Activities of human recombination protein Rad51

Abstract

Homologous pairing and strand exchange, which are catalyzed by Escherichia coli RecA protein, are central to homologous recombination. Homologs of this protein are found in eukaryotes; however, little has been reported on the recombinase activities of the mammalian homologs, including the human protein, denoted HsRad51. For the studies described here, we purified HsRad51 from E. coli. Although the activities of HsRad51 and RecA were qualitatively similar in the presence of ATP, there were also striking differences. The stoichiometry of binding to DNA and the rate of renaturation of complementary strands were similar for the two proteins, but rates of ATP hydrolysis, homologous pairing, and subsequent strand exchange promoted by HsRad51 were less than 1 ⁄10 those of RecA. In addition, HsRad51 bound ␥-thio-ATP and formed stable presynaptic complexes that promoted renaturation as rapidly as RecA, but the recombinant human protein catalyzed neither strand exchange nor homologous pairing of a single strand with duplex DNA in the presence of the ATP analog. By contrast, RecA promoted both of the latter reactions in control experiments. These observations suggest that among RecA-like proteins, HsRad51 may be a variant in which homologous pairing and strand exchange are more closely linked to the hydrolysis of ATP.

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).…”

“…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). We included an unlabeled ssDNA component, the 67-mer oligonucleotide 1, and the homologous 32 P-labeled duplex into the reaction mixtures.…”

“…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.…”

“…The eukaryotic counterpart of RecA, the Rad51 protein, was biochemically characterized both from yeast (Sung and Robberson, 1995) and from man (Baumann et al, 1996;Gupta et al, 1997Gupta et al, , 1999aBaumann and West, 1999). Like RecA also human Rad51 (hRad51) binds to single-and double-stranded DNAs (ssDNA and dsDNA), assembles cooperatively into helical nucleoprotein ®laments, synapses ssDNA with homologous dsDNA, and catalyzes strand exchange.…”

“…However, hRad51 hydrolyzes ATP and promotes strand exchange at least 10-fold less e ciently than RecA, so that the length of heteroduplex DNA formed is limited to 1.3 kb (Baumann and West, 1999). Rad51 fails to generate stable joint molecules in the absence of ATP, although it still binds cooperatively and with high a nity to DNA and can renature complementary DNA strands (Baumann et al, 1996;Gupta et al, 1997;Kelley De Zutter and Knight, 1999). Opposite to the polarity of the bacterial recombinase, hRad51 seems to preferentially start transfer at the 5' end of the linear duplex (Sung and Robberson, 1995;Baumann et al, 1996;West, 1997, 1999).…”