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Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein
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Published on: March 31, 2010

Engineered disulfide-forming amino acid substitutions interfere with a conformational change in the mismatch

Victoria V Hargreaves1, Christopher D Putnam, Richard D Kolodner

  • 1Ludwig Institute for Cancer Research, Department of Medicine, Moores-University of California San Diego Cancer Center, and Institute of Genomic Medicine, University of California School of Medicine, San Diego, La Jolla, California 92093-0669, USA.

The Journal of Biological Chemistry
|October 10, 2012
PubMed

Insights

Adenosine triphosphate (ATP) binding induces a conformational change in the Msh6 protein, which is crucial for DNA mismatch repair. This change allows the Msh2-Msh6 complex to interact with other repair proteins and function effectively.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • DNA mismatch repair (MMR) is essential for maintaining genomic stability.
  • The Msh2-Msh6 complex recognizes DNA mismatches, and its interaction with Mlh1-Pms1 is critical for downstream repair.
  • The role of ATP in Msh2-Msh6 conformational changes and MMR remains incompletely understood.

Purpose of the Study:

  • To elucidate the specific conformational changes induced by ATP binding in the Msh2-Msh6 complex.
  • To determine the functional significance of these ATP-driven conformational changes in DNA mismatch repair.

Main Methods:

  • Site-directed mutagenesis to engineer a disulfide bond in the Msh6 C-terminal region.
  • Biochemical assays to assess Msh2-Msh6 complex binding, sliding, and interaction with Mlh1-Pms1.
  • In vivo experiments to evaluate the impact of the engineered disulfide bond on mismatch repair efficiency.

Main Results:

  • ATP binding induces a conformational change in the Msh6 C-terminal region, protecting a specific trypsin cleavage site.
  • An engineered disulfide bond preventing this conformational change abolished ATP-dependent sliding and Mlh1-Pms1 interaction.
  • The engineered disulfide bond significantly impaired in vivo DNA mismatch repair efficiency.

Conclusions:

  • The ATP-driven conformational change in Msh6 is essential for the Msh2-Msh6 complex's ability to form sliding clamps and interact with Mlh1-Pms1.
  • This conformational change plays a critical role in the overall efficiency of the DNA mismatch repair pathway.

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