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Hydrolytically deficient MutS E694A is defective in the MutL-dependent activation of MutH and in the mismatch-dependent assembly of the MutS.MutL.heteroduplex complex.

Publication ,  Journal Article
Baitinger, C; Burdett, V; Modrich, P
Published in: J Biol Chem
December 5, 2003

The roles of ATP binding and hydrolysis by MutS in mismatch repair are poorly understood. MutS E694A, in which Glu-694 of the Walker B motif is substituted with alanine, is defective in hydrolysis of bound ATP and has been reported to support MutL-dependent activation of the MutH d(GATC) endonuclease in a trans DNA activation assay (Junop, M. S., Obmolova, G., Rausch, K., Hsieh, P., and Yang, W. (2001) Mol. Cell 7, 1-12). Because the MutH trans activation assay used in these previous studies was characterized by high background and low efficiency, we have re-evaluated the activities of MutS E694A. In contrast to native MutS, which can be isolated in a nucleotide-free form, purified MutS E694A contains 1.0 mol of bound ATP per dimer equivalent, and substoichiometric levels of bound ADP (0.08-0.58 mol/dimer), consistent with the suggestion that the ADP.MutS.ATP complex comprises a significant fraction of the protein in solution (Bjornson, K. P. and Modrich, P. (2003) J. Biol. Chem. 278, 18557-18562). In the presence of Mg2+, endogenous ATP is hydrolyzed with a rate constant of 0.12 min-1 at 30 degrees C, and hydrolysis yields a protein that displays increased specificity for heteroduplex DNA. As observed with wild type MutS, ATP can promote release of MutS E694A from a mismatch. However, the mutant protein is defective in the methyl-directed, mismatch- and MutL-dependent cis activation of MutH endonuclease on a 6.4-kilobase pair heteroduplex, displaying only 1 to 2% of the activity of wild type MutS. The mutant protein also fails to support normal assembly of the MutS.MutL.DNA ternary complex. Although a putative ternary complex can be observed in the presence of MutS E694A, assembly of this structure displays little if any dependence on a mismatched base pair.

Duke Scholars

Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

December 5, 2003

Volume

278

Issue

49

Start / End Page

49505 / 49511

Location

United States

Related Subject Headings

  • MutS DNA Mismatch-Binding Protein
  • MutL Proteins
  • Hydrolysis
  • Escherichia coli Proteins
  • Enzyme Activation
  • Endodeoxyribonucleases
  • Dimerization
  • DNA-Binding Proteins
  • DNA Repair Enzymes
  • Biochemistry & Molecular Biology
 

Citation

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Baitinger, C., Burdett, V., & Modrich, P. (2003). Hydrolytically deficient MutS E694A is defective in the MutL-dependent activation of MutH and in the mismatch-dependent assembly of the MutS.MutL.heteroduplex complex. J Biol Chem, 278(49), 49505–49511. https://doi.org/10.1074/jbc.M308738200
Baitinger, Celia, Vickers Burdett, and Paul Modrich. “Hydrolytically deficient MutS E694A is defective in the MutL-dependent activation of MutH and in the mismatch-dependent assembly of the MutS.MutL.heteroduplex complex.J Biol Chem 278, no. 49 (December 5, 2003): 49505–11. https://doi.org/10.1074/jbc.M308738200.
Baitinger, Celia, et al. “Hydrolytically deficient MutS E694A is defective in the MutL-dependent activation of MutH and in the mismatch-dependent assembly of the MutS.MutL.heteroduplex complex.J Biol Chem, vol. 278, no. 49, Dec. 2003, pp. 49505–11. Pubmed, doi:10.1074/jbc.M308738200.

Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

December 5, 2003

Volume

278

Issue

49

Start / End Page

49505 / 49511

Location

United States

Related Subject Headings

  • MutS DNA Mismatch-Binding Protein
  • MutL Proteins
  • Hydrolysis
  • Escherichia coli Proteins
  • Enzyme Activation
  • Endodeoxyribonucleases
  • Dimerization
  • DNA-Binding Proteins
  • DNA Repair Enzymes
  • Biochemistry & Molecular Biology