CHARACTERIZATION OF RECA MEDIATED HOMOLOGOUS PAIRING ON NITROCELLULOSE MEMBRANE

Reactions between a single-stranded DNA (ssDNA) and a double-stranded DNA (dsDNA) provide an efficient model to study RecA promoted homologo us recombination. We have devised an assay in which the ssDNA is first bound to a nitrocellulose membrane. RecA protein is loaded on this me mbrane (loading step) which is then incubated with a labelled homologo us dsDNA (incubation step). Since this assay can be used for study of mutant RecA proteins or RecA-like activities in crude extracts from ot her organisms, we have characterized the reaction promoted on the memb rane. Under these new conditions, the reaction keeps the main characte ristics observed with classical assays performed in solution: increasi ng NaCl concentration destabilized the RecA-DNA complex, ATP gamma S w as required for formation of stable RecA-DNA complex, initiation of th e reaction exhibits the same polarity as in classical assays, a comple te strand exchange with a 44 bp long duplex oligonucleotide has been r ecorded under our conditions. Moreover, our results indicate that the binding of RecA protein itself to the nitrocellulose membrane did not impair its ability to promote homologous pairing. Pairing reactions in volving long dsDNA (6407 bp) were more efficient with hydrolysable ATP than with ATP gamma S only when the ssDNA was bound to the membrane. Furthermore, ATP hydrolysis was not required when using short dsDNA(44 bp). These results constitute experimental support for a new role for the ATPase activity of RecA protein: the energy produced could favor the initiation of RecA mediated recombination involving long stretches of DNA which have restricted freedom to rotation.