RecA protein from Escherichia coli. a very rapid and simple purification procedure: binding of adenosine 5'-triphosphate and adenosine 5'-diphosphate by the homogeneous protein.

Abstract

The recA protein from Escherichia coli may be rapidly purified to homogeneity by a simple procedure involving only selective precipitation and one gel filtration step. The binding of ATP to the homogeneous protein has been measured by nonequilibrium dialysis. At pH 8.1 and 25 degrees C, the stoichiometry of the recA X ATP complex is 1:1 and the dissociation constant 24 microM. The binding of ADP to the enzyme and its complexes with single-stranded (ss) DNA and double-stranded (ds) DNA has been measured by equilibrium dialysis. In the absence of DNA, the binding is similar to that observed for ATP. The addition of ssDNA weakens the binding 3-fold. The addition of dsDNA causes a significant drop in the stoichiometry, suggesting an asymmetric distribution of active sites in the complex.