Two mutant RecA proteins possessing pH-dependent strand exchange activity exhibit pH-dependent presynaptic filament...

In previous studies it was shown that the mutant RecA proteins, [G160N]RecA and [H163A]RecA, are unable to catalyze ATP-dependent DNA strand exchanges at pH 7.5, but are active at pH 6.0 to 6.8. Here, we have used electron microscopy to follow the assembly of these mutant proteins onto single-stranded DNA at pH 7.5 and pH 6.2. In the absence of ATP, the filaments formed by the mutant proteins were similar to those formed by the wild-type protein, at both pH 7.5 and pH 6.2. In the presence of ATP, however, the filaments formed by the wild-type protein at pH 7.5 were extended and were stable in the presence of saturating SSB protein, whereas the filaments formed by the mutant proteins were shorter and unstable in the presence of SSB protein. At pH 6.2, in contrast, the filaments formed by the mutant proteins in the presence of ATP were of the same contour length as the wild-type RecA protein filaments and were stable in the presence of SSB protein. In the presence of the non-hydrolyzable ATP analog, ATP gamma S, and SSB protein, the mutant proteins formed full-length filaments at pH 7.5 that had a helical periodicity identical with that of the wild-type filaments (and characteristic of the strand exchange-active open conformational state); if SSB protein was omitted, the mutant protein filaments still exhibited the open helical periodicity, but were shorter and of highly variable length, presumably because of an improper threading of the ssDNA into the mutant filament. To account for these results, we propose that: (1) the mutant proteins are unable to isomerize efficiently to the open conformational state at pH 7.5 in the presence of ATP, but are able to do so in the presence of ATP gamma S; this indicates that the mechanistic defect is related to ATP hydrolysis rather than ATP binding; and (2) the mutant proteins are able to isomerize to the open conformational state in the presence of ATP at pH 6.2, indicating that protonation of the mutant filaments is sufficient to relieve the mechanistic deficiency.