP or p160). Recombinant hMutSalpha bound a G/T mismatch-containing oligonucleotide with an apparent dissociation constant Kd = 2.6 nM, while its affinity for a homoduplex substrate was >20-fold lower. In the presence of ATP, hMutSalpha dissociated from mismatched oligonucleotide substrates, and this reaction was attenuated by mutating the conserved lysine in the ATP-binding domains of hMSH6, hMSH2 or both to arginine. Surprisingly, this reaction required only ATP binding, not hydrolysis. The ATPase activity of hMutSalpha variants carrying the Lys-->Arg mutation in hMSH2 or in hMSH6 was severely affected, but these mutants were still proficient in mismatch binding and were able to complement, albeit to different extents, mismatch repair-deficient cell extracts. The mismatch binding-proficient, ATPase-deficient double mutant was inactive in the complementation assay and its presence in repair-proficient extracts was inhibitory. We conclude that although the ATPase activity of hMutSalpha is dispensible for mismatch binding, it is required for mismatch correction.
Iaccarino
I
I
Institute of Medical Radiobiology, P.O. Box 424, August Forel-Strasse 7, CH-8029, Zürich, Switzerland.
Marra
G
G
Palombo
F
F
Jiricny
J
J
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D051718:MutS Homolog 2 Protein; D000074081:MutS Homolog 3 Protein; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D016415:Sequence Alignment; D013296:Streptococcus pneumoniae / Q000378:metabolism
D000251:Adenosine Triphosphatases; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D051718:MutS Homolog 2 Protein; D000074081:MutS Homolog 3 Protein; D009838:Oligodeoxyribonucleotides; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; C094443:G-T mismatch-binding protein; C078444:MSH3 protein, human; C497172:MSH2 protein, human; C421109:multidrug resistance-associated protein 1