Saccharomyces cerevisiae Msh2p and Msh6p ATPase activities are both required during mismatch repair.
In the Saccharomyces cerevisiae Msh2p-Msh6p complex, mutations that were predicted to disrupt ATP binding, ATP hydrolysis, or both activities in each subunit were created. Mutations in either subunit resulted in a mismatch repair defect, and overexpression of either mutant subunit in a wild-type strain resulted in a dominant negative phenotype. Msh2p-Msh6p complexes bearing one or both mutant subunits were analyzed for binding to DNA containing base pair mismatches. None of the mutant complexes displayed a significant defect in mismatch binding; however, unlike wild-type protein, all mutant combinations continued to display mismatch binding specificity in the presence of ATP and did not display ATP-dependent conformational changes as measured by limited trypsin protease digestion. Both wild-type complex and complexes defective in the Msh2p ATPase displayed ATPase activities that were modulated by mismatch and homoduplex DNA substrates. Complexes defective in the Msh6p ATPase, however, displayed weak ATPase activities that were unaffected by the presence of DNA substrate. The results from these studies suggest that the Msh2p and Msh6p subunits of the Msh2p-Msh6p complex play important and coordinated roles in postmismatch recognition steps that involve ATP hydrolysis. Furthermore, our data support a model whereby Msh6p uses its ATP binding or hydrolysis activity to coordinate mismatch binding with additional mismatch repair components.
Studamire
B
B
Section of Genetics and Development, Cornell University, Ithaca, New York 14853-2703, USA.
Quach
T
T
Alani
E
E
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000595:Amino Acid Sequence; D020137:Base Pair Mismatch / Q000235:genetics*; D001667:Binding, Competitive; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D008969:Molecular Sequence Data; D051718:MutS Homolog 2 Protein; D009154:Mutation / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010641:Phenotype; D011485:Protein Binding; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C098790:MSH6 protein, S cerevisiae; D009838:Oligodeoxyribonucleotides; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases; C489751:MSH2 protein, S cerevisiae; D051718:MutS Homolog 2 Protein