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

Julie K. Mirshad, Stephen C. Kowalczykowski

Research output: Contribution to journalArticle

19 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)5945-5954
Number of pages10
JournalBiochemistry
Volume42
Issue number19
DOIs
StatePublished - May 20 2003

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Rec A Recombinases
Mutant Proteins
Single-Stranded DNA
DNA
Genetic Recombination
Hydrolysis
Adenosine Triphosphate
Binding Sites
Polynucleotides
Proteins
Escherichia coli Proteins
DNA Repair
Escherichia coli
Ion exchange
Repair
Substitution reactions
Thermodynamic properties
Association reactions
Phenotype

ASJC Scopus subject areas

  • Biochemistry

Cite this

Biochemical characterization of a mutant RecA protein altered in DNA-binding loop 1. / Mirshad, Julie K.; Kowalczykowski, Stephen C.

In: Biochemistry, Vol. 42, No. 19, 20.05.2003, p. 5945-5954.

Research output: Contribution to journalArticle

Mirshad, Julie K. ; Kowalczykowski, Stephen C. / Biochemical characterization of a mutant RecA protein altered in DNA-binding loop 1. In: Biochemistry. 2003 ; Vol. 42, No. 19. pp. 5945-5954.
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