Characterization of RecA1332 in vivo and in vitro. A role for α-helix E as a liaison between the subunit-subunit interface and the DNA and ATP binding domains of RecA protein

Piero R. Bianco, George M. Weinstock

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

Background: The RecA protein of Escherichia coli is essential for homologous recombination and induction of the SOS response. RecA has three cysteines located at positions 90, 116 and 129. Chemical modification of these residues abolishes ATP hydrolysis and repressor cleavage, and causes a reduction in the DNA strand exchange and DNA strand annealing activities. Several mutants at each of these positions were isolated and partially characterized. One of these, recA1332, replaces cysteine 129 with methionine. Although this is a relatively conservative mutation based on hydrophobicity, recA1332 was completely defective for DNA repair but the purified protein was active for ATPase in vitro. Results: In vivo, strains containing this mutant allele were shown to be defective when assayed for all RecA-dependent activities. In vitro, RecA1332 protein possessed DNA-dependent ATP hydrolysis activity that showed an increased sensitivity to inhibition by monovalent cations, and whose k(cat) was reduced 3- to 12-fold. In addition, RecA1332 was unable to use oligodeoxyribonucleotides as ssDNA cofactors in the ATPase reaction. RecA1332 showed altered binding to single- and double-stranded DNA and, although it was able to perform DNA strand exchange, it was slowed in its ability to both form joint molecule intermediates and to convert these species to product. Conclusions: Our results are consistent with a defect in intermolecular interactions between RecA monomers. We propose that α-helix E (which includes C129M) is a liaison that connects the subunit-subunit interactions to DNA and ATP binding, thereby creating filament stability and cooperativity.

Original languageEnglish (US)
Pages (from-to)79-97
Number of pages19
JournalGenes to Cells
Volume3
Issue number2
DOIs
StatePublished - 1998

ASJC Scopus subject areas

  • Genetics
  • Cell Biology

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