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DNA mismatch correction in Haemophilus influenzae: characterization of MutL, MutH and their interaction

Nimesh Joseph1, Ritwick Sawarkar, Desirazu N Rao

  • 1Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.

DNA Repair
|October 12, 2004
PubMed

Insights

This study characterizes Haemophilus influenzae DNA mismatch repair proteins (MutS, MutL, MutH). MutH binding specificity is enhanced by MutL, and key residues are crucial for its function and DNA repair activity.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • DNA mismatch repair (MMR) is crucial for maintaining genomic stability.
  • Haemophilus influenzae possesses MutS, MutL, and MutH proteins involved in MMR.
  • Understanding the function of these proteins is key to comprehending bacterial DNA repair mechanisms.

Purpose of the Study:

  • To functionally characterize the DNA mismatch repair proteins MutS, MutL, and MutH from Haemophilus influenzae.
  • To investigate the DNA binding properties and activation mechanisms of H. influenzae MutH.
  • To explore the role of specific amino acids in MutH function and the impact on MutS activity.

Main Methods:

  • Complementation assays using Escherichia coli mutant strains.
  • DNA binding studies of H. influenzae MutH, including non-specific and MutL-mediated binding.
  • Site-directed mutagenesis of conserved amino acids in MutH (Ile213, Leu214).
  • Analysis of MutS ATPase activity in the presence of different DNA substrates.

Main Results:

  • H. influenzae MutS, MutL, and MutH genes complemented E. coli MMR-deficient strains.
  • MutH binds DNA non-specifically, but MutL and ATP confer specificity for hemi-methylated DNA.
  • Mutated MutH proteins (Ile213Ala, Leu214Ala) showed reduced DNA binding, nicking, and MutL-mediated activation.
  • MutH failed to nick HU-bound DNA, unlike other methyl-specific endonucleases.
  • MutS ATPase activity decreased with mismatched circular DNA, suggesting MutS clamp trapping.

Conclusions:

  • The study provides functional characterization of H. influenzae MMR proteins.
  • MutL plays a critical role in conferring DNA sequence specificity to MutH.
  • Conserved residues in MutH are essential for its DNA binding and nicking activities.
  • MutS interaction with mismatched DNA involves a trapping mechanism, impacting ATPase activity.

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