DISSOCIATION OF NON-COMPLEMENTARY 2ND-DNA FROM RECA FILAMENT WITHOUT ATP HYDROLYSIS - MECHANISM OF SEARCH FOR HOMOLOGOUS DNA

RecA protein catalyzes the DNA annealing and mimics the DNA strand exc hange reaction in vitro in the presence of ATP or its non-hydrolyzable analog, adenosine 5'-O-3-thiotriphosphate (ATP gamma S). For these ac tivities RecA coordinates two DNA molecules [Takahashi, M. and Norden, B. (1994) Adv. Biophys, 30, 1-35]. In order to get a better understan ding of how RecA performs the search for sequence complementarity or h omology between two DNA molecules, the association and dissociation ki netics oa second DNA molecule to and from RecA in the presence of ATP gamma S have been investigated, The kinetics were monitored by fluores cence measurements of partly etheno-modified poly(dA) assisted by line ar dichroism measurements of the flow-oriented complex. The associatio n of the second DNA is fast;, regardless of whether the sequence is co mplementary or not. Bg contrast, the dissociation kinetics is strongly dependent on sequence complementarity, If the second DNA is complemen tary 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 im portant in the search for homology by RecA.