Contribution of human mlh1 and pms2 ATPase activities to DNA mismatch repair.
MutLalpha, a heterodimer composed of Mlh1 and Pms2, is the major MutL activity in mammalian DNA mismatch repair. Highly conserved motifs in the N termini of both subunits predict that the protein is an ATPase. To study the significance of these motifs to mismatch repair, we have expressed in insect cells wild type human MutLalpha and forms altered in conserved glutamic acid residues, predicted to catalyze ATP hydrolysis of Mlh1, Pms2, or both. Using an in vitro assay, we showed that MutLalpha proteins altered in either glutamic acid residue were each partially defective in mismatch repair, whereas the double mutant showed no detectable mismatch repair. Neither strand specificity nor directionality of repair was affected in the single mutant proteins. Limited proteolysis studies of MutLalpha demonstrated that both Mlh1 and Pms2 N-terminal domains undergo ATP-induced conformational changes, but the extent of the conformational change for Mlh1 was more apparent than for Pms2. Furthermore, Mlh1 was protected at lower ATP concentrations than Pms2, suggesting Mlh1 binds ATP with higher affinity. These findings imply that ATP hydrolysis is required for MutLalpha activity in mismatch repair and that this activity is associated with differential conformational changes in Mlh1 and Pms2.
Tomer
Guy
G
Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, Oregon 97201, USA.
Buermeyer
Andrew B
AB
Nguyen
Megan M
MM
Liskay
R Michael
RM
D048868:Adaptor Proteins, Signal Transducing; D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D020137:Base Pair Mismatch; D002352:Carrier Proteins; D002384:Catalysis; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004260:DNA Repair; D045643:DNA Repair Enzymes; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007313:Insecta; D051379:Mice; D000070976:Mismatch Repair Endonuclease PMS2; D008969:Molecular Sequence Data; D000070957:MutL Protein Homolog 1; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000737:chemistry*; D009687:Nuclear Proteins; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C501486:MLH1 protein, human; C501485:Mlh1 protein, mouse; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D011506:Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D014357:Trypsin; D000251:Adenosine Triphosphatases; C094934:PMS2 protein, human; C499533:Pms2 protein, mouse; D000070976:Mismatch Repair Endonuclease PMS2; D000070957:MutL Protein Homolog 1; D045643:DNA Repair Enzymes