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1.
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.  相似文献   

2.
The BRCA2 tumor suppressor is important in maintaining genomic stability. BRCA2 is proposed to control the availability, cellular localization and DNA binding activity of the central homologous recombination protein, RAD51, with loss of BRCA2 resulting in defective homologous recombination. Nevertheless, the roles of BRCA2 in regulating RAD51 and how other proteins implicated in RAD51 regulation, such as RAD52 and RAD54 function relative to BRCA2 is not known. In this study, we tested whether defective homologous recombination in Brca2-depleted mouse hybridoma cells could be rectified by expression of mouse Rad51 or the Rad51-interacting mouse proteins, Rad52 and Rad54. In the Brca2-depleted cells, defective homologous recombination can be restored by over-expression of wild-type mouse Rad51, but not mouse Rad52 or Rad54. Correction of the homologous recombination defect requires Rad51 ATPase activity. A sizeable fraction ( approximately 50%) of over-expressed wild-type Rad51 is nuclear localized. The restoration of homologous recombination in the presence of a low (i.e., non-functional) level of Brca2 by wild-type Rad51 over-expression is unexpected. We suggest that Rad51 may access the nuclear compartment in a Brca2-independent manner and when Rad51 is over-expressed, the normal requirement for Brca2 control over Rad51 function in homologous recombination is dispensable. Our studies support loss of Rad51 function as a critical underlying factor in the homologous recombination defect in the Brca2-depleted cells.  相似文献   

3.
The BRCA2 breast cancer tumor suppressor is involved in the repair of double strand breaks and broken replication forks by homologous recombination through its interaction with DNA repair protein Rad51. Cells defective in BRCA2.FANCD1 are extremely sensitive to mitomycin C (MMC) similarly to cells deficient in any of the Fanconi anemia (FA) complementation group proteins (FANC). These observations suggest that the FA pathway and the BRCA2 and Rad51 repair pathway may be linked, although a functional connection between these pathways in DNA damage signaling remains to be determined. Here, we systematically investigated the interaction between these pathways. We show that in response to DNA damage, BRCA2-dependent Rad51 nuclear focus formation was normal in the absence of FANCD2 and that FANCD2 nuclear focus formation and mono-ubiquitination appeared normal in BRCA2-deficient cells. We report that the absence of BRCA2 substantially reduced homologous recombination repair of DNA breaks, whereas the absence of FANCD2 had little effect. Furthermore, we established that depletion of BRCA2 or Rad51 had a greater effect on cell survival in response to MMC than depletion of FANCD2 and that depletion of BRCA2 in FANCD2 mutant cells further sensitized these cells to MMC. Our results suggest that FANCD2 mediates double strand DNA break repair independently of Rad51-associated homologous recombination.  相似文献   

4.
After DNA damage, caspases cleave and activate proteins involved in cell death by apoptosis but also cleave and inactivate proteins implicated in DNA repair. Here we report a rapid onset of Rad51 cleavage by caspase 3 in BRCA2-defective mouse and human cells. This rapid cleavage was reduced markedly by transfer of full-length human BRCA2 into BRCA2-defective mouse or human cells, which also blocked the association of caspase 3 and Rad51 proteins. Overall caspase 3 activity was increased in BRCA2-defective cells, but the time course was much slower than that for Rad51 cleavage. We further showed that caspase 3 cleavage of Rad51 resulted in a functional decrease in Rad51 strand exchange activity and that inhibition of caspase 3 activity increased Rad51 protein levels and Rad51 foci. These findings indicate that BRCA2 inhibits Rad51 cleavage and subsequent apoptosis.  相似文献   

5.
The BRCA1 protein is known to participate in multiple cellular processes. In these experiments, we resolved four distinct BRCA1-containing complexes. We found BRCA1 associated with the RNA polymerase II holoenzyme (holo-pol), a large mass complex called the fraction 5 complex, the Rad50-Mre11-Nbs1 complex, and a complex that has not been described previously. We observed this new complex after treating cells with hydroxyurea, suggesting that the hydroxyurea-induced complex (HUIC) is involved with the response to DNA replication blockage. After hydroxyurea treatment of cells, BRCA1 content decreased in the holo-pol and the fraction 5 complex, and BRCA1 was redistributed to the HUIC. The HUIC was shown not to contain a number of holo-pol components or the Rad50-Mre11-Nbs1 complex but was associated with the BRCA1-associated RING domain protein BARD1. These data suggest that BRCA1 participates in multiple cellular processes by multiple protein complexes and that the BRCA1 content of these complexes is dynamically altered after DNA replication blockage.  相似文献   

6.
BRCA2 is a breast tumor suppressor with a potential function in the cellular response to DNA damage. BRCA2 binds to Rad51 through its BRC repeats. In support of the biological significance of this interaction, we found that the complex of BRCA2 and Rad51 in breast cancer MCF-7 cells was diminished upon conditional expression of a wild-type, but not a mutated, BRC4 repeat using the tetracycline-inducible system. Cells expressing a wild-type BRC4 repeat showed hypersensitivity to gamma-irradiation, an inability to form Rad51 radiation-induced foci, and a failure of radiation-induced G(2)/M, but not G(1)/S, checkpoint control. These results strongly suggest that the interaction between BRCA2 and Rad51 mediated by BRC repeats is critical for the cellular response to DNA damage.  相似文献   

7.
A rare hereditary disorder, Fanconi anemia (FA), is caused by mutations in an array of genes, which interact in a common FA pathway/network. These genes encode components of the FA "core" complex, a key factor FancD2, the familial breast cancer suppressor BRCA2/FancD1, and Brip1/FancJ helicase. Although BRCA2 is known to play a pivotal role in homologous recombination repair by regulating Rad51 recombinase, the precise functional relationship between BRCA2 and the other FA genes is unclear. Here we show that BRCA2-dependent chromatin loading of Rad51 after mitomycin C treatment was not compromised by disruption of FANCC or FANCD2. Rad51 and FancD2 form colocalizing subnuclear foci independently of each other. Furthermore, we created a conditional BRCA2 truncating mutation lacking the C-terminal conserved domain (CTD) (brca2DeltaCTD), and disrupted the FANCC gene in this background. The fancc/brca2DeltaCTD double mutant revealed an epistatic relationship between FANCC and BRCA2 CTD in terms of x-ray sensitivity. In contrast, levels of cisplatin sensitivity and mitomycin C-induced chromosomal aberrations were increased in fancc/brca2DeltaCTD cells relative to either single mutant. Taken together, these results indicate that FA proteins work together with BRCA2/Rad51-mediated homologous recombination in double strand break repair, whereas the FA pathway plays a role that is independent of the CTD of BRCA2 in interstrand cross-link repair. These results provide insights into the functional interplay between the classical FA pathway and BRCA2.  相似文献   

8.
The Rad51 paralogs are required for homologous recombination (HR) and the maintenance of genomic stability. The molecular mechanisms by which the five vertebrate Rad51 paralogs regulate HR and genomic integrity remain unclear. The Rad51 paralogs associate with one another in two distinct complexes: Rad51B-Rad51C-Rad51D-XRCC2 (BCDX2) and Rad51C-XRCC3 (CX3). We find that the BCDX2 and CX3 complexes act at different stages of the HR pathway. In response to DNA damage, the BCDX2 complex acts downstream of BRCA2 recruitment but upstream of Rad51 recruitment. In contrast, the CX3 complex acts downstream of Rad51 recruitment but still has a marked impact on the measured frequency of homologous recombination. Both complexes are epistatic with BRCA2 and synthetically lethal with Rad52. We conclude that human Rad51 paralogs facilitate BRCA2-Rad51-dependent homologous recombination at different stages in the pathway and function independently of Rad52.  相似文献   

9.
The tumor suppressor BRCA2 protein plays a major role in the regulation of Rad51-catalyzed homologous recombination. BRCA2 interacts with monomeric Rad51 primarily via conserved BRC domains and coordinates the formation of Rad51 filaments at double-stranded DNA (dsDNA) breaks. A number of cancer-associated mutations in BRC4 and BRC2 domains have been reported. To elucidate their effects on homologous recombination, we studied Rad51 filament formation on single-stranded DNA and dsDNA substrates and Rad51-catalyzed strand exchange, in the presence of wild-type and mutated peptides of either BRC4 or BRC2. While the wild-type BRC2 and BRC4 peptides inhibited filament formation and, thus, strand exchange, the mutated forms decreased significantly these inhibitory effects. These results are consistent with a three-dimensional model for the interface between individual BRC repeats and Rad51. We suggest that mutations at sites crucial for the association between Rad51 and BRC domains impair the ability of BRCA2 to recruit Rad51 to dsDNA breaks, hampering recombinational repair.  相似文献   

10.
Fanconi anemia (FA) is a cancer susceptibility disorder characterized by chromosomal instability and hypersensitivity to DNA cross-linking agents. So far 11 complementation groups have been identified, from which only FA-D1/BRCA2 and FA-J are defective downstream of the central FANCD2 protein as cells from these groups are capable of monoubiquitinating FANCD2. In this study we show that cells derived from patients from the new complementation groups, FA-I, FA-J and FA-L are all proficient in DNA damage induced Rad51 foci formation, making the cells from FA-D1/BRCA2 patients that are defective in this process the sole exception. Although FA-B patient HSC230 was previously reported to also have biallelic BRCA2 mutations, we found normal Rad51 foci formation in cells from this patient, consistent with the recent identification of an X-linked gene being mutated in four unrelated FA-B patients. Thus, our data show that none of the FA proteins, except BRCA2, are required to sequester Rad51 into nuclear foci. Since cells from the FA-D1 and FA-J patient groups are both able to monoubiquitinate FANCD2, the "Rad51 foci phenotype" provides a convenient assay to distinguish between these two groups. Our results suggest that FANCJ and FANCD1/BRCA2 are part of the integrated FANC/BRCA DNA damage response pathway or, alternatively, that they represent sub-pathways in which only FANCD1/BRCA2 is directly connected to the process of homologous recombination.  相似文献   

11.
12.
The BRCA2 tumour suppressor works in DNA recombination and repair pathways to preserve genome integrity. Recent progress provides fresh insights into its role as a regulator of the Rad51 recombination protein, underpinning a model in which BRCA2's involvement in chromosome stability and tumour suppression arises from its participation in recombinational processes essential for DNA replication.  相似文献   

13.
Fission yeast checkpoint protein Rad17 is required for the DNA integrity checkpoint responses. A fraction of Rad17 is chromatin bound independent of the other checkpoint proteins throughout the cell cycle. Here we show that in response to DNA damage induced by either methyl methanesulfonate treatment or ionizing radiation, increased levels of Rad17 bind to chromatin. Following S-phase stall induced by hydroxyurea or a cdc22 mutation, the chromatin-bound Rad17 progressively dissociates from the chromatin. After S-phase arrest by hydroxyurea in cds1Delta or rad3Delta cells or by replication mutants, Rad17 remains chromatin bound. Rad17 is able to complex in vivo with an Rfc small subunit, Rfc2, but not with Rfc1. Furthermore, cells with rfc1Delta are checkpoint proficient, suggesting that Rfc1 does not have a role in checkpoint function. A checkpoint-defective mutant protein, Rad17(K118E), which has similar nuclear localization to that of the wild type, is unable to bind ATP and has reduced ability in chromatin binding. Mutant Rad17(K118E) protein also has reduced ability to complex with Rfc2, suggesting that Lys(118) of Rad17 plays a role in Rad17-Rfc small-subunit complex formation and chromatin association. However, in the rad17.K118E mutant cells, Cds1 can be activated by hydroxyurea. Together, these results suggest that Rad17 binds to chromatin in response to an aberrant genomic structure generated from DNA damage, replication mutant arrest, or hydroxyurea arrest in the absence of Cds1. Rad17 is not required to bind chromatin when genomic structures are protected by hydroxyurea-activated Cds1. The possible checkpoint events induced by chromatin-bound Rad17 are discussed.  相似文献   

14.
BRCA2 likely exerts its tumor suppressor function by enhancing the efficiency of the homology-directed repair of injured chromosomes. To help define the DNA repair role of BRCA2, we expressed and purified a polypeptide, BRC3/4-DBD, that harbors its BRC3 and BRC4 repeats and DNA binding domain. BRC3/4-DBD interacted with hRad51 and bound DNA with a distinct preference for single-stranded (ss) DNA. Importantly we demonstrated by biochemical means and electron microscopy that BRC3/4-DBD nucleates hRad51 onto ssDNA and acts as a recombination mediator in enabling hRad51 to utilize replication protein A-coated ssDNA as recombination substrate. These functions of BRC3/4-DBD required both the BRC repeats and the BRCA2 DNA binding domain. The results thus clarify the role of BRCA2 in Rad51-dependent DNA recombination and repair, and the experimental strategies described herein should be valuable for systematically deciphering this BRCA2 function.  相似文献   

15.
Homologous recombination is of major importance for the prevention of genomic instability during chromosome duplication and repair of DNA damage, especially double-strand breaks. Biochemical experiments have revealed that during the process of homologous recombination the RAD52 group proteins, including Rad51, Rad52 and Rad54, are involved in an essential step: formation of a joint molecule between the broken DNA and the intact repair template. Accessory proteins for this reaction include the Rad51 paralogs and BRCA2. The significance of homologous recombination for the cell is underscored by the evolutionary conservation of the Rad51, Rad52 and Rad54 proteins from yeast to humans. Upon treatment of cells with ionizing radiation, the RAD52 group proteins accumulate at the sites of DNA damage into so-called foci. For the yeast Saccharomyces cerevisiae, foci formation of Rad51 and Rad54 is abrogated in the absence of Rad52, while Rad51 foci formation does occur in the absence of the Rad51 paralog Rad55. By contrast, we show here that in mammalian cells, Rad52 is not required for foci formation of Rad51 and Rad54. Furthermore, radiation-induced foci formation of Rad51 and Rad54 is impaired in all Rad51 paralog and BRCA2 mutant cell lines tested, while Rad52 foci formation is not influenced by a mutation in any of these recombination proteins. Despite their evolutionary conservation and biochemical similarities, S. cerevisiae and mammalian Rad52 appear to differentially contribute to the DNA-damage response.  相似文献   

16.
Archaeal RadA or Rad51 recombinases are close homologues of eukaryal Rad51 and DMC1. These and bacterial RecA orthologues play a key role in DNA repair by forming helical nucleoprotein filaments in which a hallmark strand exchange reaction between homologous DNA substrates occurs. Recent studies have discovered the stimulatory role by calcium on human and yeast recombinases. Here we report that the strand exchange activity but not the ATPase activity of an archaeal RadA/Rad51 recombinase from Methanococcus voltae (MvRadA) is also subject to calcium stimulation. Crystallized MvRadA filaments in the presence of CaCl(2) resemble that of the recently reported ATPase active form in the presence of an activating dose of KCl. At the ATPase center, one Ca(2+) ion takes the place of two K(+) ions in the K(+)-bound form. The terminal phosphate of the nonhydrolyzable ATP analogue is in a staggered conformation in the Ca(2+)-bound form. In comparison, an eclipsed conformation was seen in the K(+)-bound form. Despite the changes in the ATPase center, both forms harbor largely ordered L2 regions in essentially identical conformations. These data suggest a unified stimulation mechanism by potassium and calcium because of the existence of a conserved ATPase center promiscuous in binding cations.  相似文献   

17.
The present report deals with the functional relationships among protein complexes which, when mutated, are responsible for four human syndromes displaying cancer proneness, and whose cells are deficient in DNA double-strand break (DSB) repair. In some of them, the cells are also unable to activate the proper checkpoint, while in the others an unduly override of the checkpoint-induced arrest occurs. As a consequence, all these patients display genome instability. In ataxia-telangiectasia, the mutated protein (ATM) is a kinase, which acts as a transducer of DNA damage signalling. The defective protein in the ataxia-telangiectasia-like disorder is a DNase (the Mre11 nuclease) that in vivo produces single-strand tails at both sides of DSBs. Mre11 is always present with the Rad50 ATPase in a protein machine: the nuclease complex. In mammals, this complex also contains nibrin, the protein mutated in the Nijmegen syndrome. Nibrin confers new abilities to the nuclease complex, and can also bind to BRCA1 (one of the two proteins mutated in familial breast cancer). BRCA1 has a central motif that binds with high affinity to cruciform DNA, a structure present in places where the DNA loops are anchored to the chromosomal axis or scaffold. The BRCA1 x cruciform DNA complex should be released to allow the nuclease complex to work in DNA recombinational repair of DSBs. BRCA1 also acts as a scaffold for the assembly of ATPases such as Rad51, responsible for the somatic homologous recombination. Loss of the BRCA1 gene prevents cell survival after exposure to cross-linkers. The BRCA1-RING domain is an E3-ubiquitin ligase. It can mono-ubiquitinate the FANCD2 protein, mutated in one of the Fanconi anemia complementation groups, to regulate it. Finally, during DNA replication, the nuclease complex and its activating ATM kinase are integrated in the BRCA1-associated surveillance complex (BASC) that contains, among others, enzymes required for mismatch excision repair. In short, the proteins missing in these syndromes have in common their BRCA1-mediated assembly into multimeric machines responsible for the surveillance of DNA replication, DSB recombinational repair, and the removal of DNA cross-links.  相似文献   

18.
Both Rad51 and Rad52 are required for homologous genetic recombination in Saccharomyces cerevisiae. Rad51 promotes heteroduplex joint formation, a general step in homologous recombination. Rad52 facilitates the binding of Rad51 to replication protein A (RPA)-coated single-stranded DNA. The requirement of RPA can be avoided in vitro, if the single-stranded DNA is short. Using short single-stranded DNA and homologous double-stranded DNA, in the absence of RPA, we found that Rad52 (optimal at three per Rad51) was still required for Rad51-promoted heteroduplex joint formation in vitro, as assayed by the formation of D-loops, suggesting another role for Rad52. Rad51 has to bind to the single-stranded DNA before the addition of double-stranded DNA for efficient D-loop formation. Immunoprecipitation and single-stranded DNA-bead precipitation analyses revealed the presence of the free and DNA-bound complexes of Rad51 and Rad52 at a 1 to 2 stoichiometry. In the presence of single-stranded DNA, in addition to Rad51, Rad52 was required for extensive untwisting that is an intermediate step toward D-loop formation. Thus, these results suggest that the formation of the stoichiometric complex of Rad52 with Rad51 on single-stranded DNA is required for the functional binding of the protein-single-stranded DNA complex to the double-stranded DNA to form D-loops.  相似文献   

19.

Background

The function of BRCA1 in response to ionizing radiation, which directly generates DNA double strand breaks, has been extensively characterized. However previous investigations have produced conflicting data on mutagens that initially induce other classes of DNA adducts. Because of the fundamental and clinical importance of understanding BRCA1 function, we sought to rigorously evaluate the role of this tumor suppressor in response to diverse forms of genotoxic stress.

Methodology/Principal Findings

We investigated BRCA1 stability and localization in various human cells treated with model mutagens that trigger different DNA damage signaling pathways. We established that, unlike ionizing radiation, either UVC or methylmethanesulfonate (MMS) (generating bulky DNA adducts or alkylated bases respectively) induces a transient downregulation of BRCA1 protein which is neither prevented nor enhanced by inhibition of PIKKs. Moreover, we found that the proteasome mediates early degradation of BRCA1, BARD1, BACH1, and Rad52 implying that critical components of the homologous recombinaion machinery need to be functionally abrogated as part of the early response to UV or MMS. Significantly, we found that inhibition of BRCA1/BARD1 downregulation is accompanied by the unscheduled recruitment of both proteins to chromatin along with Rad51. Consistently, treatment of cells with MMS engendered complete disassembly of Rad51 from pre-formed ionizing radiation-induced foci. Following the initial phase of BRCA1/BARD1 downregulation, we found that the recovery of these proteins in foci coincides with the formation of RPA and Rad51 foci. This indicates that homologous recombination is reactivated at later stage of the cellular response to MMS, most likely to repair DSBs generated by replication blocks.

Conclusion/Significance

Taken together our results demonstrate that (i) the stabilities of BRCA1/BARD1 complexes are regulated in a mutagen-specific manner, and (ii) indicate the existence of mechanisms that may be required to prevent the simultaneous recruitment of conflicting signaling pathways to sites of DNA damage.  相似文献   

20.
Brh2, a member of the BRCA2 family of proteins, governs homologous recombination in the fungus Ustilago maydis through interaction with Rad51. Brh2 serves at an early step in homologous recombination to mediate Rad51 nucleoprotein filament formation and also has the capability to function at a later step in recombination through its inherent DNA annealing activity. Rec2, a Rad51 paralogue, and Rad52 are additional components of the homologous recombination system, but the absence of either is less critical than Brh2 for operational activity. Here we tested a variety of mutant forms of Brh2 for activity in recombinational repair as measured by DNA repair proficiency. We found that a mutant of Brh2 deleted of the non-canonical DNA-binding domain within the N-terminal region is dependent upon the presence of Rad52 for DNA repair activity. We also determined that a motif first identified in human BRCA2 as important in binding DMC1 also contributes to DNA repair proficiency and cooperates with the BRC element in Rad51 binding.  相似文献   

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