Biochemical characterization of a mutant RecA protein altered in DNA-binding loop 1

Biochemistry. 2003 May 20;42(19):5945-54. doi: 10.1021/bi027233i.

Abstract

The double substitution of Glu156 with Leu and Gly157 with Val in the Escherichia coli RecA protein results in a severely reduced level of recombination and constitutive coprotease behavior. Here we present our examination of the biochemical properties of this mutant protein, RecA N99, in an effort to understand its phenotype and the role of loop 1 (L1) in RecA function. We find that RecA N99 protein has reduced single-stranded DNA (ssDNA)-dependent ATP hydrolysis activity, which is not as sensitive to the presence of SSB protein as wild-type RecA protein. RecA N99 protein is also nearly unable to utilize duplex DNA as a polynucleotide cofactor for ATP hydrolysis, and it shows both a decreased rate of association with ssDNA and a diminished capacity to bind DNA in the secondary binding site. The mutant protein has a corresponding reduction in DNA strand exchange activity, which probably results in the decrease in recombination activity in vivo. The constitutive induction of the SOS response may be a consequence of the impaired ability to repair damaged DNA, resulting in unrepaired ssDNA which can act as a cofactor for the cleavage of LexA repressor. These findings point to an involvement of L1 in both the primary and secondary DNA binding sites of the RecA protein.

Publication types

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

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Amino Acid Substitution
  • Bacterial Proteins / metabolism
  • Binding Sites / genetics
  • DNA, Bacterial / genetics
  • DNA, Bacterial / metabolism
  • DNA, Single-Stranded / genetics
  • DNA, Single-Stranded / metabolism
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / genetics*
  • Escherichia coli Proteins / metabolism
  • Hydrolysis
  • Kinetics
  • Mutagenesis, Site-Directed
  • Nucleoproteins / metabolism
  • Protein Binding
  • Rec A Recombinases / chemistry*
  • Rec A Recombinases / genetics*
  • Rec A Recombinases / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Recombination, Genetic
  • Serine Endopeptidases / metabolism

Substances

  • Bacterial Proteins
  • DNA, Bacterial
  • DNA, Single-Stranded
  • Escherichia coli Proteins
  • LexA protein, Bacteria
  • Nucleoproteins
  • Recombinant Proteins
  • Adenosine Triphosphate
  • Rec A Recombinases
  • Serine Endopeptidases