BCCIP regulates homologous recombination by distinct domains and suppresses spontaneous DNA damage

Homologous recombination (HR) is critical for maintaining genome stability through precise repair of DNA double-strand breaks (DSBs) and restarting stalled or collapsed DNA replication forks. HR is regulated by many proteins through distinct mechanisms. Some proteins have direct enzymatic roles in HR reactions, while others act as accessory factors that regulate HR enzymatic activity or coordinate HR with other cellular processes such as the cell cycle. The breast cancer susceptibility gene BRCA2 encodes a critical accessory protein that interacts with the RAD51 recombinase and this interaction fluctuates during the cell cycle. We previously showed that a BRCA2- and p21-interacting protein, BCCIP, regulates BRCA2 and RAD51 nuclear focus formation, DSB-induced HR and cell cycle progression. However, it has not been clear whether BCCIP acts exclusively through BRCA2 to regulate HR and whether BCCIP also regulates the alternative DSB repair pathway, non-homologous end joining. In this study, we found that BCCIP fragments that interact with BRCA2 or with p21 each inhibit DSB repair by HR. We further show that transient down-regulation of BCCIP in human cells does not affect non-specific integration of transfected DNA, but significantly inhibits homology-directed gene targeting. Furthermore, human HT1080 cells with constitutive down-regulation of BCCIP display increased levels of spontaneous single-stranded DNA (ssDNA) and DSBs. These data indicate that multiple BCCIP domains are important for HR regulation, that BCCIP is unlikely to regulate non-homologous end joining, and that BCCIP plays a critical role in resolving spontaneous DNA damage.     

RAD-51-dependent and -independent roles of a Caenorhabditis elegans BRCA2-related protein during DNA double-strand break repair

The BRCA2 tumor suppressor is implicated in DNA double-strand break (DSB) repair by homologous recombination (HR), where it regulates the RAD51 recombinase. We describe a BRCA2-related protein of Caenorhabditis elegans (CeBRC-2) that interacts directly with RAD-51 via a single BRC motif and that binds preferentially to single-stranded DNA through an oligonucleotide-oligosaccharide binding fold. Cebrc-2 mutants fail to repair meiotic or radiation-induced DSBs by HR due to inefficient RAD-51 nuclear localization and a failure to target RAD-51 to sites of DSBs. Genetic and cytological comparisons of Cebrc-2 and rad-51 mutants revealed fundamental phenotypic differences that suggest a role for Cebrc-2 in promoting the use of an alternative repair pathway in the absence of rad-51 and independent of nonhomologous end joining (NHEJ). Unlike rad-51 mutants, Cebrc-2 mutants also accumulate RPA-1 at DSBs, and abnormal chromosome aggregates that arise during the meiotic prophase can be rescued by blocking the NHEJ pathway. CeBRC-2 also forms foci in response to DNA damage and can do so independently of rad-51. Thus, CeBRC-2 not only regulates RAD-51 during HR but can also function independently of rad-51 in DSB repair processes.     

Ring Finger Nuclear Factor RNF168 Is Important for Defects in Homologous Recombination Caused by Loss of the Breast Cancer Susceptibility Factor BRCA1

The RING finger nuclear factor RNF168 is required for recruitment of several DNA damage response factors to double strand breaks (DSBs), including 53BP1 and BRCA1. Because 53BP1 and BRCA1 function antagonistically during the DSB repair pathway homologous recombination (HR), the influence of RNF168 on HR has been unclear. We report that RNF168 depletion causes an elevated frequency of two distinct HR pathways (homology-directed repair and single strand annealing), suppresses defects in HR caused by BRCA1 silencing, but does not suppress HR defects caused by disruption of CtIP, RAD50, BRCA2, or RAD51. Furthermore, RNF168-depleted cells can form ionizing radiation-induced foci of the recombinase RAD51 without forming BRCA1 ionizing radiation-induced foci, indicating that this loss of BRCA1 recruitment to DSBs does not reflect a loss of function during HR. Additionally, we find that RNF168 and 53BP1 have a similar influence on HR. We suggest that RNF168 is important for HR defects caused by BRCA1 loss.     

The cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair

The essential checkpoint kinase Chk1 is required for cell-cycle delays after DNA damage or blocked DNA replication. However, it is unclear whether Chk1 is involved in the repair of damaged DNA. Here we establish that Chk1 is a key regulator of genome maintenance by the homologous recombination repair (HRR) system. Abrogation of Chk1 function with small interfering RNA or chemical antagonists inhibits HRR, leading to persistent unrepaired DNA double-strand breaks (DSBs) and cell death after replication inhibition with hydroxyurea or DNA-damage caused by camptothecin. After hydroxyurea treatment, the essential recombination repair protein RAD51 is recruited to DNA repair foci performing a vital role in correct HRR. We demonstrate that Chk1 interacts with RAD51, and that RAD51 is phosphorylated on Thr 309 in a Chk1-dependent manner. Consistent with a functional interplay between Chk1 and RAD51, Chk1-depleted cells failed to form RAD51 nuclear foci after exposure to hydroxyurea, and cells expressing a phosphorylation-deficient mutant RAD51(T309A) were hypersensitive to hydroxyurea. These results highlight a crucial role for the Chk1 signalling pathway in protecting cells against lethal DNA lesions through regulation of HRR.     

BRCA2 diffuses as oligomeric clusters with RAD51 and changes mobility after DNA damage in live cells

Genome maintenance by homologous recombination depends on coordinating many proteins in time and space to assemble at DNA break sites. To understand this process, we followed the mobility of BRCA2, a critical recombination mediator, in live cells at the single-molecule level using both single-particle tracking and fluorescence correlation spectroscopy. BRCA2-GFP and -YFP were compared to distinguish diffusion from fluorophore behavior. Diffusive behavior of fluorescent RAD51 and RAD54 was determined for comparison. All fluorescent proteins were expressed from endogenous loci. We found that nuclear BRCA2 existed in oligomeric clusters, and exhibited heterogeneous mobility. DNA damage increased BRCA2 transient binding, presumably including binding to damaged sites. Despite its very different size, RAD51 displayed mobility similar to BRCA2, which indicates physical interaction between these proteins both before and after induction of DNA damage. We propose that BRCA2-mediated sequestration of nuclear RAD51 serves to prevent inappropriate DNA interactions and that all RAD51 is delivered to DNA damage sites in association with BRCA2.     

Full-length archaeal Rad51 structure and mutants: mechanisms for RAD51 assembly and control by BRCA2

To clarify RAD51 interactions controlling homologous recombination, we report here the crystal structure of the full-length RAD51 homolog from Pyrococcus furiosus. The structure reveals how RAD51 proteins assemble into inactive heptameric rings and active DNA-bound filaments matching three-dimensional electron microscopy reconstructions. A polymerization motif (RAD51-PM) tethers individual subunits together to form assemblies. Subunit interactions support an allosteric 'switch' promoting ATPase activity and DNA binding roles for the N-terminal domain helix-hairpin-helix (HhH) motif. Structural and mutational results characterize RAD51 interactions with the breast cancer susceptibility protein BRCA2 in higher eukaryotes. A designed P.furiosus RAD51 mutant binds BRC repeats and forms BRCA2-dependent nuclear foci in human cells in response to gamma-irradiation-induced DNA damage, similar to human RAD51. These results show that BRCA2 repeats mimic the RAD51-PM and imply analogous RAD51 interactions with RAD52 and RAD54. Both BRCA2 and RAD54 may act as antagonists and chaperones for RAD51 filament assembly by coupling RAD51 interface exchanges with DNA binding. Together, these structural and mutational results support an interface exchange hypothesis for coordinated protein interactions in homologous recombination.     

Stabilization of RAD51 nucleoprotein filaments by the C-terminal region of BRCA2

The human breast cancer susceptibility gene BRCA2 is required for the regulation of RAD51-mediated homologous recombinational repair. BRCA2 interacts with RAD51 monomers, as well as nucleoprotein filaments, primarily though the conserved BRC motifs. The unrelated C-terminal region of BRCA2 also interacts with RAD51. Here we show that the BRCA2 C terminus interacts directly with RAD51 filaments, but not monomers, by binding an interface created by two adjacent RAD51 protomers. These interactions stabilize filaments so that they cannot be dissociated by association with BRC repeats. Interaction of the BRCA2 C terminus with the RAD51 filament causes a large movement of the flexible RAD51 N-terminal domain that is important in regulating filament dynamics. We suggest that interactions of the BRCA2 C-terminal region with RAD51 may facilitate efficient nucleation of RAD51 multimers on DNA and thereby stimulate recombination-mediated repair.     

RAD51 localization and activation following DNA damage

The efficient repair of double-strand breaks in DNA is critical for the maintenance of genome stability. In response to ionizing radiation and other DNA-damaging agents, the RAD51 protein, which is essential for homologous recombination, relocalizes within the nucleus to form distinct foci that can be visualized by microscopy and are thought to represent sites where repair reactions take place. The formation of RAD51 foci in response to DNA damage is dependent upon BRCA2 and a series of proteins known as the RAD51 paralogues (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3), indicating that the components present within foci assemble in a carefully orchestrated and ordered manner. By contrast, RAD51 foci that form spontaneously as cells undergo DNA replication at S phase occur without the need for BRCA2 or the RAD51 paralogues. It is known that BRCA2 interacts directly with RAD51 through a series of degenerative motifs known as the BRC repeats. These interactions modulate the ability of RAD51 to bind DNA. Taken together, these observations indicate that BRCA2 plays a critical role in controlling the actions of RAD51 at both the microscopic (focus formation) and molecular (DNA binding) level.     

FIGL1 coordinates with dosage-sensitive BRCA2 in modulating meiotic recombination in maize

Meiotic crossover(CO) formation between homologous chromosomes ensures their subsequent proper segregation and generates genetic diversity among offspring. In maize, however, the mechanisms that modulate CO formation remain poorly characterized. Here, we found that both maize BREAST CANCER SUSCEPTIBILITY PROTEIN 2(BRCA2) and AAA-ATPase FIDGETIN-LIKE-1(FIGL1)act as positive factors of CO formation by controlling the assembly or/and stability of two conserved DNA recombinases RAD51 and DMC1 filame...     

Structural basis for recruitment of BRCA2 by PALB2

The breast cancer 2, early onset protein (BRCA2) is central to the repair of DNA damage by homologous recombination. BRCA2 recruits the recombinase RAD51 to sites of damage, regulates its assembly into nucleoprotein filaments and thereby promotes homologous recombination. Localization of BRCA2 to nuclear foci requires its association with the partner and localizer of BRCA2 (PALB2), mutations in which are associated with cancer predisposition, as well as subtype N of Fanconi anaemia. We have determined the structure of the PALB2 carboxy‐terminal β‐propeller domain in complex with a BRCA2 peptide. The structure shows the molecular determinants of this important protein–protein interaction and explains the effects of both cancer‐associated truncating mutants in PALB2 and missense mutations in the amino‐terminal region of BRCA2.     

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.     

Identification of Rad51 regulation by BRCA2 using Caenorhabditis elegans BRCA2 and bimolecular fluorescence complementation analysis

BRCA2 is involved in double-stranded DNA break repair by binding and regulating Rad51-mediated homologous recombination. Insights as to how BRCA2 regulates Rad51-mediated DNA repair arose from in vitro biochemical studies on fragments of BRCA2. However, the large 400-kDa BRCA2 protein has hampered our ability to understand the entire process by which full-length BRCA2 regulates Rad51. Here, we show that CeBRC-2, which is only one tenth the size of mammalian BRCA2, complemented BRCA2-deficiency in Rad51 regulation. CeBRC-2 was able to bind to mammalian Rad51 (mRad51) and form distinct nuclear foci when they interacted. In our bimolecular fluorescence complementation analysis (BiFC), we show that the strength of the interaction between CeBRC-2 and mRad51 increased markedly after DNA damage. The BRC motif of CeBRC-2 was responsible for binding mRad51, but without the OB fold, the complex was unable to target damaged DNA. When CeBRC-2 was introduced into BRCA2-deficient cells, it restored Rad51 foci after DNA damage. Our study suggests a mode of action for BRCA2 with regard to DNA repair.     

Interaction with the BRCA2 C terminus protects RAD51-DNA filaments from disassembly by BRC repeats

BRCA2 has an essential function in DNA repair by homologous recombination, interacting with RAD51 via short motifs in the middle and at the C terminus of BRCA2. Here, we report that a conserved 36-residue sequence of human BRCA2 encoded by exon 27 (BRCA2Exon27) interacts with RAD51 through the specific recognition of oligomerized RAD51 ATPase domains. BRCA2Exon27 binding stabilizes the RAD51 nucleoprotein filament against disassembly by BRC repeat 4. The protection is specific for RAD51 filaments formed on single-stranded DNA and is lost when BRCA2Exon27 is phosphorylated on Ser3291. We propose that productive recombination results from the functional balance between the different RAD51-binding modes [corrected] of the BRC repeat and exon 27 regions of BRCA2. Our results further suggest a mechanism in which CDK phosphorylation of BRCA2Exon27 at the G2-M transition alters the balance in favor of RAD51 filament disassembly, thus terminating recombination.     

c-ABL tyrosine kinase stabilizes RAD51 chromatin association

The assembly of RAD51 recombinase on DNA substrates at sites of breakage is essential for their repair by homologous recombination repair (HRR). The signaling pathway that triggers RAD51 assembly at damage sites to form subnuclear foci is unclear. Here, we provide evidence that c-ABL, a tyrosine kinase activated by DNA damage which phosphorylates RAD51 on Tyr-315, works at a previously unrecognized, proximal step to initiate RAD51 assembly. We first show that c-ABL associates with chromatin after DNA damage in a manner dependent on its kinase activity. Using RAD51 mutants that are unable to oligomerize to form a nucleoprotein filament, we separate RAD51 assembly on DNA to form foci into two steps: stable chromatin association followed by oligomerization. We show that phosphorylation on Tyr-315 by c-ABL is required for chromatin association of oligomerization-defective RAD51 mutants, but is insufficient to restore oligomerization. Our findings suggest a new model for the regulation of early steps of HRR.     

Double-strand break repair-independent role for BRCA2 in blocking stalled replication fork degradation by MRE11

Breast cancer suppressor BRCA2 is critical for maintenance of genomic integrity and resistance to agents that damage DNA or collapse replication forks, presumably through homology-directed repair of double-strand breaks (HDR). Using single-molecule DNA fiber analysis, we show here that nascent replication tracts created before fork stalling with hydroxyurea are degraded in the absence of BRCA2 but are stable in wild-type cells. BRCA2 mutational analysis reveals that a conserved C-terminal site involved in stabilizing RAD51 filaments, but not in loading RAD51 onto DNA, is essential for this fork protection but dispensable for HDR. RAD51 filament disruption in wild-type cells phenocopies BRCA2 deficiency. BRCA2 prevents chromosomal aberrations on replication stalling, which are alleviated by inhibition of MRE11, the nuclease responsible for this form of fork instability. Thus, BRCA2 prevents rather than repairs nucleolytic lesions at stalled replication forks to maintain genomic integrity and hence likely suppresses tumorigenesis through this replication-specific function.     

Functional analysis of Drosophila melanogaster BRCA2 in DNA repair

The human BRCA2 cancer susceptibility protein functions in double-strand DNA break repair by homologous recombination and this pathway is conserved in the fly Drosophila melanogaster. Although a potential Drosophila melanogaster BRCA2 orthologue (dmbrca2; CG30169) has been identified by sequence similarity, no functional data addressing the role of this protein in DNA repair is available. Here, we demonstrate that depletion of dmbrca2 from Drosophila cells induces sensitivity to DNA damage induced by irradiation or treatment with hydroxyurea. Dmbrca2 physically interacts with dmrad51 (spnA) and the two proteins become recruited to nuclear foci after DNA damage. A functional assay for DNA repair demonstrated that in flies dmbrca2 plays a role in double-strand break repair by gene conversion. Finally, we show that depletion of dmbrca2 in cells is synthetically lethal with deficiency in other DNA repair proteins including dmparp. The conservation of the function of BRCA2 in Drosophila will allow the analysis of this key DNA repair protein in a genetically tractable organism potentially illuminating mechanisms of carcinogenesis and aiding the development of therapeutic agents.