Abstract
The DNA-dependent ATPase activity of the recA protein of Escherichia coli shows a complex dependence on ATP concentration. With a single-stranded (SS) DNA cofactor, the Hill coefficient for ATP is 3.3 at pH 8.1 and 1.4 at pH 6.2. With a double-stranded (DS) DNA cofactor, the Hill coefficient is 3.3 at pH 6.2 (no reaction is detectable at pH 8.1). In the presence of SS DNA, the Km for ATP is 20 microM, independent of pH, while with DS DNA at pH 6.2, KmATP is 100 microM. These and other observations indicate that the interaction of recA protein with ATP is influenced by both pH and DNA cofactor. ADP, UTP, dTTP, and GTP are competitive inhibitors of the ATPase activity of recA protein, indicating that there is a single binding site for nucleoside triphosphates. Nucleoside triphosphates, but not ADP, reduce the Hill coefficient for ATP hydrolysis and thus can contribute to the cooperative effect of ATP.
Highlights
The DNA-dependent ATPasaectivity of the recA protein of Escherichia coli shows a complex dependenceon ATP concentration
DNA at pH 6.2, KmATiPs 100 These and other observations indicate that the interaction of recA protein with ATP is influenced by both pH and DNA cofactor
The binding of recA protein to DS DNA requires ATP and SS DNA ( 4 ) and results inunwinding of the duplex (5-7); the requirement for SS DNA is obviated at pH 6.2 (6, 7)
Summary
(Received for publication, December 22, 1980, and in revised form, April 27, 1981). From the Departmentof Biochemistry, Stanford University School of Medicine, Stanford,California 94305. Once paired, recA protein may align DNA molecules at homologous sequences These are clearly complex processes,remarkable for a protein of molecular weight 38,000. Both types of hybridization reactions require ATP hydrolysis. Not required for SS DNA binding, ATP influences the stability of recA protein-SS DNA complexes (6, 7). This dependence is complex in that it is affected both by pH and the DNA factor These factors account for the different sensitivities to ADP of SS and DS DNA-dependent ATP hydrolysis that we had observed previously (8).In addition, we have found that UTP, which is hydrolyzed by the recA protein, is a competitive inhibitor of the ATPase activity, consistent with the notion of a single (or overlapping) active site for nucleoside triphosphate hydrolysis. All velocities were determined by measuring the time course of the reaction
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