Alteration of the nucleoside triphosphate (NTP) catalytic domain within Escherichia coli recA protein attenuates NTP hydrolysis but not joint molecule formation.

The hydrolysis of the nucleoside triphosphates, such as ATP or GTP, plays a central role in a variety of biochemical processes; but, in most cases, the specific mechanism of energy transduction is unclear. DNA strand exchange promoted by the Escherichia coli recA protein is normally associated with ATP hydrolysis. However, we advanced the idea that the observed ATP hydrolysis is not obligatorily linked to the exchange of DNA strands (Menetski, J. P., Bear, D. G., and Kowalczykowski, S. C. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 21-25); instead, ATP binding resulting in an allosteric transition to an active form of the recA protein is sufficient. In this paper, we extend this conclusion by introducing a mutation within a highly conserved region of the recA protein that, on the basis of sequence similarity, is proposed to interact with the pyrophosphate moiety of a bound NTP molecule. The conservative substitution of an arginine for the invariant lysine at position 72 reduces NTP hydrolysis by approximately 600-850-fold. This mutation does not significantly alter the capacity of the mutant recA (K72R) protein either to bind nucleotide cofactors and single-stranded DNA or to respond allosterically to nucleotide cofactor binding. Despite the dramatic attenuation in NTP hydrolysis, the recA (K72R) protein retains the ability to promote homologous pairing and extensive exchange of DNA strands (up to 1.5 kilobase pairs). These results both identify a component of the catalytic domain for NTP hydrolysis and demonstrate that the recA protein-promoted pairing and exchange of DNA strands mechanistically require the allosteric transition induced by NTP cofactor binding, but not the energy educed from NTP hydrolysis.