Mutations within the hMLH1 and hPMS2 subunits of the human MutLalpha mismatch repair factor affect its ATPase activity, but not its ability to interact with hMutSalpha.
The MutL family of mismatch repair proteins belongs to the GHKL class of ATPases, which contains also type II topoisomerases, HSP90, and histidine kinases. The nucleotide binding domains of these polypeptides are highly conserved, but this similarity has failed to help us understand the biological role of the ATPase activity of the MutL proteins in mismatch repair. hMutLalpha is a heterodimer of the human MutL homologues hMLH1 and hPMS2, and we decided to exploit its asymmetry to study this function. We now show that although the two subunits contribute differently to the ATPase activity of the heterodimer, hMutLalpha variants in which one subunit was able to bind but not hydrolyze ATP displayed similarly reduced mismatch repair activities in vitro. In contrast, variants in which either subunit was unable to bind the nucleotide were inactive. Mutation of the catalytic sites of both subunits abolished repair without altering the ability of these peptides to interact with one another. Since the binding of the nucleotide in hMutLalpha was not required for the formation of ternary complexes with the mismatch recognition factor hMutSalpha bound to a heteroduplex substrate, we propose that the ATPase activity of hMutLalpha is required downstream from this process.
Räschle
Markus
M
Institute of Medical Radiobiology, August Forel-Strasse 7, Zürich 8008, Switzerland.
Dufner
Patrick
P
Marra
Giancarlo
G
Jiricny
Josef
J
D048868:Adaptor Proteins, Signal Transducing; D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D020137:Base Pair Mismatch; D001665:Binding Sites; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004252:DNA Mutational Analysis; D004260:DNA Repair; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006868:Hydrolysis; D007313:Insecta; D008274:Magnesium / Q000378:metabolism; D000070976:Mismatch Repair Endonuclease PMS2; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D000070957:MutL Protein Homolog 1; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics*; D009687:Nuclear Proteins; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014466:Ultraviolet Rays
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C501486:MLH1 protein, human; C409221:MSH4 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D009841:Oligonucleotides; D011506:Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; C094934:PMS2 protein, human; D000070976:Mismatch Repair Endonuclease PMS2; D000070957:MutL Protein Homolog 1; D045643:DNA Repair Enzymes; D008274:Magnesium