The Saccharomyces cerevisiae RAD51 and RAD54 genes are both required for the occurrence of homologous recombination and for the repair of double-stranded DNA breaks. Previous studies have indicated that Rad51 protein, together with the single-stranded DNA-binding factor replication protein A (RPA), can promote the formation of heteroduplex DNA, which is a key intermediate in homologous recombination. Here we report the purification of the Rad54 protein to near homogeneity and the biochemical testing of its molecular function. We find that Rad54 protein possesses a double-stranded DNA-dependent ATPase activity, and that it interacts with the Rad51 protein. Addition of Rad54 protein to reactions containing Rad51 strongly stimulates the rate of pairing between homologous single-stranded and double-stranded DNA molecules. We conclude that Rad54 acts to overcome kinetic impediments that would limit homologous DNA pairing between recombining chromosomes in vivo.

Recognition of modified histones by “reader” proteins plays a critical role in the regulation of chromatin1. H3K36 trimethylation (H3K36me3) is deposited onto the nucleosomes in the transcribed regions following RNA polymerase II (Pol II) elongation. In yeast, this mark in turn recruits epigenetic regulators to reset the chromatin to a relatively repressive state thus suppressing cryptic transcription2. However, much less is known about the role of H3K36me3 in transcription regulation in mammals. This is further complicated by the transcription-coupled incorporation of the histone variant H3.3 in gene bodies3. Here we show that the candidate tumor suppressor ZMYND11 specifically recognizes H3K36me3 on H3.3 (H3.3K36me3) and regulates Pol II elongation. Structural studies reveal that in addition to the trimethyl-lysine binding by an aromatic cage within the PWWP domain, the H3.3-dependent recognition is mediated by the encapsulation of the H3.3-specific “Ser31” residue in a composite pocket formed by the tandem bromo-PWWP domains of ZMYND11. ChIP-sequencing analyses reveal a genome-wide colocalization of ZMYND11 with H3K36me3 and H3.3 in gene bodies, and its occupancy requires the pre-deposition of H3.3K36me3. Although ZMYND11 is associated with highly expressed genes, it functions as an unconventional transcription corepressor via modulating Pol II at the elongation stage. ZMYND11 is critical for the repression of a transcriptional program that is essential for tumor cell growth; low expression level of ZMYND11 in breast cancer patients correlates with worse prognosis. Consistently, overexpression of ZMYND11 suppresses cancer cell growth in vitro and tumor formation in mice. Together, this study identifies ZMYND11 as an H3.3-specific reader of H3K36me3 that links the histone variant-mediated transcription elongation control to tumor suppression.

Yeast Rad51 recombinase has only minimal ability to form D loop. Addition of Rad54 renders D loop formation by Rad51 efficient, even when topologically relaxed DNA is used as substrate. Treatment of the nucleoprotein complex of Rad54 and relaxed DNA with topoisomerases reveals dynamic DNA remodeling to generate unconstrained negative and positive supercoils. DNA remodeling requires ATP hydrolysis by Rad54 and is stimulated by Rad51-DNA nucleoprotein complex. A marked sensitivity of DNA undergoing remodeling to P1 nuclease indicates that the negative supercoils produced lead to transient DNA strand separation. Thus, a specific interaction of Rad54 with the Rad51-ssDNA complex enhances the ability of the former to remodel DNA and allows the latter to harvest the negative supercoils generated for DNA joint formation.

Saccharomyces cerevisiae RAD51 gene is required for genetic recombination and recombinational repair of DNA strand breaks. Rad51 protein has a DNA-dependent ATPase activity, and it catalyzes ATP-dependent pairing and strand exchange between homologous DNA molecules. We show here that the rad51 Arg-191 protein, which is devoid of ATPase activity, mediates the pairing and strand exchange reaction upon binding ATP. In addition, the wild type Rad51 protein can catalyze pairing and strand exchange in the presence of the nonhydrolyzable ATP analogues adenylyl-imidodiphosphate and adenosine 5-O-thiotriphosphate. Thus, homologous pairing and the unidirectional transfer of greater than 5 kilobases of DNA can occur efficiently without the need for nucleotide hydrolysis. Consistent with the results from the biochemical analyses, expression of the rad51 Arg-191 protein in a rad51 null mutant confers normal cellular resistance to the DNA damaging agent methylmethane sulfonate, suggesting that nucleotide binding by Rad51 is sufficient for biological function.Saccharomyces cerevisiae RAD51 gene is a member of the RAD52 epistasis group that is required for meiotic and mitotic recombination and for the recombinational repair of DNA double-stranded breaks (reviewed in Ref. 1). The RAD51 structure and function have been conserved to a remarkable degree among eukaryotes including humans. The RAD51 encoded product and its counterparts from other eukaryotes are structurally and functionally related to the Escherichia coli recombination protein RecA (reviewed in Ref. 2).A number of biochemical activities have been identified in RecA protein including a DNA-dependent ATPase activity and an ability to catalyze the formation of heteroduplex DNA between homologous ssDNA 1 and dsDNA molecules in a reaction termed homologous pairing and strand exchange (reviewed in Refs. 2-5). In the presence of ATP, RecA polymerizes on both ssDNA and dsDNA to form nucleoprotein filaments in which the DNA is stretched to Ϸ150% the length of uncoated DNA. A substantial body of evidence indicates that pairing and strand exchange occur within the confines of the RecA-ssDNA nucleoprotein filament, with the formation of heteroduplex DNA initiating from the 3Ј end of the DNA strand in the duplex molecule that is complementary to the ssDNA bound in the RecA filament (2-5). A central but as yet unresolved question concerns the molecular role of ATP in the RecA catalyzed pairing and strand exchange reactions (see "Discussion"). Earlier work on RecA protein had suggested that although homologous pairing occurs efficiently in the presence of the nonhydrolyzable analogue ATP␥S (6, 7), the branch migration or strand exchange phase of the reaction requires continuous ATP hydrolysis (6). More recently, it was found that under certain reaction conditions, nucleotide binding alone can in fact enable RecA protein to carry out a limited amount of strand exchange (8 -10). The function of ATP hydrolysis in RecAmediated strand exchange remains a subject of intense debate (see "D...

Human Rad51 (hRad51), a member of a conserved family of general recombinases, is shown here to have an avid capability to make DNA joints between homologous DNA molecules and promote highly efficient DNA strand exchange of the paired molecules over at least 5.4 kilobase pairs. Furthermore, maximal efficiency of homologous DNA pairing and strand exchange is strongly dependent on the heterotrimeric single-stranded DNA binding factor hRPA and requires conditions that lessen interactions of the homologous duplex with the hRad51-single-stranded DNA nucleoprotein filament. The homologous DNA pairing and strand exchange system described should be valuable for dissecting the action mechanism of hRad51 and for deciphering its functional interactions with other recombination factors.Genetic studies in various eukaryotic organisms have indicated that homologous recombination processes are mediated by a group of evolutionarily conserved genes known as the RAD52 epistasis group. As revealed in studies on meiotic recombination and mating type switching in Saccharomyces cerevisiae, DNA double-strand breaks are formed and then processed exonucleolytically to yield long single-stranded tails with a 3Ј extremity. Nucleation of various RAD52 group proteins onto these ssDNA 1 tails renders them recombinogenic, leading to the search for a homologous DNA target (sister chromatid or homologous chromosome), formation of DNA joints with the target, and an exchange of genetic information with it. The repair by recombination of DNA double-strand breaks induced by ionizing radiation and other DNA damaging agents very likely follows the same mechanistic route, as it too is dependent on genes of the RAD52 epistasis group (reviewed in Refs. 1 and 2).Among members of the RAD52 group, the RAD51-encoded product is of particular interest because of its structural and functional similarities to the Escherichia coli recombination protein RecA (2-5). RecA promotes the pairing and strand exchange between homologous DNA molecules to form heteroduplex DNA (4, 5), an enzymatic activity believed to be germane for the central role of RecA in recombination and DNA repair processes. Likewise, homologous DNA pairing and strand exchange activities have been shown for S. cerevisiae Rad51 (yRad51) (6). Under optimized conditions, the length of heteroduplex DNA joints formed by yRad51 and RecA can extend over quite a few kilobase pairs (4, 5, 7).In published studies, human Rad51 (hRad51) was found to have the ability to make DNA joints but the maximal potential for forming only about 1 kilobase pairs of heteroduplex DNA (8 -11). Furthermore, while yRad51 and RecA require their cognate single-strand DNA binding factors, SSB and yRPA, for optimal recombinase activity, hRPA has been suggested to stimulate the hRad51-mediated homologous pairing and strand exchange reaction only when the hRad51 concentration is suboptimal (9, 10).Given the central role of hRad51 in recombination processes and the fact that the activities of hRad51 are apparently subject to modulation...