Evidence for the coupling of ATP hydrolysis to the final (extension) phase of RecA protein-mediated DNA strand exchange

Abstract

RecA protein promotes a limited DNA strand exchange reaction, without ATP hydrolysis, that typically results in formation of short (1-2 kilobase pairs) regions of hybrid DNA. This nascent hybrid DNA is extended in a reaction that can be coupled to ATP hydrolysis. When ATP is hydrolyzed, the extension phase is progressive and its rate is 380 ± 20 bp min-1 at 37 °C. A single RecA nucleoprotein filament can participate in multiple DNA strand exchange reactions concurrently (involving duplex DNA fragments that are homologous to different segments of the DNA within a nucleoprotein filament), with no effect on the observed rate of ATP hydrolysis. The ATP hydrolytic and hybrid DNA extension activities exhibit a dependence on temperature between 25 and 45 °C that is, within experimental error, identical. This provides new evidence that the two processes are coupled. Arrhenius activation energies derived from the work are 13.3 ± 1.1 kcal mole-1 for DNA strand exchange, and 14.4 ± 1.4 kcal mole-1 for ATP hydrolysis during strand exchange. The rate of branch movement in the extension phase (base pair min-1) is related to the k(cat) for ATP hydrolysis during strand exchange (min-1) by a factor equivalent to 18 bp throughout the temperature range examined. The 18-base pair factor conforms to a quantitative prediction derived from a model in which ATP hydrolysis is coupled to a facilitated rotation of the DNA substrates. RecA filaments possess an intrinsic capacity for DNA strand exchange, mediated by binding energy rather than ATP hydrolysis, that is augmented by an ATP-dependent molecular motor.