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

Abstract

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.

Role of heteroduplex joints in the functional interactions between human Rad51 and wild-type p53

“…Given that Rad51 represents the initial strand transferase in eukaryotes (Benson et al, 1994;Sung and Robberson, 1995;Gupta et al, 1997) we studied strand exchange by human Rad51 (hRad51) in the presence or absence of human p53. Both hRad51 expressed in E. coli and p53 expressed in insect cells were puri®ed to homogeneity by chromatographic procedures (Benson et al, 1994;Mummenbrauer et al, 1996). As p53 binds to early recombination intermediates, we focused on the early phase of DNA exchange by use of oligonucleotide substrates, similar to the assay system described by Gupta et al (1997).…”

“…In the analysis presented here, we carried out hRad51-dependent strand exchange reactions, to analyse functional interactions with p53. Short oligonucleotide substrate DNAs allowed us to concentrate on the initial steps of homology recognition and strand invasion, for which hRad51 is essential and su cient (Benson et al, 1994;Sung and Robberson, 1995;Gupta et al, 1997), and which create heteroduplex joints recognized by p53 (DudenhoÈ er et al, 1998(DudenhoÈ er et al, , 1999. Under these conditions the p53-associated exonuclease rapidly digested the input DNAs.…”

“…Overexpression and purification of proteins hRad51 hRad51 was overproduced from plasmid pFB530 (Benson et al, 1994) after transfection of the recA 7 E. coli strain BLR(DE3)pLysS (Novagen). Transgenic bacteria were cultured in 4 1 of Luria broth containing 50 mg/ml ampicillin, 25 mg/ml chloramphenicol, and 12.5 mg/ml tetracyclin.…”

BRCA2: a universal recombinase regulator

“…Recombinant RAD51 and DMC1 proteins form oligomeric ring structures of seven or eight monomers, respectively (Shin et al, 2003;Kinebuchi et al, 2004). However, they are functionally active when associated with DNA in the form of a highly ordered right-handed helical nucleoprotein filament (Figure 3b) (Benson et al, 1994;Conway et al, 2004;Sehorn et al, 2004). It is within this nucleoprotein filament that DNA interactions take place between homologous sequences.…”

Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

“…Subsequently, RAD51 is recruited to the DNA overhang to form a right‐handed helical nucleoprotein filament (NPF) in an ATP‐dependent manner (Bianco et al , 1998). The NPF is responsible for DNA sequence homology recognition in a duplex DNA, usually the sister chromatid, and formation of a joint intermediate that will serve as a priming site for DNA synthesis needed to copy the missing information (Benson et al , 1994). In the cell, HR is tightly regulated, for example by the tumour‐suppressor protein BRCA2 (Sung & Klein, 2006), which is known to mediate the loading of hRAD51 onto RPA‐coated ssDNA, making use of its ability to bind hRAD51 with its BRC‐repeat domain (Wong et al , 1997; Chen et al , 1998; Carreira et al , 2009).…”