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RAD51 assembly on single-stranded (ss)DNAs is a crucial step in the homology-dependent repair of DNA damage for genomic stability. The formation of the RAD51 filament is promoted by various RAD51-interacting proteins including RAD51 paralogues. However, the mechanisms underlying the differential control of RAD51-filament dynamics by these factors remain largely unknown. Here, we report a role for the human RAD51 paralogue, SWSAP1, as a novel regulator of RAD51 assembly. Swsap1-deficient cells show defects in DNA damage-induced RAD51 assembly during both mitosis and meiosis. Defective RAD51 assembly in SWSAP1-depleted cells is suppressed by the depletion of FIGNL1, which binds to RAD51 as well as SWSAP1. Purified FIGNL1 promotes the dissociation of RAD51 from ssDNAs. The dismantling activity of FIGNL1 does not require its ATPase but depends on RAD51-binding. Purified SWSAP1 inhibits the RAD51-dismantling activity of FIGNL1. Taken together, our data suggest that SWSAP1 protects RAD51 filaments by antagonizing the anti-recombinase, FIGNL1.
The RAD51 recombinase plays critical roles in safeguarding genome integrity, which is fundamentally important for all living cells. While interphase functions of RAD51 in maintaining genome stability are well-characterised, its role in mitosis remains contentious. In this study, we show that RAD51 protects under-replicated DNA in mitotic human cells and, in this way, promotes mitotic DNA synthesis (MiDAS) and successful chromosome segregation. In cells experiencing mild replication stress, MiDAS was detected irrespective of mitotically generated DNA damage. MiDAS broadly required de novo RAD51 recruitment to single-stranded DNA, which was supported by the phosphorylation of RAD51 by the key mitotic regulator Polo-like kinase 1. Importantly, acute inhibition of MiDAS delayed anaphase onset and induced centromere fragility, suggesting a mechanism that prevents the satisfaction of the spindle assembly checkpoint while chromosomal replication remains incomplete. This study hence identifies an unexpected function of RAD51 in promoting genomic stability in mitosis.
Homologous recombination mediated by RAD51 recombinase helps eliminate chromosomal lesions, such as DNA double-strand breaks induced by radiation or arising from injured DNA replication forks. The tumor suppressors BRCA2 and PALB2 act together to deliver RAD51 to chromosomal lesions to initiate repair. Here we document a new function of PALB2: to enhance RAD51's ability to form the D loop. We show that PALB2 binds DNA and physically interacts with RAD51. Notably, although PALB2 alone stimulates D-loop formation, it has a cooperative effect with RAD51AP1, an enhancer of RAD51. This stimulation stems from the ability of PALB2 to function with RAD51 and RAD51AP1 to assemble the synaptic complex. Our results demonstrate the multifaceted role of PALB2 in chromosome damage repair. Because PALB2 mutations can cause cancer or Fanconi anemia, our findings shed light on the mechanism of tumor suppression in humans.
The protozoan parasite responsible for human amoebiasis is Entamoeba histolytica. An important facet of the life cycle of E. histolytica involves the conversion of the mature trophozoite to a cyst. This transition is thought to involve homologous recombination (HR), which is dependent upon the Rad51 recombinase. Here, a biochemical characterization of highly purified ehRad51 protein is presented. The ehRad51 protein preferentially binds ssDNA, forms a presynaptic filament and possesses ATP hydrolysis activity that is stimulated by the presence of DNA. Evidence is provided that ehRad51 catalyzes robust DNA strand exchange over at least 5.4 kilobase pairs. Although the homologous DNA pairing activity of ehRad51 is weak, it is strongly enhanced by the presence of two HR accessory cofactors, calcium and Hop2-Mnd1. The biochemical system described herein was used to demonstrate the potential for targeting ehRad51 with two small molecule inhibitors of human RAD51. We show that 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited ehRad51 by interfering with DNA binding and attenuated encystation in Entamoeba invadens, while B02 had no effect on ehRad51 strand exchange activity. These results provide insight into the underlying mechanism of homology-directed DNA repair in E. histolytica.
Homologous recombination (HR) provides an efficient mechanism for error-free repair of DNA double-strand breaks (DSBs). However, HR can be also harmful as inappropriate or untimely HR events can give rise to lethal recombination intermediates and chromosome rearrangements. A critical step of HR is the formation of a RAD51 filament on single-stranded (ss)DNA, which mediates the invasion of a homologous DNA molecule. In mammalian cells, several DNA helicases have been implicated in the regulation of this process. RECQ5, a member of the RecQ family of DNA helicases, interacts physically with the RAD51 recombinase and disrupts RAD51 presynaptic filaments in a reaction dependent on ATP hydrolysis. Here, we have precisely mapped the RAD51-interacting domain of RECQ5 and generated mutants that fail to interact with RAD51. We show that although these mutants retain normal ATPase activity, they are impaired in their ability to displace RAD51 from ssDNA. Moreover, we show that ablation of RECQ5-RAD51 complex formation by a point mutation alleviates the inhibitory effect of RECQ5 on HR-mediated DSB repair. These findings provide support for the proposal that interaction with RAD51 is critical for the anti-recombinase attribute of RECQ5.
Osteoporosis is a metabolic bone disease that significantly affects the quality of life and can even lead to death. In this study, we aimed to investigate the role of RAD51 recombinase (RAD51) in osteoblast and osteoclast differentiation. We analyzed differentially expressed genes using microarray analysis. The osteogenic differentiation capability was analyzed by alkaline phosphatase (ALP) staining and alizarin red staining assays. Osteogenesis and osteoclast related genes expression was detected using quantitative real-time PCR (qPCR) and Western blotting. The phosphorylation of Ataxia-telangiectasia mutated (ATM) and ATR serine/threonine kinase (ATR) was tested using Western blotting. The effect of RAD51 on osteoporosis was also explored in vivo. The results showed that RAD51 was downregulated in osteoporosis, but upregulated in differentiated osteoblasts. Overexpression of RAD51 enhanced the differentiation of osteoblasts and suppressed the formation of osteoclasts. Furthermore, p-ATM and p-ATR levels were upregulated in osteoblasts and downregulated in osteoclasts. RAD51 expression was reduced by the ATM/ATR pathway inhibitor AZ20. AZ20 treatment inhibited osteoblastogenesis and promoted osteoclastogenesis, whereas RAD51 reversed the effects induced by AZ20. Moreover, RAD51 improved bone microarchitecture in vivo. Taken together, ATM/ATR signaling-mediated RAD51 promoted osteogenic differentiation and suppressed osteoclastogenesis. These findings reveal a critical role for RAD51 in osteoporosis.
The homologous recombination (HR) pathway has been implicated as the predominant mechanism for the repair of chromosomal DNA double-strand breaks (DSBs) of the malarial parasite. Although the extrachromosomal mitochondrial genome of this parasite experiences a greater number of DSBs due to its close proximity to the electron transport chain, nothing is known about the proteins involved in the repair of the mitochondrial genome. We investigated the involvement of nucleus-encoded HR proteins in the repair of the mitochondrial genome, as this genome does not code for any DNA repair proteins. Here, we provide evidence that the nucleus-encoded "recombinosome" of the parasite is also involved in mitochondrial genome repair. First, two crucial HR proteins, namely, Plasmodium falciparum Rad51 (PfRad51) and P. falciparum Bloom helicase (PfBlm) are located in the mitochondria. They are recruited to the mitochondrial genome at the schizont stage, a stage that is prone to DSBs due to exposure to various endogenous and physiologic DNA-damaging agents. Second, the recruitment of these two proteins to the damaged mitochondrial genome coincides with the DNA repair kinetics. Moreover, both the proteins exit the mitochondrial DNA (mtDNA) once the genome is repaired. Most importantly, the specific chemical inhibitors of PfRad51 and PfBlm block the repair of UV-induced DSBs of the mitochondrial genome. Additionally, overexpression of these two proteins resulted in a kinetically faster repair. Given the essentiality of the mitochondrial genome, blocking its repair by inhibiting the HR pathway could offer a novel strategy for curbing malaria. IMPORTANCE The impact of malaria on global public health and the world economy continues to surge despite decades of vaccine research and drug development efforts. An alarming rise in resistance toward all the commercially available antimalarial drugs and the lack of an effective malaria vaccine brings us to the urge to identify novel intervention strategies for curbing malaria. Here, we uncover the molecular mechanism behind the repair of the most deleterious form of DNA lesions on the parasitic mitochondrial genome. Given that the single-copy mitochondrion is an indispensable organelle of the malaria parasite, we propose that targeting the mitochondrial DNA repair pathways should be exploited as a potential malaria control strategy. The establishment of the parasitic homologous recombination machinery as the predominant repair mechanism of the mitochondrial DNA double-strand breaks underscores the importance of this pathway as a novel druggable target.
Detection of genetic exchange has been a limiting factor to deepen the knowledge on the mechanisms by which Trypanosoma cruzi is able to generate progeny and genetic diversity. Here we show that incorporation of halogenated thymidine analogues, followed by immunostaining, is a reliable method not only to detect T. cruzi fused-cell hybrids, but also to quantify their percentage in populations of this parasite. Through this approach, we were able to detect and quantify fused-cell hybrids of T. cruzi clones CL Brener and Y. Given the increased detection of fused-cell hybrids in naturally-occurring hybrid CL Brener strain, which displays increased levels of RAD51 and BRCA2 transcripts, we further investigated the role of Rad51 - a recombinase involved in homologous recombination - in the process of genetic exchange. We also verified that the detection of fused-cell hybrids in T. cruzi overexpressing RAD51 is increased when compared to wild-type cells, suggesting a key role for Rad51 either in the formation or in the stabilization of fused-cell hybrids in this organism.
The budding yeast Srs2 is the archetype of helicases that regulate several aspects of homologous recombination (HR) to maintain genomic stability. Srs2 inhibits HR at replication forks and prevents high frequencies of crossing-over. Additionally, sensitivity to DNA damage and synthetic lethality with replication and recombination mutants are phenotypes that can only be attributed to another role of Srs2: the elimination of lethal intermediates formed by recombination proteins. To shed light on these intermediates, we searched for mutations that bypass the requirement of Srs2 in DNA repair without affecting HR. Remarkably, we isolated rad52-L264P, a novel allele of RAD52, a gene that encodes one of the most central recombination proteins in yeast. This mutation suppresses a broad spectrum of srs2Δ phenotypes in haploid cells, such as UV and γ-ray sensitivities as well as synthetic lethality with replication and recombination mutants, while it does not significantly affect Rad52 functions in HR and DNA repair. Extensive analysis of the genetic interactions between rad52-L264P and srs2Δ shows that rad52-L264P bypasses the requirement for Srs2 specifically for the prevention of toxic Rad51 filaments. Conversely, this Rad52 mutant cannot restore viability of srs2Δ cells that accumulate intertwined recombination intermediates which are normally processed by Srs2 post-synaptic functions. The avoidance of toxic Rad51 filaments by Rad52-L264P can be explained by a modification of its Rad51 filament mediator activity, as indicated by Chromatin immunoprecipitation and biochemical analysis. Remarkably, sensitivity to DNA damage of srs2Δ cells can also be overcome by stimulating Rad52 sumoylation through overexpression of the sumo-ligase SIZ2, or by replacing Rad52 by a Rad52-SUMO fusion protein. We propose that, like the rad52-L264P mutation, sumoylation modifies Rad52 activity thereby changing the properties of Rad51 filaments. This conclusion is strengthened by the finding that Rad52 is often associated with complete Rad51 filaments in vitro.
RecA-family recombinase-catalyzed ATP-dependent homologous joint formation is critical for homologous recombination, in which RecA or Rad51 binds first to single-stranded (ss)DNA and then interacts with double-stranded (ds)DNA. However, when RecA or Rad51 interacts with dsDNA before binding to ssDNA, the homologous joint-forming activity of RecA or Rad51 is quickly suppressed. We found that under these and adenosine diphosphate (ADP)-generating suppressive conditions for the recombinase activity, RecA or Rad51 at similar optimal concentrations enhances the DNA ligase-catalyzed dsDNA end-joining (DNA ligation) about 30- to 40-fold. The DNA ligation enhancement by RecA or Rad51 transforms most of the substrate DNA into multimers within 2-5 min, and for this enhancement, ADP is the common and best cofactor. Adenosine triphosphate (ATP) is effective for RecA, but not for Rad51. Rad51/RecA-enhanced DNA ligation depends on dsDNA-binding, as shown by a mutant, and is independent of physical interactions with the DNA ligase. These observations demonstrate the common and unique activities of RecA and Rad51 to juxtapose dsDNA-ends in preparation for covalent joining by a DNA ligase. This new in vitro function of Rad51 provides a simple explanation for our genetic observation that Rad51 plays a role in the fidelity of the end-joining of a reporter plasmid DNA, by yeast canonical non-homologous end-joining (NHEJ) in vivo.
Human RAD51 protein catalyzes DNA pairing and strand exchange reactions that are central to homologous recombination and homology-directed DNA repair. Successful recombination/repair requires the formation of a presynaptic filament of RAD51 on ssDNA. Mutations in BRCA2 and other proteins that control RAD51 activity are associated with human cancer. Here we describe a set of mutations associated with human breast tumors that occur in a common structural motif of RAD51. Tumor-associated D149N, R150Q and G151D mutations map to a Schellman loop motif located on the surface of the RecA homology domain of RAD51. All three variants are proficient in DNA strand exchange, but G151D is slightly more sensitive to salt than wild-type (WT). Both G151D and R150Q exhibit markedly lower catalytic efficiency for adenosine triphosphate hydrolysis compared to WT. All three mutations alter the physical properties of RAD51 nucleoprotein filaments, with G151D showing the most dramatic changes. G151D forms mixed nucleoprotein filaments with WT RAD51 that have intermediate properties compared to unmixed filaments. These findings raise the possibility that mutations in RAD51 itself may contribute to genome instability in tumor cells, either directly through changes in recombinase properties, or indirectly through changes in interactions with regulatory proteins.
FANCD2 protein, a key coordinator and effector of the interstrand crosslink repair pathway, is also required to prevent excessive nascent strand degradation at hydroxyurea-induced stalled forks. The RAD51 recombinase has also been implicated in regulation of resection at stalled replication forks. The mechanistic contributions of these proteins to fork protection are not well understood. Here, we used purified FANCD2 and RAD51 to study how each protein regulates DNA resection at stalled forks. We characterized three mechanisms of FANCD2-mediated fork protection: (1) The N-terminal domain of FANCD2 inhibits the essential DNA2 nuclease activity by directly binding to DNA2 accounting for over-resection in FANCD2 defective cells. (2) Independent of dimerization with FANCI, FANCD2 itself stabilizes RAD51 filaments to inhibit multiple nucleases, including DNA2, MRE11 and EXO1. (3) Unexpectedly, we uncovered a new FANCD2 function: by stabilizing RAD51 filaments, FANCD2 acts to stimulate the strand exchange activity of RAD51. Our work biochemically explains non-canonical mechanisms by which FANCD2 and RAD51 protect stalled forks. We propose a model in which the strand exchange activity of FANCD2 provides a simple molecular explanation for genetic interactions between FANCD2 and BRCA2 in the FA/BRCA fork protection pathway.
The breast and ovarian cancer suppressor protein BRCA2 controls the RAD51 recombinase in reactions that lead to homologous DNA recombination (HDR). BRCA2 binds RAD51 via eight conserved BRC repeat motifs of approximately 35 amino acids, each with a varying capacity to bind RAD51. BRC repeats both promote and inhibit RAD51 assembly on different DNA substrates to regulate HDR, but the structural basis for these functions is unclear. Here, we demarcate two tetrameric clusters of hydrophobic residues in the BRC repeats, interacting with distinct pockets in RAD51, and show that the co-location of both modules within a single BRC repeat is necessary for BRC-RAD51 binding and function. The two modules comprise the sequence FxxA, known to inhibit RAD51 assembly by blocking the oligomerization interface, and a previously unrecognized tetramer with the consensus sequence LFDE, which binds to a RAD51 pocket distinct from this interface. The LFDE motif is essential in BRC repeats for modes of RAD51 binding both permissive and inhibitory to RAD51 oligomerization. Targeted insertion of point mutations in RAD51 that disrupt the LFDE-binding pocket impair its assembly at DNA damage sites in living cells. Our findings suggest a model for the modular architecture of BRC repeats that provides fresh insight into the mechanisms regulating homologous DNA recombination.
Rad51 protein promotes homologous recombination in eukaryotes. Recombination activities are activated by Rad51 filament assembly on ssDNA. Previous studies of yeast Rad51 showed that His352 occupies an important position at the filament interface, where it could relay signals between subunits and active sites. To investigate, we characterized yeast Rad51 H352A and H352Y mutants, and solved the structure of H352Y. H352A forms catalytically competent but salt-labile complexes on ssDNA. In contrast, H352Y forms salt-resistant complexes on ssDNA, but is defective in nucleotide exchange, RPA displacement and strand exchange with full-length DNA substrates. The 2.5 A crystal structure of H352Y reveals a right-handed helical filament in a high-pitch (130 A) conformation with P6(1) symmetry. The catalytic core and dimer interface regions of H352Y closely resemble those of DNA-bound Escherichia coli RecA protein. The H352Y mutation stabilizes Phe187 from the adjacent subunit in a position that interferes with the gamma-phosphate-binding site of the Walker A motif/P-loop, potentially explaining the limited catalysis observed. Comparison of Rad51 H352Y, RecA-DNA and related structures reveals that the presence of bound DNA correlates with the isomerization of a conserved cis peptide near Walker B to the trans configuration, which appears to prime the catalytic glutamate residue for ATP hydrolysis.
The Shu complex, which contains RAD51 paralogues, is involved in the decision between homologous recombination and error-prone repair. We discovered a link to ribosomal DNA (rDNA) recombination when we found an interaction between one member of the Shu complex, SHU1, and UAF30, a component of the upstream activating factor complex (UAF), which regulates rDNA transcription. In the absence of Uaf30, rDNA copy number increases, and this increase depends on several functional subunits of the Shu complex. Furthermore, in the absence of Uaf30, we find that Shu1 and Srs2, an anti-recombinase DNA helicase with which the Shu complex physically interacts, act in the same pathway regulating rDNA recombination. In addition, Shu1 modulates Srs2 recruitment to both induced and spontaneous foci correlating with a decrease in Rad51 foci, demonstrating that the Shu complex is an important regulator of Srs2 activity. Last, we show that Shu1 regulation of Srs2 to double-strand breaks is not restricted to the rDNA, indicating a more general function for the Shu complex in the regulation of Srs2. We propose that the Shu complex shifts the balance of repair toward Rad51 filament stabilization by inhibiting the disassembly reaction of Srs2.
In eukaryotes, DNA strand exchange is the central reaction of homologous recombination, which is promoted by Rad51 recombinases forming a right-handed nucleoprotein filament on single-stranded DNA, also known as a presynaptic filament. Accessory proteins known as recombination mediators are required for the formation of the active presynaptic filament. One such mediator in the fission yeast Schizosaccharomyces pombe is the Swi5-Sfr1 complex, which has been identified as an activator of Rad51 that assists in presynaptic filament formation and stimulates its strand exchange reaction. Here, we determined the 1:1 binding stoichiometry between the two subunits of the Swi5-Sfr1 complex using analytical ultracentrifugation and electrospray ionization mass spectrometry. Small-angle x-ray scattering experiments revealed that the Swi5-Sfr1 complex displays an extremely elongated dogleg-shaped structure in solution, which is consistent with its exceptionally high frictional ratio (f/f(0)) of 2.0 ± 0.2 obtained by analytical ultracentrifugation. Furthermore, we determined a rough topology of the complex by comparing the small-angle x-ray scattering-based structures of the Swi5-Sfr1 complex and four Swi5-Sfr1-Fab complexes, in which the Fab fragments of monoclonal antibodies were specifically bound to experimentally determined sites of Sfr1. We propose a model for how the Swi5-Sfr1 complex binds to the Rad51 filament, in which the Swi5-Sfr1 complex fits into the groove of the Rad51 filament, leading to an active and stable presynaptic filament.
The efficiency of the knock-in process is very important to successful gene editing in domestic animals. Recently, it was reported that transient loosening of the nucleosomal folding of transcriptionally inactive chromatin might have the potential to enhance homologous recombination efficiency. The objective of this study was to determine whether histone deacetylases (HDAC) inhibitor and RAD51 recombinase (RAD51) expression were associated with increased knock-in efficiency on the β-casein (bCSN2) gene locus in mammary alveolar-large T antigen (MAC-T) cells using the transcription activator-like effector nucleases (TALEN) system.
Present-day oncology sees at least two-thirds of cancer patients receiving radiation therapy as a part of their anticancer treatment. The objectives of the current study were to investigate the effects of the small molecule inhibitors of Wee1 kinase II (681641) and Rad51 (RI-1) on cell cycle progression, DNA double-strand breaks repair and apoptosis following ionizing radiation exposure in human leukemic T-cells Jurkat and MOLT-4. Pre-treatment with the Wee1 681641 or Rad51 RI-1 inhibitor alone increased the sensitivity of Jurkat cells to irradiation, however combining both inhibitors together resulted in a further enhancement of apoptosis. Jurkat cells pre-treated with inhibitors were positive for γH2AX foci 24h upon irradiation. MOLT-4 cells were less affected by inhibitors application prior to ionizing radiation exposure. Pre-treatment with Rad51 RI-1 had no effect on apoptosis induction; however Wee1 681641 increased ionizing radiation-induced cell death in MOLT-4 cells.
Fork reversal is a conserved mechanism to prevent stalled replication forks from collapsing. Formation and protection of reversed forks are two crucial steps in ensuring fork integrity and stability. Five RAD51 paralogs, namely, RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, which share sequence and structural similarity to the recombinase RAD51, play poorly defined mechanistic roles in these processes. Here, using purified BCDX2 (RAD51BCD-XRCC2) and CX3 (RAD51C-XRCC3) complexes and in vitro reconstituted biochemical systems, we mechanistically dissect their functions in forming and protecting reversed forks. We show that both RAD51 paralog complexes lack fork reversal activities. Whereas CX3 exhibits modest fork protection activity, BCDX2 significantly synergizes with RAD51 to protect DNA against attack by the nucleases MRE11 and EXO1. DNA protection is contingent upon the ability of RAD51 to form a functional nucleoprotein filament on DNA. Collectively, our results provide evidence for a hitherto unknown function of RAD51 paralogs in synergizing with RAD51 nucleoprotein filament to prevent degradation of stressed replication forks.
The RAD51 recombinase assembles as helical nucleoprotein filaments on single-stranded DNA (ssDNA) and mediates invasion and strand exchange with homologous duplex DNA (dsDNA) during homologous recombination (HR), as well as protection and restart of stalled replication forks. Strand invasion by RAD51-ssDNA complexes depends on ATP binding. However, RAD51 can bind ssDNA in non-productive ADP-bound or nucleotide-free states, and ATP-RAD51-ssDNA complexes hydrolyse ATP over time. Here, we define unappreciated mechanisms by which the RAD51 paralog complex RFS-1/RIP-1 limits the accumulation of RAD-51-ssDNA complexes with unfavorable nucleotide content. We find RAD51 paralogs promote the turnover of ADP-bound RAD-51 from ssDNA, in striking contrast to their ability to stabilize productive ATP-bound RAD-51 nucleoprotein filaments. In addition, RFS-1/RIP-1 inhibits binding of nucleotide-free RAD-51 to ssDNA. We propose that 'nucleotide proofreading' activities of RAD51 paralogs co-operate to ensure the enrichment of active, ATP-bound RAD-51 filaments on ssDNA to promote HR.
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