Modulation of MutS ATP hydrolysis by DNA cofactors

Biochemistry. 2000 Mar 21;39(11):3176-83. doi: 10.1021/bi992286u.


Escherichia coli MutS protein, which is required for mismatch repair, has a slow ATPase activity that obeys Michalelis-Menten kinetics. At 37 degrees C, the steady-state turnover rate for ATP hydrolysis is 1.0 +/- 0.3 min(-1) per monomer equivalent with a K(m) of 33 +/- 6 microM. Hydrolysis is competitively inhibited by the ATP analogues AMPPNP and ATPgammaS, with K(i) values of 4 microM in both cases, and by ADP with a K(i) of 40 microM. The rate of ATP hydrolysis is stimulated 2-5-fold by short hetero- and homoduplex DNAs. The concentration of DNA cofactor that yields half-maximal stimulation is lowest for oligodeoxynucleotide duplexes that contain a mismatched base pair. Pre-steady-state chemical quench analysis has demonstrated a substoichiometric initial burst of ADP formation by free MutS that is governed by a rate constant of 78 min(-1), indicating that the rate-limiting step for the steady-state reaction occurs after hydrolysis. Prebinding of MutS to homoduplex DNA does not alter the burst kinetics or amplitude but only increases the steady-state rate. In contrast, binding of the protein to heteroduplex DNA abolishes the burst of ADP formation, indicating that the rate-limiting step now occurs before hydrolysis. Gel filtration analysis indicates that the MutS dimer assembles into higher order oligomers in a concentration-dependent manner, and that ATP binding shifts this equilibrium to favor assembly. These results, together with kinetic findings, indicate nonequivalence of subunits within a MutS oligomer with respect to ATP hydrolysis and DNA binding.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphatases / chemistry
  • Adenosine Triphosphatases / metabolism*
  • Adenosine Triphosphate / chemistry
  • Adenosine Triphosphate / metabolism*
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Base Pair Mismatch
  • DNA Repair
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / metabolism*
  • DNA-Binding Proteins*
  • Dimerization
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Escherichia coli Proteins*
  • Hydrolysis
  • Kinetics
  • MutS DNA Mismatch-Binding Protein
  • Nucleic Acid Heteroduplexes / chemistry


  • Bacterial Proteins
  • DNA, Bacterial
  • DNA-Binding Proteins
  • Escherichia coli Proteins
  • Nucleic Acid Heteroduplexes
  • Adenosine Triphosphate
  • Adenosine Triphosphatases
  • MutS DNA Mismatch-Binding Protein
  • MutS protein, E coli