Functional studies on the candidate ATPase domains of Saccharomyces cerevisiae MutLalpha.
Saccharomyces cerevisiae MutL homologues Mlh1p and Pms1p form a heterodimer, termed MutLalpha, that is required for DNA mismatch repair after mismatch binding by MutS homologues. Recent sequence and structural studies have placed the NH(2) termini of MutL homologues in a new family of ATPases. To address the functional significance of this putative ATPase activity in MutLalpha, we mutated conserved motifs for ATP hydrolysis and ATP binding in both Mlh1p and Pms1p and found that these changes disrupted DNA mismatch repair in vivo. Limited proteolysis with purified recombinant MutLalpha demonstrated that the NH(2) terminus of MutLalpha undergoes conformational changes in the presence of ATP and nonhydrolyzable ATP analogs. Furthermore, two-hybrid analysis suggested that these ATP-binding-induced conformational changes promote an interaction between the NH(2) termini of Mlh1p and Pms1p. Surprisingly, analysis of specific mutants suggested differential requirements for the ATPase motifs of Mlh1p and Pms1p during DNA mismatch repair. Taken together, these results suggest that MutLalpha undergoes ATP-dependent conformational changes that may serve to coordinate downstream events during yeast DNA mismatch repair.
Department of Molecular and Medical Genetics, Oregon Health Sciences University, Portland, Oregon 97201, USA.
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D048868:Adaptor Proteins, Signal Transducing; D000225:Adenine / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004252:DNA Mutational Analysis; D004260:DNA Repair; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D008954:Models, Biological; D008969:Molecular Sequence Data; D000070957:MutL Protein Homolog 1; D000070956:MutL Proteins; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques; D001616:beta-Galactosidase / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D005656:Fungal Proteins; C088859:MLH1 protein, S cerevisiae; C074770:MutL protein, E coli; C088858:PMS1 protein, S cerevisiae; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D001616:beta-Galactosidase; D000251:Adenosine Triphosphatases; D000070957:MutL Protein Homolog 1; D000070956:MutL Proteins; D000225:Adenine