ADP-dependent DNA strand exchange by the mutant [P67G/E68A]RecA protein - Evidence for an involvement of ADP in RecA protein-mediated branch migration

We have prepared a mutant RecA protein in which proline 67 and glutamic aci d 68 in the NTP binding site were replaced by a glycine and alanine residue , respectively, The [P67G/E68A]RecA protein catalyzes the single-stranded D NA-dependent hydrolysis of ATP and is able to promote the standard ATP-depe ndent three-strand exchange reaction between a circular bacteriophage phi X 174 (phiX) single-stranded DNA molecule and a homologous linear phiX double -stranded (ds) DNA molecule (5.4 kilobase pairs). The strand exchange activ ity differs from that of the wild type RecA protein, however, in that it is (i) completely inhibited by an ATP regeneration system, and (ii) strongly stimulated by the addition of high concentrations of ADP to the reaction so lution. These results indicate that the strand exchange activity of the [P6 7G/E68A]RecA protein is dependent on the presence of both ATP and ADP. The ADP dependence of the reaction is reduced or eliminated when (i) a shorter linear phiX dsDNA fragment (1.1 kilobase pairs) is substituted for the full -length linear phiX dsDNA substrate, or (ii) the Mg2+ concentration is redu ced to a level just sufficient to complex the ATP present in the reaction s olution. These results indicate that it is the branch migration phase (and not the initial pairing step) of the [P67G/E68A]RecA protein-promoted stran d exchange reaction that is dependent on ADP. It is likely that the [P67G/E 68A]RecA mutation has revealed a requirement for ADP that also exists (but is not as readily apparent) in the strand exchange reaction of the wild typ e RecA protein.