PURIFICATION AND CHARACTERIZATION OF THE HUMAN RAD51 PROTEIN, AN ANALOG OF ESCHERICHIA-COLI RECA

In bacteria, genetic recombination is catalysed by RecA protein, the p roduct of the recA gene, A human gene that shares homology with Escher ichia coli recA (and its yeast homologue RAD51) has been cloned from a testis cDNA library, and its 37 kDa product (hRad51) purified to homo geneity, The human Rad51 protein binds to single- and double-stranded DNA and exhibits DNA-dependent ATPase activity, Using a topological as say, we demonstrate that hRad51 underwinds duplex DNA, in a reaction d ependent upon the presence of ATP or its non-hydrolysable analogue ATP gamma S, Complexes formed with single- and double-stranded DNA have b een observed by electron microscopy following negative staining, With nicked duplex DNA, hRad51 forms helical nucleoprotein filaments which exhibit the striated appearance characteristic of RecA or yeast Rad51 filaments, Contour length measurements indicate that the DNA is underw ound and extended within the nucleoprotein complex, In contrast to yea st Rad51 protein, human Rad51 forms filaments with single-stranded DNA in the presence of ATP/ATP gamma S, These resemble the inactive form of the RecA filament which is observed in the absence of a nucleotide cofactor.