Control of sister chromatid recombination by histone H2AX

Histone H2AX has a role in suppressing genomic instability and cancer. However, the mechanisms by which it performs these functions are poorly understood. After DNA breakage, H2AX is phosphorylated on serine 139 in chromatin near the break. We show here that H2AX serine 139 enforces efficient homologous recombinational repair of a chromosomal double-strand break (DSB) by using the sister chromatid as a template. BRCA1, Rad51, and CHK2 contribute to recombinational repair, in part independently of H2AX. H2AX(-/-) cells show increased use of single-strand annealing, an error-prone deletional mechanism of DSB repair. Therefore, the chromatin response around a chromosomal DSB, in which H2AX serine 139 phosphorylation plays a central role, "shapes" the repair process in favor of potentially error-free interchromatid homologous recombination at the expense of error-prone repair. H2AX phosphorylation may help set up a favorable disposition between sister chromatids.     

Brca2 (XRCC11) Deficiency Results in Radioresistant DNA Synthesis and a Higher Frequency of Spontaneous Deletions

We show here that the radiosensitive Chinese hamster cell mutant (V-C8) of group XRCC11 is defective in the breast cancer susceptibility gene Brca2. The very complex phenotype of V-C8 cells is complemented by a single human chromosome 13 providing the BRCA2 gene, as well as by the murine Brca2 gene. The Brca2 deficiency in V-C8 cells causes hypersensitivity to various DNA-damaging agents with an extreme sensitivity toward interstrand DNA cross-linking agents. Furthermore, V-C8 cells show radioresistant DNA synthesis after ionizing radiation, suggesting that Brca2 deficiency affects cell cycle checkpoint regulation. In addition, V-C8 cells display tremendous chromosomal instability and a high frequency of abnormal centrosomes. The mutation spectrum at the hprt locus showed that the majority of spontaneous mutations in V-C8 cells are deletions, in contrast to wild-type V79 cells. A mechanistic explanation for the genome instability phenotype of Brca2-deficient cells is provided by the observation that the nuclear localization of the central DNA repair protein in homologous recombination, Rad51, is reduced in V-C8 cells.     

Analysis of DNA repair and recombination responses in mouse cells depleted for Brca2 by SiRNA

The tumor suppressor BRCA2 is considered to play an important role in the maintenance of genome integrity through the repair of DNA lesions by homologous recombination. A mechanistic understanding of BRCA2 has been complicated by the embryonic lethality of mice bearing allelic knockouts of Brca2, and by variation in the DNA damage response in cells bearing BRCA2 deficiencies. It would be advantageous to develop approaches that avoid the cell lethality associated with complete inactivation of the gene, or the use of established tumor cell lines in which other genes in addition to BRCA2 may be mutant. In this study, SiRNA was used in stable transformation assays to knockdown Brca2 in mouse hybridoma cells by at least 75%. The Brca2-depleted cells were analyzed with respect to cell growth, sensitivity to DNA damaging agents (mitomycin C, methylmethane sulfonate, or ionizing radiation), intrachromosomal homologous recombination and gene targeting. Although the effect of Brca2-depletion on cell growth and sensitivity to DNA damaging agents was modest, the Brca2-depleted cells did show a significant shift in homologous recombination from gene conversion to single-strand annealing and a significant decrease in the efficiency of gene targeting. Both of these phenotypes are consistent with the proposed role of Brca2 in DNA repair and recombination.     

BRCA1 facilitates microhomology-mediated end joining of DNA double strand breaks

BRCA1 is critical for the maintenance of genomic stability, in part through its interaction with the Rad50.Mre11.Nbs1 complex, which occupies a central role in DNA double strand break repair mediated by nonhomologous end joining (NHEJ) and homologous recombination. BRCA1 has been shown to be required for homology-directed recombination repair. However, the role of BRCA1 in NHEJ, a critical pathway for the repair of double strand breaks and genome stability in mammalian cells, remains elusive. Here, we established a pair of mouse embryonic fibroblasts (MEFs) derived from 9.5-day-old embryos with genotypes Brca1(+/+):p53(-/-) or Brca1(-/-):p53(-/-). The Brca1(-/-):p53(-/-) MEFs appear to be extremely sensitive to ionizing radiation. The contribution of BRCA1 in NHEJ was evaluated in these cells using three different assay systems. First, transfection of a linearized plasmid in which expression of the reporter gene required precise end joining indicated that Brca1(-/-) MEFs display a moderate deficiency when compared with Brca1(+/+) cells. Second, using a retrovirus infection assay dependent on NHEJ, a 5-10-fold reduction in retroviral integration efficiency was observed in Brca1(-/-) MEFs when compared with the Brca1(+/+) MEFs. Third, Brca1(-/-) MEFs exhibited a 50-100-fold deficiency in microhomology-mediated end-joining activity of a defined chromosomal DNA double strand break introduced by a rare cutting endonuclease I-SceI. These results provide evidence that Brca1 has an essential role in microhomology-mediated end joining and suggest a novel molecular basis for its caretaker role in the maintenance of genome integrity.     

The role of the BRCA1 tumor suppressor in DNA double-strand break repair

The tumor suppressor gene BRCA1 was cloned in 1994 based on its linkage to early-onset breast and ovarian cancer. Although the BRCA1 protein has been implicated in multiple cellular functions, the precise mechanism that determines its tumor suppressor activity is not defined. Currently, the emerging picture is that BRCA1 plays an important role in maintaining genomic integrity by protecting cells from double-strand breaks (DSB) that arise during DNA replication or after DNA damage. The DSB repair pathways available in mammalian cells are homologous recombination and nonhomologous end-joining. BRCA1 function seems to be regulated by specific phosphorylations in response to DNA damage and we will focus this review on the roles played by BRCA1 in DNA repair and cell cycle checkpoints. Finally, we will explore the idea that tumor suppression by BRCA1 depends on its control of DNA DSB repair, resulting in the promotion of error-free and the inhibition of error-prone recombinational repair.     

Drosophila brca2 is required for mitotic and meiotic DNA repair and efficient activation of the meiotic recombination checkpoint

Heterozygous mutations in the tumor suppressor BRCA2 confer a high risk of breast and other cancers in humans. BRCA2 maintains genome stability in part through the regulation of Rad51-dependent homologous recombination. Much about its precise function in the DNA damage responses is, however, not yet known. We have made null mutations in the Drosophila homolog of BRCA2 and measured the levels of homologous recombination, non-homologous end-joining, and single-strand annealing in the pre-meiotic germline of Drosophila males. We show that repair by homologous recombination is dramatically decreased in Drosophila brca2 mutants. Instead, large flanking deletions are formed, and repair by the non-conservative single-strand annealing pathway predominates. We further show that during meiosis, Drosophila Brca2 has a dual role in the repair of meiotic double-stranded breaks and the efficient activation of the meiotic recombination checkpoint. The eggshell patterning defects that result from activation of the meiotic recombination checkpoint in other meiotic DNA repair mutants can be strongly suppressed by mutations in brca2. In addition, Brca2 co-immunoprecipitates with the checkpoint protein Rad9, suggesting a direct role for Brca2 in the transduction of the meiotic recombination checkpoint signal.     

Male Fertility Defect Associated with Disrupted BRCA1-PALB2 Interaction in Mice

PALB2 links BRCA1 and BRCA2 in homologous recombinational repair of DNA double strand breaks (DSBs). Mono-allelic mutations in PALB2 increase the risk of breast, pancreatic, and other cancers, and biallelic mutations cause Fanconi anemia (FA). Like Brca1 and Brca2, systemic knock-out of Palb2 in mice results in embryonic lethality. In this study, we generated a hypomorphic Palb2 allele expressing a mutant PALB2 protein unable to bind BRCA1. Consistent with an FA-like phenotype, cells from the mutant mice showed hypersensitivity and chromosomal breakage when treated with mitomycin C, a DNA interstrand crosslinker. Moreover, mutant males showed reduced fertility due to impaired meiosis and increased apoptosis in germ cells. Interestingly, mutant meiocytes showed a significant defect in sex chromosome synapsis, which likely contributed to the germ cell loss and fertility defect. Our results underscore the in vivo importance of the PALB2-BRCA1 complex formation in DSB repair and male meiosis.     

Chk2 phosphorylation of BRCA1 regulates DNA double-strand break repair

The pathway determining malignant cellular transformation, which depends upon mutation of the BRCA1 tumor suppressor gene, is poorly defined. A growing body of evidence suggests that promotion of DNA double-strand break repair by homologous recombination (HR) may be the means by which BRCA1 maintains genomic stability, while a role of BRCA1 in error-prone nonhomologous recombination (NHR) processes has just begun to be elucidated. The BRCA1 protein becomes phosphorylated in response to DNA damage, but the effects of phosphorylation on recombinational repair are unknown. In this study, we tested the hypothesis that the BRCA1-mediated regulation of recombination requires the Chk2- and ATM-dependent phosphorylation sites. We studied Rad51-dependent HR and random chromosomal integration of linearized plasmid DNA, a subtype of NHR, which we demonstrate to be dependent on the Mre11-Rad50-Nbs1 complex. Prevention of Chk2-mediated phosphorylation via mutation of the serine 988 residue of BRCA1 disrupted both the BRCA1-dependent promotion of HR and the suppression of NHR. Similar results were obtained when endogenous Chk2 kinase activity was inhibited by expression of a dominant-negative Chk2 mutant. Surprisingly, the opposing regulation of HR and NHR did not require the ATM phosphorylation sites on serines 1423 and 1524. Together, these data suggest a functional link between recombination control and breast cancer predisposition in carriers of Chk2 and BRCA1 germ line mutations. We propose a dual regulatory role for BRCA1 in maintaining genome integrity, whereby BRCA1 phosphorylation status controls the selectivity of repair events dictated by HR and error-prone NHR.     

BRCA2 is required for neurogenesis and suppression of medulloblastoma

Defective DNA damage responses in the nervous system can result in neurodegeneration or tumorigenesis. Despite the importance of DNA damage signalling, the neural function of many critical DNA repair factors is unclear. BRCA2 is necessary for homologous recombination repair of DNA and the prevention of diseases including Fanconi Anemia and cancer. We determined the role of BRCA2 during brain development by inactivating murine Brca2 throughout neural tissues. In striking contrast to early embryonic lethality after germ-line inactivation, Brca2(LoxP/LoxP);Nestin-cre mice were viable. However, Brca2 loss profoundly affected neurogenesis, particularly during embryonic and postnatal neural development. These neurological defects arose from DNA damage as Brca2(LoxP/LoxP);Nestin-cre mice showed extensive gammaH2AX in neural tissue and p53 deficiency restored brain histology but lead to rapid formation of medulloblastoma brain tumors. In contrast, loss of the Atm kinase did not markedly attenuate apoptosis after Brca2 loss, but did partially restore cerebellar morphology, supporting a genomic surveillance function for ATM during neurogenesis. These data illustrate the importance of Brca2 during nervous system development and underscore the tissue-specific requirements for DNA repair factors.     

DNA double-strand break repair pathway choice and cancer

Since DNA double-strand breaks (DSBs) contribute to the genomic instability that drives cancer development, DSB repair pathways serve as important mechanisms for tumor suppression. Thus, genetic lesions, such as BRCA1 and BRCA2 mutations, that disrupt DSB repair are often associated with cancer susceptibility. In addition, recent evidence suggests that DSB “mis-repair”, in which DSBs are resolved by an inappropriate repair pathway, can also promote genomic instability and presumably tumorigenesis. This notion has gained currency from recent cancer genome sequencing studies which have uncovered numerous chromosomal rearrangements harboring pathological DNA repair signatures. In this perspective, we discuss the factors that regulate DSB repair pathway choice and their consequences for genome stability and cancer.     

BRCA1-Ku80 Protein Interaction Enhances End-joining Fidelity of Chromosomal Double-strand Breaks in the G1 Phase of the Cell Cycle

Quality control of DNA double-strand break (DSB) repair is vital in preventing mutagenesis. Non-homologous end-joining (NHEJ), a repair process predominant in the G₁ phase of the cell cycle, rejoins DSBs either accurately or with errors, but the mechanisms controlling its fidelity are poorly understood. Here we show that BRCA1, a tumor suppressor, enhances the fidelity of NHEJ-mediated DSB repair and prevents mutagenic deletional end-joining through interaction with canonical NHEJ machinery during G₁. BRCA1 binds and stabilizes Ku80 at DSBs through its N-terminal region, promotes precise DSB rejoining, and increases cellular resistance to radiation-induced DNA damage in a G₁ phase-specific manner. These results suggest that BRCA1, as a central player in genome integrity maintenance, ensures high fidelity repair of DSBs by not only promoting homologous recombination repair in G₂/M phase but also facilitating fidelity of Ku80-dependent NHEJ repair, thus preventing deletional end-joining of chromosomal DSBs during G₁.     

Evidence that extrachromosomal double-strand break repair can be coupled to the repair of chromosomal double-strand breaks in mammalian cells

Transfected linear DNA molecules are substrates for double-strand break (DSB) repair in mammalian cells. The DSB repair process can involve recombination between the transfected DNA molecules, between the transfected molecules and chromosomal DNA, or both. In order to determine whether these different types of repair events are linked, we devised assays enabling us to follow the fate of linear extrachromosomal DNA molecules involved in both interplasmid and chromosome-plasmid recombination, in the presence or absence of a pre-defined chromosomal DSB. Plasmid-based vectors were designed that could either recombine via interplasmid recombination or chromosome-plasmid recombination to produce a functional beta-galactosidase (betagal) fusion gene. By measuring the frequency of betagal+ cells at 36 h post-transfection versus the frequency of betagal+ clones after 14 days, we found that the number of cells containing extrachromosomal recombinant DNA molecules at 36 h (i.e., betagal+), either through interplasmid or chromosome-plasmid recombination, was nearly the same as the number of cells integrating these recombinant molecules. Furthermore, when a predefined DSB was created at a chromosomal site, the extrachromosomal recombinant DNA molecules were shown to integrate preferentially at that site by Southern and fiber-FISH (fluorescence in situ hybridization) analysis. Together these data indicate that the initial recombination event can potentiate or commit extrachromosomal DNA to integration in the genome at the site of a chromosomal DSB. The efficiency at which extrachromosomal recombinant molecules are used as substrates in chromosomal DSB repair suggests extrachromosomal DSB repair can be coupled to the repair of chromosomal DSBs in mammalian cells.     

Characterization of Brca2-deficient plants excludes the role of NHEJ and SSA in the meiotic chromosomal defect phenotype

In somatic cells, three major pathways are involved in the repair of DNA double-strand breaks (DBS): Non-Homologous End Joining (NHEJ), Single-Strand Annealing (SSA) and Homologous Recombination (HR). In somatic and meiotic HR, DNA DSB are 5' to 3' resected, producing long 3' single-stranded DNA extensions. Brca2 is essential to load the Rad51 recombinase onto these 3' overhangs. The resulting nucleofilament can thus invade a homologous DNA sequence to copy and restore the original genetic information. In Arabidopsis, the inactivation of Brca2 specifically during meiosis by an RNAi approach results in aberrant chromosome aggregates, chromosomal fragmentation and missegregation leading to a sterility phenotype. We had previously suggested that such chromosomal behaviour could be due to NHEJ. In this study, we show that knock-out plants affected in both BRCA2 genes show the same meiotic phenotype as the RNAi-inactivated plants. Moreover, it is demonstrated that during meiosis, neither NHEJ nor SSA compensate for HR deficiency in BRCA2-inactivated plants. The role of the plant-specific DNA Ligase6 is also excluded. The possible mechanism(s) involved in the formation of these aberrant chromosomal bridges in the absence of HR during meiosis are discussed.     

Similar effects of Brca2 truncation and Rad51 paralog deficiency on immunoglobulin V gene diversification in DT40 cells support an early role for Rad51 paralogs in homologous recombination

BRCA2 is a tumor suppressor gene that is linked to hereditary breast and ovarian cancer. Although the Brca2 protein participates in homologous DNA recombination (HR), its precise role remains unclear. From chicken DT40 cells, we generated BRCA2 gene-deficient cells which harbor a truncation at the 3' end of the BRC3 repeat (brca2tr). Comparison of the characteristics of brca2tr cells with those of other HR-deficient DT40 clones revealed marked similarities with rad51 paralog mutants (rad51b, rad51c, rad51d, xrcc2, or xrcc3 cells). The phenotypic similarities include a shift from HR-mediated diversification to single-nucleotide substitutions in the immunoglobulin variable gene segment and the partial reversion of this shift by overexpression of Rad51. Although recent evidence supports at least Xrcc3 and Rad51C playing a role late in HR, our data suggest that Brca2 and the Rad51 paralogs may also contribute to HR at the same early step, with their loss resulting in the stimulation of an alternative, error-prone repair pathway.     

Identification of a Saccharomyces cerevisiae Ku80 homologue: roles in DNA double strand break rejoining and in telomeric maintenance.

Ku is a heterodimer of polypeptides of approximately 70 and 80 kDa (Ku70 and Ku80, respectively) that binds to DNA ends. Mammalian cells lacking Ku are defective in DNA double-strand break (DSB) repair and in site-specific V(D)J recombination. Here, we describe the identification and characterisation of YKU80, the gene for the Saccharomyces cerevisiae Ku80 homologue. Significantly, we find that YKU80 disruption enhances the radiosensitivity of rad52 mutant strains, suggesting that YKU80 functions in a DNA DSB repair pathway that does not rely on homologous recombination. Indeed, through using an in vivo plasmid rejoining assay, we find that YKU80 plays an essential role in illegitimate recombination events that result in the accurate repair of restriction enzyme generated DSBs. Interestingly, in the absence of YKU80function, residual repair operates through an error-prone pathway that results in recombination between short direct repeat elements. This resembles closely a predominant DSB repair pathway in vertebrates. Together, our data suggest that multiple, evolutionarily conserved mechanisms for DSB repair exist in eukaryotes. Furthermore, they imply that Ku binds to DSBs in vivo and promotes repair both by enhancing accurate DNA end joining and by suppressing alternative error-prone repair pathways. Finally, we report that yku80 mutant yeasts display dramatic telomeric shortening, suggesting that, in addition to recognising DNA damage, Ku also binds to naturally occurring chromosomal ends. These findings raise the possibility that Ku protects chromosomal termini from nucleolytic attack and functions as part of a telomeric length sensing system.     

BRCA1 functions independently of homologous recombination in DNA interstrand crosslink repair

Brca1 is required for DNA repair by homologous recombination (HR) and normal embryonic development. Here we report that deletion of the DNA damage response factor 53BP1 overcomes embryonic lethality in Brca1-nullizygous mice and rescues HR deficiency, as measured by hypersensitivity to polyADP-ribose polymerase (PARP) inhibition. However, Brca1,53BP1 double-deficient cells are hypersensitive to DNA interstrand crosslinks (ICLs), indicating that BRCA1 has an additional role in DNA crosslink repair that is distinct from HR. Disruption of the nonhomologous end-joining (NHEJ) factor, Ku, promotes DNA repair in Brca1-deficient cells; however deletion of either Ku or 53BP1 exacerbates genomic instability in cells lacking FANCD2, a mediator of the Fanconi anemia pathway for ICL repair. BRCA1 therefore has two separate roles in ICL repair that can be modulated by manipulating NHEJ, whereas FANCD2 provides a key activity that cannot be bypassed by ablation of 53BP1 or Ku.     

Genetic steps of mammalian homologous repair with distinct mutagenic consequences

Repair of chromosomal breaks is essential for cellular viability, but misrepair generates mutations and gross chromosomal rearrangements. We investigated the interrelationship between two homologous-repair pathways, i.e., mutagenic single-strand annealing (SSA) and precise homology-directed repair (HDR). For this, we analyzed the efficiency of repair in mammalian cells in which double-strand break (DSB) repair components were disrupted. We observed an inverse relationship between HDR and SSA when RAD51 or BRCA2 was impaired, i.e., HDR was reduced but SSA was increased. In particular, expression of an ATP-binding mutant of RAD51 led to a >90-fold shift to mutagenic SSA repair. Additionally, we found that expression of an ATP hydrolysis mutant of RAD51 resulted in more extensive gene conversion, which increases genetic loss during HDR. Disruption of two other DSB repair components affected both SSA and HDR, but in opposite directions: SSA and HDR were reduced by mutation of Brca1, which, like Brca2, predisposes to breast cancer, whereas SSA and HDR were increased by Ku70 mutation, which affects nonhomologous end joining. Disruption of the BRCA1-associated protein BARD1 had effects similar to those of mutation of BRCA1. Thus, BRCA1/BARD1 has a role in homologous repair before the branch point of HDR and SSA. Interestingly, we found that Ku70 mutation partially suppresses the homologous-repair defects of BARD1 disruption. We also examined the role of RAD52 in homologous repair. In contrast to yeast, Rad52(-)(/)(-) mouse cells had no detectable HDR defect, although SSA was decreased. These results imply that the proper genetic interplay of repair factors is essential to limit the mutagenic potential of DSB repair.     

BRC-1 acts in the inter-sister pathway of meiotic double-strand break repair

The breast and ovarian cancer susceptibility protein BRCA1 is evolutionarily conserved and functions in DNA double-strand break (DSB) repair through homologous recombination, but its role in meiosis is poorly understood. By using genetic analysis, we investigated the role of the Caenorhabditis elegans BRCA1 orthologue (brc-1) during meiotic prophase. The null mutant in the brc-1 gene is viable, fertile and shows the wild-type complement of six bivalents in most diakinetic nuclei, which is indicative of successful crossover recombination. However, brc-1 mutants show an abnormal increase in apoptosis and RAD-51 foci at pachytene that are abolished by loss of spo-11 function, suggesting a defect in meiosis rather than during premeiotic DNA replication. In genetic backgrounds in which chiasma formation is abrogated, such as him-14/MSH4 and syp-2, loss of brc-1 leads to chromosome fragmentation suggesting that brc-1 is dispensable for crossing over but essential for DSB repair through inter-sister recombination.     

Recognition of DNA double strand breaks by the BRCA1 tumor suppressor network

DNA double-strand breaks (DSBs) occur in response to both endogenous and exogenous genotoxic stress. Inappropriate repair of DSBs can lead to either loss of viability or to chromosomal alterations that increase the likelihood of cancer development. In strong support of this assertion, many cancer predisposition syndromes stem from germline mutations in genes involved in DNA DSB repair. Among the most prominent of such tumor suppressor genes are the Breast Cancer 1 and Breast Cancer 2 genes (BRCA1 and BRCA2), which are mutated in familial forms of breast and ovarian cancer. Recent findings implicate BRCA1 as a central component of several distinct macromolecular protein complexes, each dedicated to distinct elements of DNA DSB repair and tumor suppression. Emerging evidence has shed light on some of the molecular recognition processes that are responsible for targeting BRCA1 and its associated partners to DNA and chromatin directly flanking DSBs. These events are required for BRCA1-dependent DNA repair and tumor suppression. Thus, a detailed temporal and spatial knowledge of how breaks are recognized and repaired has profound implications for understanding processes related to the genesis of malignancy and to its treatment.