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Functional studies on the candidate ATPase domains of Saccharomyces cerevisiae MutLalpha
1Department of Molecular and Medical Genetics, Oregon Health Sciences University, Portland, Oregon 97201, USA.
Abstract:
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.
Insights
Mutations disrupting ATPase activity in MutLalpha (Mlh1p-Pms1p) impaired DNA mismatch repair in yeast. ATP binding induces conformational changes, suggesting a role in coordinating repair processes.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA mismatch repair (MMR) is crucial for maintaining genomic stability.
- The MutLalpha heterodimer (Mlh1p-Pms1p) is essential for MMR in Saccharomyces cerevisiae.
- MutL homologues possess conserved ATPase motifs, suggesting a functional role.
Purpose of the Study:
- To investigate the functional significance of the putative ATPase activity of MutLalpha in DNA mismatch repair.
- To determine the role of ATP binding and hydrolysis in MutLalpha function and interactions.
Main Methods:
- Site-directed mutagenesis of conserved ATP hydrolysis and binding motifs in Mlh1p and Pms1p.
- In vivo DNA mismatch repair assays.
- Limited proteolysis of purified recombinant MutLalpha.
- Yeast two-hybrid analysis.
Main Results:
- Mutations in ATPase motifs disrupted DNA mismatch repair in vivo.
- ATP and non-hydrolyzable ATP analogs induced conformational changes in the MutLalpha N-terminus.
- ATP-binding induced conformational changes promoted N-terminal interactions between Mlh1p and Pms1p.
- Differential requirements for Mlh1p and Pms1p ATPase motifs were observed.
Conclusions:
- MutLalpha undergoes ATP-dependent conformational changes essential for its function in DNA mismatch repair.
- These conformational changes likely coordinate downstream events in the yeast MMR pathway.
- The ATPase activity of MutLalpha plays a critical regulatory role in DNA repair.