Human Rad51 Protein Can Form Homologous Joints in the Absence of Net Strand Exchange

Abstract

The eukaryotic homologs of RecA protein are central enzymes of recombination and repair, and notwithstanding a high degree of conservation they differ sufficiently from RecA to offer insights into mechanisms and biological roles. The yield of DNA strand exchange reactions driven by both Escherichia coli RecA protein and its human homolog HsRad51 protein was inversely related to the GC content of oligonucleotide substrates, but at any given GC composition, HsRad51 promoted less exchange than RecA. When 40% of bases were GC pairs, the rate constant for strand exchange by HsRad51 was unmeasurable, whereas the rate constants for homologous pairing were unaltered relative to more AT-rich DNA. The ability of HsRad51 to form joints in the absence of net strand exchange was confirmed by experiments in which heterologous blocks at both ends of linear duplex oligonucleotides produced joints that instantly dissociated upon deproteinization. These findings suggest that HsRad51 acting alone on human DNA in vivo is a pairing protein that cannot form extensive heteroduplex DNA.Human Rad51 protein (HsRad51) is a member of the universally distributed class of RecA proteins that play important roles in homologous recombination and recombinational repair (1). In prokaryotes, the RecA proteins play roles in recombination, postreplication repair, and the repair of double-strand breaks (2). In eukaryotes, members of this class play roles in meiotic recombination, double-strand break repair (3, 4), and possibly immunoglobulin switch recombination (5). In the mouse, the requirement of Rad51 for embryonic survival (6, 7) reveals a vital role that has not been found in prokaryotes and lower eukaryotes.RecA protein from Escherichia coli promotes a search for homology by a single strand of DNA, and initiates an exchange between that strand and homologous duplex DNA. To carry out those complicated interactions, RecA protein forms a helical nucleoprotein filament on single-stranded DNA; and Rad51 from Saccharomyces cerevisiae and Homo sapiens form nucleoprotein filaments that resemble the one formed by RecA (8,9). Once the nucleoprotein filament has been formed, RecA requires no cofactors other than ATP to promote a rapid search for homology and an extensive strand exchange. Yeast, frog, and human Rad51, as well as human Dmc1, a homolog that is specifically expressed in meiosis, are DNA-dependent ATPases that carry out homologous pairing and strand exchange reactions that resemble those catalyzed by RecA protein (10 -14). However, the eukaryotic homologs differ from RecA in several notable respects. None of the eukaryotic enzymes appears to manifest the kinetic barrier to binding to duplex DNA (15), which in the case of RecA favors the loading of protein on single-stranded DNA; all hydrolyze ATP at a rate that is at least an order of magnitude lower than hydrolysis by RecA, and all promote recombination reactions much more slowly than RecA (10,11,12,14). Observations on human Rad51 showed that both phases of the recombination reaction, homol...

The synaptic activity of HsDmc1, a human recombination protein specific to meiosis

“…The results described here and previously (20,31,35) provide insight into functional differences between E. coli RecA protein and several of its eukaryotic homologs. It was immediately apparent in various studies that eukaryotic homologs of RecA hydrolyze ATP at rates that are between one and two orders of magnitude slower (14,27,35,36).…”

“…2 A). However, we have observed previously that deproteinization diminishes part of the signal generated in the pairing assay but has no effect on the signal generated in the assay for strand exchange, which we have taken as evidence of dissociation of synaptic complexes (31).…”

“…Previous studies of Rad51, by using FRET assays, produced evidence that a three-stranded synaptic intermediate exists in which stable homologous recognition is accomplished by the partial switching of base pairs (20). As might be expected, deproteinization of this complex leads in some measure to the regeneration of substrates, whereas deproteinization of the duplex product of the reaction has no effect (31). Similarly, the failure of deproteinization to reverse strand exchange promoted by Dmc1 argues that reversal of the pairing assay is because of the presence of synaptic intermediate which that assay detects.…”

“…The His-tagged Dmc1 protein hydrolyzed ATP in a DNA-dependent manner with a catalytic rate constant (k cat ) of 0.7 mols͞mol͞min, similar to the k cat for nontagged protein, described before (27). In previous studies, we have used assays based on FRET to distinguish and kinetically characterize two phases of recombination reactions catalyzed by members of the RecA family of proteins (20,30,31). These phases are the recognition of homology and the exchange of strands.…”

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

“…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.…”