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Evidence for sequential action of two ATPase active sites in yeast Msh2-Msh6
Karin Drotschmann1, Wei Yang, Thomas A Kunkel
1Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
Abstract:
Bacterial MutS homodimers contain two ATPase active sites that have non-equivalent functions in DNA mismatch repair. The homologous Msh2-Msh6 complex in eukaryotes also has intrinsic ATPase activity that is essential for mismatch repair. Here, we investigate differences in the two putative ATPase active sites by examining the properties of heterodimers containing alanine substituted for an invariant glutamic acid in the active site of either Msh2, Msh6 or both. Mutation rates in wild type versus Glu-->Ala mutant haploid yeast strains indicate that both ATPase active sites are essential for mismatch repair activity in vivo. The properties of purified heterodimers suggest that the ATPase active site in Msh6 binds ATP with higher affinity and hydrolyzes ATP faster and with higher efficiency than does the ATPase active site in Msh2. This suggests sequential action of the two ATPase active sites, in which ATP binds to Msh6 first to trigger downstream events in mismatch repair.
Insights
Both ATPase active sites in the Msh2-Msh6 complex are crucial for DNA mismatch repair in yeast. The Msh6 site shows higher affinity and faster ATP hydrolysis, suggesting a sequential action mechanism.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Bacterial MutS homodimers possess two distinct ATPase active sites vital for DNA mismatch repair.
- The eukaryotic Msh2-Msh6 complex, homologous to MutS, also relies on ATPase activity for mismatch repair.
Purpose of the Study:
- To investigate the functional differences between the two putative ATPase active sites in the Msh2-Msh6 complex.
- To determine the role of each ATPase active site in DNA mismatch repair in vivo and in vitro.
Main Methods:
- Site-directed mutagenesis was used to create Msh2 and Msh6 variants with alanine substitutions in their ATPase active sites.
- Mutation rates were assessed in haploid yeast strains expressing wild-type or mutant Msh2-Msh6 heterodimers.
- Biochemical assays were performed on purified heterodimers to analyze ATP binding affinity and hydrolysis rates.
Main Results:
- Mutation rates in yeast strains revealed that both ATPase active sites are essential for DNA mismatch repair.
- Purified Msh2-Msh6 heterodimers exhibited differential properties: the Msh6 ATPase site binds ATP with higher affinity and hydrolyzes it more efficiently than the Msh2 site.
- These findings indicate distinct functional roles for the two ATPase active sites within the Msh2-Msh6 complex.
Conclusions:
- The study demonstrates that both ATPase active sites of the Msh2-Msh6 complex are indispensable for DNA mismatch repair in yeast.
- The Msh6 ATPase active site appears to act first, binding ATP with higher affinity and faster kinetics, initiating downstream repair events.
- This suggests a sequential mechanism for ATP utilization by the Msh2-Msh6 complex during DNA mismatch repair.