Florence Larminat

Deficiency in BRCA2 leads to increase in non-conservative homologous recombination

The BRCA2 tumor suppressor has been implicated in the maintenance of genomic integrity through a function in cellular responses to DNA damage. The BRCA2 protein directly associates with Rad51, that is essential for repair of double-strand breaks (DSBs) by homologous recombination (HR). In this report, we study the BRCA2-defective Chinese hamster cell mutant V-C8 for its ability to perform homology-directed repair (HDR) between repeated sequences. V-C8 cells were recently shown to be defective in Rad51 foci formation in response to DNA damage. Strikingly, we find that these BRCA2 mutant cells exhibit a strong stimulation of HDR activity compared to the V79 parental cells, which harbor a wild-type BRCA2. Furthermore, molecular characterization of the HDR products shows that loss of BRCA2 in V-C8 cells leads to significant reduction in Rad51-dependent gene conversion but strong enhancement of Rad51-independent single-strand annealing (SSA) events frequency. These data imply that, when HDR by conservative gene conversion is impaired, DSBs usually repaired by this pathway are instead resolved by other non-conservative HDR subpathways. Therefore, high chromosomal instability in BRCA2-deficient cells presumably results from enhancement of error-prone repair mechanisms, such as SSA.

Modulation of the SOS response by truncated RecA proteins.

SummaryRecA protein plays several key roles in the SOS response. We have constructed truncated proteins and examined their capacity to accomplish Weigle reactivation and mutagenesis of bacteriophage lambda and recombination in Escherichia coli. Our data indicate that the 17 carboxyl terminal amino acids are not essential to RecA function. However in the presence of wild-type RecA protein, the truncated protein reduces the efficiency of recombination without affecting either mutagenesis or induction of an SOS gene or Weigle reactivation. The data presented here suggest that activation of RecA protein does not involve mixed multimers or is not affected by their presence.

Mitochondrial proteomic approach reveals galectin-7 as a novel BCL-2 binding protein in human cells

Our results reveal a network of new potential Bcl-2 partners identified through the Bcl-2 immunocapture and mass spectrometry approach and analyzed by gene ontology mining. Importantly, we report for the first time the identification of galectin-7, a member of a family of β-galactoside-binding lectins, as a new mitochondrial Bcl-2 interacting partner.

Overexpression of Metallothionein-II Sensitizes Rodent Cells to Apoptosis Induced by DNA Cross-linking Agent through Inhibition of NF-κB Activation

DNA cross-linking agents such as mitomycin C (MMC) and cisplatin are used as chemotherapeutic agents in cancer treatment. However, the molecular mechanism underlying their antitumor activity is not entirely clear. Critical steps in cytotoxicity toward cross-linking agents can involve DNA repair efficiency, inhibition of replication, cell-cycle checkpoints, regulation, and induction of apoptosis. The complexity of the mechanisms of the mammalian cell defense against cross-linking agents is reflected by the existence of many complementation groups identified in rodent cells that are specifically sensitive to MMC. We recently showed that increased induction of apoptosis contributes to the MMC sensitivity of the group represented by the V-H4 hamster mutant cell line. In this study, through the analyses of a substractive library, we discovered that sensitive V-H4 cells display a 40-fold increase of steady-state expression of metallothionein II (MT-II) mRNA compared with resistant parental V79 cells. Down-regulation of MT-II by antisense oligonucleotides partially restores MMC resistance in V-H4 cells, indicating that MT-II overexpression is directly involved in MMC hypersensitivity of these cells. MTs have been reported to regulate the activation of NF-κB, one of the key proteins that modulates the apoptotic response. Here we found that NF-κB activation by MMC is impaired in V-H4 cells and is partially restored following down-regulation of MT-II by antisense oligonucleotides. All these data suggest that the overexpression of MT-II in V-H4 cells impairs NF-κB activation by MMC, resulting in decreased cell survival and enhanced induction of apoptosis.

Lack of correlation between repair of DNA interstrand cross-links and hypersensitivity of hamster cells towards mitomycin C and cisplatin

The ability to repair DNA interstrand cross‐links may be an important factor contributing to mitomycin C (MMC) and cisplatin cytotoxicities. We have assessed the repair of interstrand cross‐links induced by MMC in two MMC‐hypersensitive hamster cell mutants and their resistant parental cell line. Using a gene‐specific repair assay, we found no evidence for repair of MMC cross‐links in either parental or mutant cells, suggesting that persistence of DNA interstrand cross‐links is not responsible for the differential toxicity of MMC towards hypersensitive cells. Repair of cisplatin‐induced interstrand cross‐links was efficient in resistant as well as in mutant cells. Therefore we concluded that a defect in excision repair of interstrand cross‐links was not responsible for the cytotoxic effects of MMC and cisplatin in these hypersensitive mutants.

Impairment of homologous recombination control in a Fanconi anemia‐like Chinese Hamster Cell mutant

Summry— DNA interstrand cross‐links (ICL)‐inducing agents such as cisplatin, mitomycin C (MMC) and nitrogen mustards are widely used as potent antitumor drugs. Although ICL repair mechanism is not yet well characterized in mammalian cells, this pathway is thought to involve a sequential action of nucleotide excision repair (NER) and homologous recombination (HR). The importance of unraveling ICL repair pathways is highlighted by the hypersensitivity to ICL‐inducing agents in cells of patients with the genetic disease Fanconi anemia (FA) and in cells mutated in the Breast Cancer susceptibility genes BRCA1 and BRCA2. To better characterize the involvement of HR in the sensitivity to ICL‐inducing agents, we examined spontaneous and ICL‐induced HR in rodent FA‐like V‐H4 cells. In this report, we show that MMC‐hypersensitive V‐H4 cells exhibit an increased spontaneous homology‐directed repair (HDR) activity compared to the resistant V79 parental cells. Elevated HDR activity results mainly in increased conservative Rad51‐dependent recombination, without affecting non‐conservative single‐strand annealing process (SSA). We also show that HDR activity is enhanced following MMC treatment in parental cells, but not in rodent FA‐like V‐H4 cells. Moreover, our data indicate that Rad51 foci formation is significantly delayed in these FA‐like cells in response to crosslinking agent. These findings provide evidence for an impairment of HR control in V‐H4 cells and emphasize the involvement of the FA pathway in HR‐mediated repair.

Polo-like kinase 1 mediates BRCA1 phosphorylation and recruitment at DNA double-strand breaks

Accurate repair of DNA double-strand breaks (DSB) caused during DNA replication and by exogenous stresses is critical for the maintenance of genomic integrity. There is growing evidence that the Polo-like kinase 1 (Plk1) that plays a number of pivotal roles in cell proliferation can directly participate in regulation of DSB repair. In this study, we show that Plk1 regulates BRCA1, a key mediator protein required to efficiently repair DSB through homologous recombination (HR). Following induction of DSB, BRCA1 concentrates in distinctive large nuclear foci at damage sites where multiple DNA repair factors accumulate. First, we found that inhibition of Plk1 shortly before DNA damage sensitizes cells to ionizing radiation and reduces DSB repair by HR. Second, we provide evidence that BRCA1 foci formation induced by DSB is reduced when Plk1 is inhibited or depleted. Third, we identified BRCA1 as a novel Plk1 substrate and determined that Ser1164 is the major phosphorylation site for Plk1 in vitro. In cells, mutation of Plk1 sites on BRCA1 significantly delays BRCA1 foci formation following DSB, recapitulating the phenotype observed upon Plk1 inhibition. Our data then assign a key function to Plk1 in BRCA1 foci formation at DSB, emphasizing Plk1 importance in the HR repair of human cells.

Loss of BRCA1 impairs centromeric cohesion and triggers chromosomal instability

In contrast to its well‐known role in the DNA damage response during interphase, the function of BRCA1 in the maintenance of chromosomal stability during mitosis remains to be defined. In this study, we uncover a novel role of BRCA1 in preserving centromere integrity in mitotic human cells. Using immunofluorescence and chromatin immunoprecipitation approaches, we report BRCA1 association with centromeric chromatin during mitosis. BRCA1 depletion impairs centromeric cohesion, leading to an increase in interkinetochore distance and in unpaired sister‐chromatids frequency during prometaphase. Moreover, BRCA1 loss partially decreased accumulation of the Aurora B kinase at the centromere. We found that proper recruitment of the DNMT3b DNA methyltransferase to satellite sequences is BRCA1‐dependent during mitosis, suggesting that DNA hypomethylation contributes to Aurora B mislocalization. BRCA1‐deficient cells exhibited decreased ability to correct improper Aurora B‐dependent chromosome‐spindle attachments and to align chromosomes at metaphase. Finally, we show that BRCA1 disruption promotes merotelic kinetochore attachments that represent a major mechanism of aneuploidy in human cells. In summary, we report here a novel function of BRCA1 in maintaining chromosomal stability through its contribution to the mitotic centromere integrity necessary for faithful segregation of sister‐chromatids during cell division.—Di Paolo, A., Racca, C., Calsou, P., and Larminat, F., Loss of BRCA1 impairs centromeric cohesion and triggers chromosomal instability. FASEB J. 28, 5250–5261 (2014). www.fasebj.org