RAD51 and other members of the RecA family of strand exchange proteins assemble on ssDNA to form presynaptic filaments, which carry out the central steps of homologous recombination. A microplate-based assay was developed for high-throughput measurement of hRAD51 filament formation on ssDNA. With this method, a 10,000 compound library was screened, leading to the identification of a small molecule (RS-1) that enhances hRAD51 binding in a wide range of biochemical conditions. Salt titration experiments showed that RS-1 can enhance filament stability. Ultrastructural analysis of filaments formed on ssDNA showed that RS-1 can increase both protein-DNA complex lengths and the pitch of helical filament turns. RS-1 stimulated hRAD51-mediated homologous strand assimilation (D-loop) activity by at least 5-to 11-fold, depending on the condition. This D-loop stimulation occurred even in the presence of Ca 2؉ or adenylyl-imidodiphosphate, indicating that the mechanism of stimulation was distinct from that conferred by Ca 2؉ and/or inhibition of ATPase. No D-loop activity was observed in the absence of a nucleotide triphosphate cofactor, indicating that the compound does not substitute for this requirement. These results indicate that RS-1 enhances the homologous recombination activity of hRAD51 by promoting the formation of active presynaptic filaments. Cell survival assays in normal neonatal human dermal fibroblasts demonstrated that RS-1 promotes a dose-dependent resistance to the cross-linking chemotherapeutic drug cisplatin. Given that RAD51-dependent recombination is a major determinant of cisplatin resistance, RS-1 seems to function in vivo to stimulate homologous recombination repair proficiency. RS-1 has many potential applications in both research and medical settings.cross-linking chemotherapy ͉ DNA repair ͉ high-throughput screen ͉ recombinase ͉ strand exchange H omologous recombination (HR) has multiple roles in DNA repair, including the repair of double strand breaks (DSBs) and recovery from the replication-blocking lesions formed by DNA cross-linking agents. HR repairs DSBs by locating a homologous stretch of DNA and replicating the missing genetic information from this homologous template. In contrast to DSB repair by nonhomologous end joining, HR repair generally occurs without mutations. Because of this, HR repair is critically important in the maintenance of genomic stability (reviewed in ref. 1). The proposed mechanism for this pathway begins with 5Ј to 3Ј nuclease activity at the DSB, resulting in a 3Ј singlestranded tail. The tail is coated by replication protein A, which is subsequently replaced by a helical filament of RAD51 protein. This displacement of replication protein A by RAD51 seems to be controlled by a number of mediator proteins, which include BRCA2, RAD52, and RAD51 paralogue complexes (2-5). The RAD51-coated 3Ј tail then locates and invades a homologous template of dsDNA. After invasion, templated DNA synthesis initiated at 3Ј ends leads to formation of branched DNA intermediates, which ...

The eukaryotic RecA homologs Rad51 and Dmc1 are essential for strand exchange between homologous chromosomes during meiosis. All members of the RecA family of recombinases polymerize on DNA to form helical nucleoprotein filaments, which is the active form of the protein. Here we compare the filament structures of the Rad51 and Dmc1 proteins from both human and budding yeast. Previous studies of Dmc1 filaments suggested that they might be structurally distinct from filaments of other members of the RecA family, including Rad51. The data presented here indicate that Rad51 and Dmc1 filaments are essentially identical with respect to several structural parameters, including persistence length, helical pitch, filament diameter, DNA base pairs per helical turn and helical handedness. These data, together with previous studies demonstrating similar in vitro recombinase activity for Dmc1 and Rad51, support the view that differences in the meiotic function of Rad51 and Dmc1 are more likely to result from the influence of distinct sets of accessory proteins than from intrinsic differences in filament structure.

DNA strand exchange is the central process of homologous recombination. In budding yeast, this reaction is catalyzed by the recombinases Rad51 and Dmc1. Rad51 is responsible for recombinational repair of DNA damage during mitosis and is also important during meiotic recombination. Dmc1 is only expressed during meiosis and is required for meiotic recombination. The discovery that these two different recombinases are needed for normal levels of meiotic recombination in budding yeast raised the question of how the functions of these proteins relate to one another. The paper by Tsubouchi and Roeder (2006) in this issue of Genes & Development represents important progress toward what is apparently a rather complex answer. Tsubouchi and Roeder (2006) report discovery of a novel meiosis-specific protein, Hed1, which appears to inhibit Rad51 when Dmc1 is absent. The authors show mutation of the HED1 gene allows cells to bypass the meiotic arrest caused by a dmc1 mutation and resolve meiosis-specific double-strand breaks (DSBs) using Rad51. A hed1 mutation can also suppress the defects caused by mutation of HOP2, which codes for a Dmc1 accessory factor. Two-hybrid experiments show that Hed1 and Rad51 can interact directly and immuno-staining shows that subnuclear foci formed by Hed1 and Rad51 colocalize. Together these results imply that Hed1's influence on Rad51 activity is direct. Furthermore, expression of Hed1 in mitotic cells provided evidence that Hed1 expression can be sufficient to inhibit Rad51-dependent repair of mitotic DNA damage.A reasonable interpretation of these results is that the meiotic recombination machinery of budding yeast is regulated such that Dmc1 carries out most strand exchange. Tsubouchi and Roeder's (2006) results also add to evidence indicating that Rad51 is capable of replacing Dmc1's function under certain circumstances. Here, we place these new findings in the context of previous observations and present a model to explain how Rad51 and Dmc1 may cooperate to promote interhomolog recombination in budding yeast, while at the same time accounting for the observed functional redundancy. We further speculate as to how the functional relationship of Rad51 and Dmc1 may have evolved.The notion that Rad51 activity is blocked during meiosis is not new. Previous work showed that Dmc1-independent recombination is inhibited by proteins associated with axial elements (Schwacha and Kleckner 1997;Xu et al. 1997;Bishop et al. 1999;Niu et al. 2005) proteinaceous structures that organize pairs of sister chromatids into two parallel sets of loops by binding at their base. The assembly of the synaptonemal complex brings an axial element with its pair of sisters into close parallel alignment with another axial element holding the homologous sister pair. Three interacting axis-associated proteins-Red1, Hop1, and Mek1-regulate recombination by suppressing Dmc1-independent recombination and, in the case of Red1 and Hop1, by enhancing the efficiency of DSB formation in a locus-specific manner (Rockmill and Roeder...

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