The alternating ATPase domains of MutS control DNA mismatch repair

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• DNA mismatch repair (MMR) maintains genomic stability by correcting DNA replication errors.
• The MutS enzyme in Escherichia coli is critical for initiating MMR.
• MutS possesses ATPase activity essential for its function, but the mechanism is unclear.

Purpose of the Study:

• To investigate the role of ATPase activity in MutS function.
• To elucidate the mechanism of ATP hydrolysis within the MutS homodimer.
• To identify key residues controlling MutS ATPase activity and its impact on DNA repair.

Main Methods:

• Biochemical assays to assess ATPase activity and ADP binding affinities.
• Site-directed mutagenesis of the MutS enzyme, specifically targeting Arg697.
• In vivo assays to evaluate the efficiency of DNA mismatch repair in wild-type and mutant strains.

Main Results:

• The two ATPase sites within the MutS homodimer exhibit asymmetric ADP binding affinities.
• These sites alternate in their ATP hydrolysis cycles.
• Mutation of a single residue, Arg697, located at the interface of ATPase domains, abolishes this asymmetry.
• Loss of asymmetry due to Arg697 mutation impairs in vivo DNA mismatch repair.

Conclusions:

• The asymmetry of ATPase domains in MutS is crucial for DNA mismatch repair.
• This asymmetry regulates the timing of sequential steps in the MMR pathway.
• Arg697 plays a key role in establishing and maintaining ATPase domain asymmetry for effective DNA repair.