The Rad51 paralog Rad51B promotes homologous recombinational repair

The highly conserved Saccharomyces cerevisiae Rad51 protein plays a central role in both mitotic and meiotic homologous DNA recombination. Seven members of the Rad51 family have been identified in vertebrate cells, including Rad51, Dmc1, and five Rad51-related proteins referred to as Rad51 paralogs, which share 20 to 30% sequence identity with Rad51. In chicken B lymphocyte DT40 cells, we generated a mutant with RAD51B/RAD51L1, a member of the Rad51 family, knocked out. RAD51B(-/-) cells are viable, although spontaneous chromosomal aberrations kill about 20% of the cells in each cell cycle. Rad51B deficiency impairs homologous recombinational repair (HRR), as measured by targeted integration, sister chromatid exchange, and intragenic recombination at the immunoglobulin locus. RAD51B(-/-) cells are quite sensitive to the cross-linking agents cisplatin and mitomycin C and mildly sensitive to gamma-rays. The formation of damage-induced Rad51 nuclear foci is much reduced in RAD51B(-/-) cells, suggesting that Rad51B promotes the assembly of Rad51 nucleoprotein filaments during HRR. These findings show that Rad51B is important for repairing various types of DNA lesions and maintaining chromosome integrity.     

Role of BRCA2 in control of the RAD51 recombination and DNA repair protein

Individuals carrying BRCA2 mutations are predisposed to breast and ovarian cancers. Here, we show that BRCA2 plays a dual role in regulating the actions of RAD51, a protein essential for homologous recombination and DNA repair. First, interactions between RAD51 and the BRC3 or BRC4 regions of BRCA2 block nucleoprotein filament formation by RAD51. Alterations to the BRC3 region that mimic cancer-associated BRCA2 mutations fail to exhibit this effect. Second, transport of RAD51 to the nucleus is defective in cells carrying a cancer-associated BRCA2 truncation. Thus, BRCA2 regulates both the intracellular localization and DNA binding ability of RAD51. Loss of these controls following BRCA2 inactivation may be a key event leading to genomic instability and tumorigenesis.     

The epistatic relationship between BRCA2 and the other RAD51 mediators in homologous recombination

RAD51 recombinase polymerizes at the site of double-strand breaks (DSBs) where it performs DSB repair. The loss of RAD51 causes extensive chromosomal breaks, leading to apoptosis. The polymerization of RAD51 is regulated by a number of RAD51 mediators, such as BRCA1, BRCA2, RAD52, SFR1, SWS1, and the five RAD51 paralogs, including XRCC3. We here show that brca2-null mutant cells were able to proliferate, indicating that RAD51 can perform DSB repair in the absence of BRCA2. We disrupted the BRCA1, RAD52, SFR1, SWS1, and XRCC3 genes in the brca2-null cells. All the resulting double-mutant cells displayed a phenotype that was very similar to that of the brca2-null cells. We suggest that BRCA2 might thus serve as a platform to recruit various RAD51 mediators at the appropriate position at the DNA-damage site.     

Interactions between BRCA2 and RAD51 for promoting homologous recombination in Leishmania infantum

In most organisms, the primary function of homologous recombination (HR) is to allow genome protection by the faithful repair of DNA double-strand breaks. The vital step of HR is the search for sequence homology, mediated by the RAD51 recombinase, which is stimulated further by proteins mediators such as the tumor suppressor BRCA2. The biochemical interplay between RAD51 and BRCA2 is unknown in Leishmania or Trypanosoma. Here we show that the Leishmania infantum BRCA2 protein possesses several critical features important for the regulation of DNA recombination at the genetic and biochemical level. A BRCA2 null mutant, generated by gene disruption, displayed genomic instability and gene-targeting defects. Furthermore, cytological studies show that LiRAD51 can no longer localize to the nucleus in this mutant. The Leishmania RAD51 and BRCA2 interact together and the purified proteins bind single-strand DNA. Remarkably, LiBRCA2 is a recombination mediator that stimulates the invasion of a resected DNA double-strand break in an undamaged template by LiRAD51 to form a D-loop structure. Collectively, our data show that LiBRCA2 and LiRAD51 promote HR at the genetic and biochemical level in L. infantum, the causative agent of visceral leishmaniasis.     

TOPORS-mediated RAD51 SUMOylation facilitates homologous recombination repair

Homologous recombination (HR) is critical for error-free repair of DNA double-strand breaks. Chromatin loading of RAD51, a key protein that mediates the recombination, is a crucial step in the execution of the HR repair. Here, we present evidence that SUMOylation of RAD51 is crucial for the RAD51 recruitment to chromatin and HR repair. We found that topoisomerase 1-binding arginine/serine-rich protein (TOPORS) induces the SUMOylation of RAD51 at lysine residues 57 and 70 in response to DNA damaging agents. The SUMOylation was facilitated by an ATM-induced phosphorylation of TOPORS at threonine 515 upon DNA damage. Knockdown of TOPORS or expression of SUMOylation-deficient RAD51 mutants caused reduction in supporting normal RAD51 functions during the HR repair, suggesting the physiological importance of the modification. We found that the SUMOylation-deficient RAD51 reduces the association with its crucial binding partner BRCA2, explaining its deficiency in supporting the HR repair. These findings altogether demonstrate a crucial role for TOPORS-mediated RAD51 SUMOylation in promoting HR repair and genomic maintenance.     

Mechanistic insights into RAD51-associated protein 1 (RAD51AP1) action in homologous DNA repair

Homologous recombination catalyzed by the RAD51 recombinase is essential for maintaining genome integrity upon the induction of DNA double strand breaks and other DNA lesions. By enhancing the recombinase activity of RAD51, RAD51AP1 (RAD51-associated protein 1) serves a key role in homologous recombination-mediated chromosome damage repair. We show here that RAD51AP1 harbors two distinct DNA binding domains that are both needed for maximal protein activity under physiological conditions. We have finely mapped the two DNA binding domains in RAD51AP1 and generated mutant variants that are impaired in either or both of the DNA binding domains. Examination of these mutants reveals that both domains are indispensable for RAD51AP1 function in cells. These and other results illuminate the mechanistic basis of RAD51AP1 action in homologous DNA repair.     

Regulation of double-strand break-induced mammalian homologous recombination by UBL1, a RAD51-interacting protein

Mammalian RAD51 protein plays essential roles in DNA homologous recombination, DNA repair and cell proliferation. RAD51 activities are regulated by its associated proteins. It was previously reported that a ubiquitin-like protein, UBL1, associates with RAD51 in the yeast two-hybrid system. One function of UBL1 is to covalently conjugate with target proteins and thus modify their function. In the present study we found that non-conjugated UBL1 forms a complex with RAD51 and RAD52 proteins in human cells. Overexpression of UBL1 down-regulates DNA double-strand break-induced homologous recombination in CHO cells and reduces cellular resistance to ionizing radiation in HT1080 cells. With or without overexpressed UBL1, most homologous recombination products arise by gene conversion. However, overexpression of UBL1 reduces the fraction of bidirectional gene conversion tracts. Overexpression of a mutant UBL1 that is incapable of being conjugated retains the ability to inhibit homologous recombination. These results suggest a regulatory role for UBL1 in homologous recombination.     

Ortholog of BRCA2-interacting protein BCCIP controls morphogenetic responses during DNA replication stress in Ustilago maydis

The BRCA2 tumor suppressor functions in repair of DNA by homologous recombination through regulating the action of Rad51. In turn, BRCA2 appears to be regulated by other interacting proteins. Dss1, a small interacting protein that binds to the C-terminal domain, has a profound effect on activity as deduced from studies on the BRCA2-related protein Brh2 in Ustilago maydis. Evidence accumulating in mammalian systems suggests that BCCIP, another small interacting protein that binds to the C-terminal domain of BRCA2, also serves to regulate homologous recombination activity. Here we were interested in testing the role of the putative U. maydis BCCIP ortholog Bcp1 in DNA repair and recombination. In keeping with the mammalian paradigm, Bcp1 bound to the C-terminal region of Brh2. Mutants deleted of the gene were extremely slow growing, showed a delay passing through S phase and exhibited sensitivity to hydroxyurea, but were otherwise normal in DNA repair and homologous recombination. In the absence of Bcp1 cells were unable to maintain the wild type morphology when challenged by a DNA replication stress. These results suggest that Bcp1 could be involved in coordinating morphogenetic events with DNA processing during replication.     

XRCC2 is a nuclear RAD51-like protein required for damage-dependent RAD51 focus formation without the need for ATP binding

The human XRCC2 gene was recently identified by its ability to complement a hamster cell line, irs1, which is sensitive to DNA-damaging agents and shows genetic instability. The XRCC2 protein is highly conserved in mammalian species and has structural features, including a putative ATP-binding domain (P-loop), consistent with membership of the RecA/RAD51 family of recombination-repair proteins. We show that a hybrid XRCC2-green fluorescent protein, which was found to be functional by complementation, localizes to the nucleus. We have established a functional link between XRCC2 and RAD51 by looking at damage-dependent RAD51 focus formation in the irs1 cell line. Little or no formation of RAD51 foci occurred in irs1. This effect was specific to the loss of XRCC2 because transfection of the gene into irs1 restored normal levels of focus formation. Surprisingly, XRCC2 genes carrying site-specific mutations in P-loop residues were found to be able to complement the XRCC2-deficient irs1 line for a number of different end points. We conclude that XRCC2 is important in the early stages of homologous recombination in mammalian cells to facilitate RAD51-dependent recombination repair but that it does not make use of ATP binding to promote this function.     

RAD51 paralogs: roles in DNA damage signalling, recombinational repair and tumorigenesis

Chromosomal double-strand breaks (DSBs) have the potential to permanently arrest cell cycle progression and endanger cell survival. They must therefore be efficiently repaired to preserve genome integrity and functionality. Homologous recombination (HR) provides an important error-free mechanism for DSB repair in mammalian cells. In addition to RAD51, the central recombinase activity in mammalian cells, a family of proteins known as the RAD51 paralogs and consisting of five proteins (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3), play an essential role in the DNA repair reactions through HR. The RAD51 paralogs act to transduce the DNA damage signal to effector kinases and to promote break repair. However, their precise cellular functions are not fully elucidated. Here we discuss recent advances in our understanding of how these factors mediate checkpoint responses and act in the HR repair process. In addition, we highlight potential functional similarities with the BRCA2 tumour suppressor, through the recently reported links between RAD51 paralog deficiencies and tumorigenesis triggered by genome instability.     

The interaction profile of homologous recombination repair proteins RAD51C,RAD51D and XRCC2 as determined by proteomic analysis

The RAD51 family of proteins is involved in homologous recombination (HR) DNA repair and maintaining chromosome integrity. To identify candidates that interact with HR proteins, the mouse RAD51C, RAD51D and XRCC2 proteins were purified using bacterial expression systems and each of them used to co‐precipitate interacting partners from mouse embryonic fibroblast cellular extracts. Mass spectroscopic analysis was performed on protein bands obtained after 1‐D SDS‐PAGE of co‐precipitation eluates from cell extracts of mitomycin C treated and untreated mouse embryonic fibroblasts. Profiling of the interacting proteins showed a clear bias toward nucleic acid binding and modification proteins. Interactions of four candidate proteins (SFPQ, NONO, MSH2 and mini chromosome maintenance protein 2) were confirmed by Western blot analysis of co‐precipitation eluates and were also verified to form ex vivo complexes with RAD51D. Additional interacting proteins were associated with cell division, embryo development, protein and carbohydrate metabolism, cellular trafficking, protein synthesis, modification or folding, and cell structure or motility functions. Results from this study are an important step toward identifying interacting partners of the RAD51 paralogs and understanding the functional diversity of proteins that assist or regulate HR repair mechanisms.     

Sequence fingerprints in BRCA2 and RAD51: implications for DNA repair and cancer

In humans, the interactions between the breast cancer susceptibility protein, BRCA2, and the RAD51 recombinase are essential for DNA repair by homologous recombination (HR), failure of which can predispose to cancer. The interactions occur through conserved BRC repeat motifs, encoded in BRCA2, binding directly to RAD51. Here, we describe full and partial BRCA2 homologues from a wide range of eukaryotes, including Drosophila melanogaster and two Plasmodium species. The crystal structure of the human BRC4-RAD51 complex allows identification of residues that are important for protein-protein interaction, and defines interaction sequence fingerprints for the BRC repeat and for RAD51. These allow us to predict that most eukaryotic RAD51 and BRC repeat orthologues should be capable of mutual interactions. We find no evidence for the presence of BRC repeats in yeast, Archaea and bacteria, and their RAD51 orthologues do not fulfil the criteria for binding the BRC repeat. Similarly, human RAD51 paralogues, including RAD51B, RAD51C, RAD51D, XRCC2, XRCC3 and DMC1, are not predicted to bind the BRC repeat. Conservation of the BRC repeat and RAD51 sequence fingerprints across a wide range of eukaryotic species substantiates the functional significance of the BRCA2-RAD51 interactions. The idea of multiple BRC repeats with binding specificity towards RAD51 leads us to suggest a possible model for the participation of BRCA2 in RAD51 nucleoprotein filament formation.     

Berberine radiosensitizes human esophageal cancer cells by downregulating homologous recombination repair protein RAD51

Background

Esophageal squamous cell carcinomas (ESCC) have poor prognosis. While combined modality of chemotherapy and radiotherapy increases survival, most patients die within five years. Development of agents that confer cancer cell-specific chemo- and radiosensitivity may improve the therapy of ESCC. We here reported the discovery of berberine as a potent radiosensitizing agent on ESCC cells.

Principal Findings

Berberine at low concentrations (<15 µM) substantially radiosensitized ESCC cells. X-ray induced DNA double-strand breaks (DSBs) persist longer in ESCC cells pretreated with berberine. Berberine pretreatment led to a significant downregulation of RAD51, a key player in homologous recombination repair, in ESCC cells, but not in non-malignant human cells. Downregulation of RAD51 by RNA interference similarly radiosensitized the cancer cells, and, conversely, introduction of exogenous RAD51 was able to significantly counteract the radiosensitizing effect of berberine, thus establishing RAD51 as a key determinant in radiation sensitivity. We also observed that RAD51 was commonly overexpressed in human ESCC tissues, suggesting that it is necessary to downregulate RAD51 to achieve high radio- or chemotherapeutic efficacy of ESCC in clinic, because overexpression of RAD51 is known to confer radio- and chemoresistance.

Conclusions/Significance

Berberine can effectively downregulate RAD51 in conferring radiosensitivity on esophageal cancer cells. Its clinical application as an adjuvant in chemotherapy and radiotherapy of esophageal cancers should be explored.     

RAD51AP1 is a structure-specific DNA binding protein that stimulates joint molecule formation during RAD51-mediated homologous recombination

Homologous recombination is essential for preserving genome integrity. Joining of homologous DNA molecules through strand exchange, a pivotal step in recombination, is mediated by RAD51. Here, we identify RAD51AP1 as a RAD51 accessory protein that specifically stimulates joint molecule formation through the combination of structure-specific DNA binding and physical contact with RAD51. At the cellular level, we show that RAD51AP1 is required to protect cells from the adverse effects of DNA double-strand break-inducing agents. At the biochemical level, we show that RAD51AP1 has a selective affinity for branched-DNA structures that are obligatory intermediates during joint molecule formation. Our results highlight the importance of structural transitions in DNA as control points in recombination. The affinity of RAD51AP1 for the central protein and DNA intermediates of recombination confers on it the ability to control the preservation of genome integrity at a number of critical mechanistic steps.     

Collaborative roles of gammaH2AX and the Rad51 paralog Xrcc3 in homologous recombinational repair

One of the earliest events in the signal transduction cascade that initiates a DNA damage checkpoint is the phosphorylation on serine 139 of histone H2AX (gammaH2AX) at DNA double-strand breaks (DSBs). However, the role of gammaH2AX in DNA repair is poorly understood. To address this question, we generated chicken DT40 cells carrying a serine to alanine mutation at position 139 of H2AX (H2AX(-/S139A)) and examined their DNA repair capacity. H2AX(-/S139A) cells exhibited defective homologous recombinational repair (HR) as manifested by delayed Rad51 focus formation following ionizing radiation (IR) and hypersensitivity to the topoisomerase I inhibitor, camptothecin (CPT), which causes DSBs at replication blockage. Deletion of the Rad51 paralog gene, XRCC3, also delays Rad51 focus formation. To test the interaction of Xrcc3 and gammaH2AX, we disrupted XRCC3 in H2AX(-/S139A) cells. XRCC3(-/-)/H2AX(-/S139A) mutants were not viable, although this synthetic lethality was reversed by inserting a transgene that conditionally expresses wild-type H2AX. Upon repression of the wild-type H2AX transgene, XRCC3(-/-)/H2AX(-/S139A) cells failed to form Rad51 foci and exhibited markedly increased levels of chromosomal aberrations after CPT treatment. These results indicate that H2AX and XRCC3 act in separate arms of a branched pathway to facilitate Rad51 assembly.