Politics and science furnish many examples of the dramatically different effects of competition and collaboration. Similar phenomena occur at the molecular level in nature, and four reports1,2,3,4 (three of them in this issue2,3,4, beginning on page 401) demonstrate the point. They show that competition between two proteins required for genetic recombination is turned into fruitful collaboration by a third participant, the Rad52 protein.
Genetic recombination, the exchange of information between DNA chains, accomplishes two seemingly conflicting tasks — generation of genetic diversity within species, and maintenance of genetic stability by repairing DNA damage. Whether recombination results in diversity or stability depends on whether the exchanges occur between homologous chromosomes during meiosis or between identical sister chromatids. Clinically, genetic recombination has attracted attention because of possible cross-talk between the breast-cancer-susceptibility genes, BRCA1 and BRCA2, and the recombination machinery5; biologically, its importance is underscored by the conservation of its salient features from fungi to humans.
At the core of recombination is the search for homologous DNA followed by exchange of DNA strands (Fig. 1). A common initiator is a DNA double-strand break that is processed to expose regions of single-stranded DNA (ssDNA). In eukaryotes, the Rad51 protein coats the ssDNA to form a filament that scans the genome for a homologous double-stranded DNA (dsDNA) sequence (in a mammalian nucleus, which contains about 6×109 base pairs, this must be an especially arduous task). On completing this quest, the ssDNA-containing filament and the intact dsDNA form a joint molecule before strand exchange can occur.
But how, in vivo, does the filament assemble despite being subject to many competing reactions? An ssDNA-binding protein known as RPA (replication protein A) is required, presumably to remove secondary structure from the ssDNA to allow for efficient filament formation by Rad51, but it also competes with Rad51 for ssDNA binding. And although dsDNA is a substrate for the reaction, it binds Rad51, thereby inhibiting filament formation. From genetic studies it was clear that another protein, Rad52, was a major player in these events, but unlike Rad51 and RPA its molecular function remained elusive. The new, biochemical, studies reveal the effect of Rad52.
Three of the reports1,2,3 deal with the budding yeast Saccharomyces cerevisiae. They show that although RPA is required for Rad51-promoted strand exchange, it inhibits exchange when incubated simultaneously with Rad51 and ssDNA. Inhibition is overcome when Rad52 is incubated together with Rad51, RPA and ssDNA, followed by the addition of homologous dsDNA.
Rad52 is ideally suited for this job as mediator between Rad51 and RPA, because it interacts with both proteins1,6 and binds ssDNA4,7. The mechanism is not yet clear, but Rad52 could function in several, not mutually exclusive, ways. First, it could increase the cooperativity of Rad51 binding to ssDNA. Second, it could enhance the dissociation of RPA from ssDNA and promote RPA transfer to the displaced strand, preventing reversion of strand exchange. Third, the ability of Rad52 to increase the annealing rate of complementary ssDNAs (ref. 7) could help Rad51 initiate joint molecule formation.
The other report4 in this issue concerns human Rad52. The authors show that joint molecule formation does not occur when human Rad51 is present at subsaturating amounts. But when ssDNA is preincubated with human Rad52, followed by the sequential addition of subsaturating amounts of human Rad51 and dsDNA, joint molecule formation proceeds efficiently. These observations support the idea that human Rad52 increases the cooperativity of human Rad51 binding to ssDNA, thereby converting discontinuous stretches of Rad51 on ssDNA into one functional contiguous filament.
These new results reveal an interesting parallel between eukaryotic and prokaryotic genetic recombination. Bacteriophage T4 also uses a mediator, the UvsY protein, to stimulate strand exchange by UvsX and the T4 ssDNA-binding protein (see Table 1)8. The requirement for a mediator, however, is not universal because the Escherichia coli RecA protein, the bacterial homologue of Rad51, does not need one. So even though strand-exchange proteins are structurally and functionally highly conserved, the details of their actions differ (see Table 1). Yeast and human Rad51, and T4 UvsX, have no clear preference for binding to ssDNA or dsDNA, whereas E. coli RecA strongly prefers ssDNA. This property can eliminate competition on two fronts — RecA can itself displace the E. coli ssDNA-binding protein without the help of a mediator, and can overcome the competition by dsDNA for filament formation.
The ramifications of the four papers1,2,3,4 go further, for S. cerevisiae contains a second RAD52 homologue, RAD59, which is required for Rad51-independent recombination between intra-chromosomal inverted repeats9. If RAD52 homologues also exist in other species, it would help explain the dramatically different effects of RAD52 mutations in S. cerevisiae, the fission yeast Schizosaccharomyces pombe and mouse cells. Although the efficiency of recombination is reduced by more than three orders of magnitude in the S. cerevisiae RAD52 mutant, it is only twofold lower in the corresponding S. pombe mutant10, and only slightly affected in mouse RAD52 knockout embryonic stem cells (T. Rijkers and A. Pastink, personal communication).
The explanation could be that some functions of S. cerevisiae Rad52 can also be assumed by Rad59 in S. pombe and mammals. Another possibility is that different Rad52 homologues act as mediators for different Rad51 homologues, for eukaryotes express multiple versions of RAD51. In S. cerevisiae, these variations on the Rad51 theme appear to assist Rad51-promoted strand exchange or to be involved in meiosis-specific processes (see Table 1). In mammals their functions are less clear. But, among other things11, they could be important at the filament ends, which might have different properties to those of the filament body; in this respect, their role would be somewhat analogous to that of the E. coli RecF and RecR proteins in post-replication repair12.
The new reports1,2,3,4 show that mediation by Rad52 is required during the critical, early steps in genetic recombination. But another question remains — because Rad51 binds with similar efficiencies to ssDNA and dsDNA, how is the inhibitory effect of dsDNA on filament formation overcome? The answer might lie in the requirement for additional protein factors. A candidate is Rad54, whose function is still unknown, but it interacts with Rad51 and genetically it is in the same epistasis group as Rad51 and Rad52. Moreover, there must be much more to come in the Rad52 story because it also features in Rad51-independent recombination.
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