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Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein
Published on: March 31, 2010
Interaction between the Msh2 and Msh6 nucleotide-binding sites in the Saccharomyces cerevisiae Msh2-Msh6 complex
Victoria V Hargreaves1, Scarlet S Shell, Dan J Mazur
1Department of Medicine and Cellular, Cancer Center, Ludwig Institute for Cancer Research, University of California San Diego School of Medicine, La Jolla, California 92093-0669, USA.
Abstract:
Indirect evidence has suggested that the Msh2-Msh6 mispair-binding complex undergoes conformational changes upon binding of ATP and mispairs, resulting in the formation of Msh2-Msh6 sliding clamps and licensing the formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes. Here, we have studied eight mutant Msh2-Msh6 complexes with defective responses to nucleotide binding and/or mispair binding and used them to study the conformational changes required for sliding clamp formation and ternary complex assembly. ATP binding to the Msh6 nucleotide-binding site results in a conformational change that allows binding of ATP to the Msh2 nucleotide-binding site, although ATP binding to the two nucleotide-binding sites appears to be uncoupled in some mutant complexes. The formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes requires ATP binding to only the Msh6 nucleotide-binding site, whereas the formation of Msh2-Msh6 sliding clamps requires ATP binding to both the Msh2 and Msh6 nucleotide-binding sites. In addition, the properties of the different mutant complexes suggest that distinct conformational states mediated by communication between the Msh2 and Msh6 nucleotide-binding sites are required for the formation of ternary complexes and sliding clamps.
Insights
The Msh2-Msh6 complex requires specific ATP binding events for conformational changes. These changes are essential for forming sliding clamps and Msh2-Msh6-Mlh1-Pms1 ternary complexes.
Area of Science:
- Molecular Biology
- Biochemistry
- DNA Repair Mechanisms
Background:
- The Msh2-Msh6 complex is crucial for DNA mismatch repair.
- Previous studies suggested ATP and mispair binding induce conformational changes in Msh2-Msh6.
- These changes are thought to facilitate sliding clamp formation and ternary complex assembly.
Purpose of the Study:
- To investigate the specific conformational changes in Msh2-Msh6 required for sliding clamp and ternary complex formation.
- To analyze the role of ATP binding at distinct sites within the Msh2-Msh6 complex.
Main Methods:
- Utilized eight mutant Msh2-Msh6 complexes with altered nucleotide and mispair binding responses.
- Assessed conformational changes in response to ATP binding at Msh2 and Msh6 nucleotide-binding sites.
- Examined the requirements for forming Msh2-Msh6 sliding clamps and Msh2-Msh6-Mlh1-Pms1 ternary complexes.
Main Results:
- ATP binding to Msh6 induces a conformational change enabling ATP binding to Msh2, though this can be uncoupled in mutants.
- Ternary complex formation requires ATP binding solely at the Msh6 site.
- Sliding clamp formation necessitates ATP binding at both Msh2 and Msh6 sites.
Conclusions:
- Distinct conformational states, regulated by communication between Msh2 and Msh6 nucleotide-binding sites, are necessary for ternary complex and sliding clamp assembly.
- ATP binding dynamics play a critical, site-specific role in Msh2-Msh6 complex function during DNA repair.
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