Homologous pairing in genetic recombination. Purification and characterization of Escherichia coli recA protein.
RecA protein, which is essential for genetic recombination in Escherichia coli, was extensively purified from a strain of E. coli which contained the recA gene cloned in a plasmid (Sancar, A., and Rupp, W. D. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 3144-3148). Using the DNA-dependent ATPase activity of recA protein as an assay, we obtained about 60 mg of purified recA protein from 100 g of cells. Ten micrograms or 1 microgram of the purified protein exhibited only one detectable band with Mr approximately = 40,000 upon sodium dodecyl sulfate-acrylamide gel electrophoresis. More than 99% of the ATPase activity of purified recA protein was dependent on single-stranded DNA. Purified recA protein had no detectable DNase, topoisomerase, or ligase activities. The enzyme was stable for a least a year when stored at 0-4 degrees C. The half-life of the ATPase activity of 25 microM recA protein was 37 min at 51 degrees C. Purified recA protein binds to single-stranded and double-stranded DNA, unwinds duplex DNA by a mechanism that is stimulated by single-stranded DNA or oligonucleotides, and pairs homologous single strands with duplex DNA.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D011938:Rec A Recombinases; D011995:Recombination, Genetic
D001426:Bacterial Proteins; D015636:Magnesium Chloride; D011938:Rec A Recombinases; D000251:Adenosine Triphosphatases; D008274:Magnesium