Dissociation of non-complementary second DNA from RecA filament without ATP hydrolysis: mechanism of search for homologous DNA.

Abstract

RecA protein catalyzes the DNA annealing and mimics the DNA strand exchange reaction in vitro in the presence of ATP or its non-hydrolyzable analog, adenosine 5'-O-3-thiotriphosphate (ATPgammaS). For these activities RecA coordinates two DNA molecules [Takahashi, M. and Nordén, B. (1994) Adv. Biophys. 30, 1-35]. In order to get a better understanding of how RecA performs the search for sequence complementarity or homology between two DNA molecules, the association and dissociation kinetics of a second DNA molecule to and from RecA in the presence of ATPgammaS have been investigated. The kinetics were monitored by fluorescence measurements of partly etheno-modified poly(dA) assisted by linear dichroism measurements of the flow-oriented complex. The association of the second DNA is fast, regardless of whether the sequence is complementary or not. By contrast, the dissociation kinetics is strongly dependent on sequence complementarity. If the second DNA is complementary to the first, dissociation is extremely slow, whilst that of non-complementary second DNA is fast. In no case does the first DNA leave the RecA fiber. Our findings indicate that the dissociation step is important in the search for homology by RecA.