Preparation and characterization of two sitespecific mutants of e. coli rec a protein

Einar Stole, Floyd Ransom Bryant

Research output: Contribution to journalArticle

Abstract

The E. coli rec A protein displays a DNA-dependent ATPase activity and is essential for homologous genetic recombination and for the postreplicative repair of damaged DNA. Previous work in this laboratory (K. L. Menge and F. R. Bryant, Biochemistry 31, 5151–5157) demonstrated that the structurally related nucleotide triphosphates (NTP), ATP, purine riboside triphosphate (PTP), inosine triphosphate (ITP) and guanosine triphosphate (GTP) are each hydrolyzed by rec A protein at the same rate. However, only ATP and PTP, which display S0.5 values of 45 and 100 uM, respectively, serve as cofactors for the rec A-promoted three-strand exchange reaction, whereas ITP and GTP, which have S0.5 values of 300 and 750 uM, respectively, do not support strand exchange. These findings complement the results obtained from studies on the recombination deficient mutant proteins, [GI60N]rec A and [HI63A]rec A (F. R. Bryant (1990) J. Biol. Chem. 263, 8716–8723; K. A. Muench and F. R. Bryant (1991) J. Biol. Chem. 266, 844–850). These mutant proteins catalyze ATP hydrolysis at a rate similar to that of the wild-type protein, but are unable to promote ATP-dependent threestrand exchange at pH 7.5. However, these two rec A mutant proteins do catalyze ATP-dependent strand exchange at pH 6.0–6.8. Thb S0.5 (ATP) values for the [G160N]rec A and the [HI63A]rec A proteins are each 150 uM, which is about 4-fold higher than the S0.5 value for wildtype rec A protein at pH 7.5. The mutant proteins exhibit S0.5 values of 20–30 uM at pH 6.2. These results suggest that the decreases in S0.5 values for the mutant rec A proteins may be responsible for their activation in strand exchange at lower pH. Thus, the studies of altered nucleotide triphosphate cofactors and of the mutant rec A proteins suggests that nucleotide triphosphates with S0.5 values greater than 100 uM are nonfunctional in strand exchange. Several mutants, based on the recently determined X-ray structure, were constructed by site directed mutagenesis in order to further explore the relationship between nucleotide binding and ATP-dependent strand exchange. Results from kinetic and physical analysis of these mutants will be presented.

Original languageEnglish (US)
Pages (from-to)52
Number of pages1
JournalProtein Engineering, Design and Selection
Volume6
DOIs
StatePublished - 1993

ASJC Scopus subject areas

  • Biochemistry
  • Biotechnology
  • Bioengineering
  • Molecular Biology

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