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Purification and characterization of the human Rad51 protein, an analogue of E. coli RecA.

By F.E. Benson, A. Stasiak and S.C. West


In bacteria, genetic recombination is catalysed by RecA protein, the product of the recA gene. A human gene that shares homology with Escherichia coli recA (and its yeast homologue RAD51) has been cloned from a testis cDNA library, and its 37 kDa product (hRad51) purified to homogeneity. The human Rad51 protein binds to single- and double-stranded DNA and exhibits DNA-dependent ATPase activity. Using a topological assay, we demonstrate that hRad51 underwinds duplex DNA, in a reaction dependent upon the presence of ATP or its non-hydrolysable analogue ATP gamma S. Complexes formed with single- and double-stranded DNA have been 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 underwound and extended within the nucleoprotein complex. In contrast to yeast 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

Topics: Adenosine Triphosphatases/metabolism; Adenosine Triphosphate/metabolism; Cloning, Molecular; DNA Topoisomerases, Type I/metabolism; DNA, Complementary; DNA, Superhelical/chemistry; DNA, Superhelical/metabolism; DNA-Binding Proteins/genetics; DNA-Binding Proteins/isolation & purification; Escherichia coli/genetics; Escherichia coli/metabolism; Gene Library; Humans; Male; Rad51 Recombinase; Rec A Recombinases/metabolism; Rec A Recombinases/ultrastructure; Testis/metabolism
Year: 1994
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