Altered nucleotide cofactor-dependent properties of the mutant [S240K]RecA protein

Scott E. Steffen, Floyd Ransom Bryant

Research output: Contribution to journalArticle

Abstract

Two mutant Escherichia coli RecA proteins were prepared in which the ATP active site residue, Ser240, was replaced with asparagine and lysine (these amino acids are found in the corresponding positions in other bacterial RecA proteins). The S240N mutation had no discernible effect on the ATP-dependent activities of the RecA protein, indicating that serine and asparagine are functionally interchangeable at position 240. The S240K mutation, in contrast, essentially eliminated the ability of the RecA protein to utilize ATP as a nucleotide cofactor. The [S240K]RecA protein was able to catalyze the hydrolysis of dATP, however, suggesting that the absence of the 2'-hydroxyl group reduced an inhibitory interaction with the Lys240 side chain. Interestingly, the [S240K]RecA protein was able to promote an efficient LexA cleavage reaction but exhibited no strand exchange activity when dATP was provided as the nucleotide cofactor. This apparent separation of function may be attributable to the elevated S 0.5 value for dATP for the [S240K]RecA protein (490μM, compared to 20-30μM for the wild type and [S240N]RecA proteins), and may reflect a differential dependence of the LexA co-protease and DNA strand exchange activities on the nucleotide cofactor-mediated stabilization of the functionally-active state of the RecA-ssDNA complex.

Original languageEnglish (US)
Pages (from-to)527-531
Number of pages5
JournalBiochemical and Biophysical Research Communications
Volume421
Issue number3
DOIs
StatePublished - May 11 2012

Fingerprint

Rec A Recombinases
Nucleotides
Adenosine Triphosphate
Asparagine
Mutation
Bacterial Proteins
Escherichia coli Proteins
Mutant Proteins
Hydroxyl Radical
Serine
Escherichia coli
Lysine
Hydrolysis
Catalytic Domain
Peptide Hydrolases
Stabilization
Amino Acids

Keywords

  • Co-protease
  • DNA strand exchange
  • LexA protein
  • RecA protein

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology
  • Molecular Biology

Cite this

Altered nucleotide cofactor-dependent properties of the mutant [S240K]RecA protein. / Steffen, Scott E.; Bryant, Floyd Ransom.

In: Biochemical and Biophysical Research Communications, Vol. 421, No. 3, 11.05.2012, p. 527-531.

Research output: Contribution to journalArticle

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