RING finger nuclear factor RNF168 is important for defects in homologous recombination caused by loss of the breast cancer susceptibility factor BRCA1.

BACKGROUND: RNF168 promotes chromosomal break localization of 53BP1 and BRCA1; 53BP1 loss rescues homologous recombination (HR) in BRCA1-deficient cells. RESULTS: RNF168 depletion suppresses HR defects caused by BRCA1 silencing; RNF168 influences HR similarly to 53BP1. CONCLUSION: RNF168 is important for HR defects caused by BRCA1 loss. SIGNIFICANCE: Although RNF168 promotes BRCA1 and 53BP1 localization to chromosomal breaks, RNF168 affects HR similarly to 53BP1. 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 secret life of Bcl-2: apoptosis-independent inhibition of DNA repair by Bcl-2 family members.

Programmed cell death and DNA repair are two fundamental biological processes that play essential roles in cell fate and genetic transmission. The canonical role of Bcl-2 family members is the regulation of programmed cell death. Strikingly, numerous studies from different laboratories have shown that although Bcl-2 increases cell survival, it also inhibits all DNA repair systems, resulting in genome instability/variability. Bcl-2 affects the mechanistically distinct DNA repair systems via different mechanisms. These effects are generally independent of the regulation of apoptosis, revealing additional roles for Bcl-2. The targets of Bcl-2 include APE1, MSH2, PARP1, Ku70 and the oncosuppressor BRCA1. Targetting BRCA1 should be of particular importance because this might impact many essential cellular processes in which BRCA1 is involved, including homologous recombination (HR), non-homologous end joining (NHEJ), base excision repair, cell-cycle regulation, cell death, ubiquitination, inactivation of the X-chromosome, transcription, and protein translation. Beside the pathological consequences, inhibition of DNA repair by Bcl-2 can be, in contrast, advantageously used in some physiological situations: (1) repression of excessive unschedule HR, thus protecting against the accumulation of toxic HR intermediates and HR-dependent genome rearrangements; (2) inhibition of NHEJ might protect against retrovirus integration; (3) it has been proposed that inhibition of mismatch repair might also favors hypermutation at immunoglobulin genes. Finally, because Bcl-2 affects the maintenance of genome stability, one can suggest Bcl-2 might play a role in molecular evolution. Bcl-2 family members control cell death through complex stochiometric equilibriums. Incorporating DNA repair proteins to such an elaborate network should allow for a fine tuning of the coordinated control of cell viability and genetic stability/instability. Relationships between DNA repair and regulation of cell death represent exciting challenges for future prospects and are essential for the development of promising new strategies against cancer.

Suberoylanilide hydroxamic acid as a radiosensitizer through modulation of RAD51 protein and inhibition of homology-directed repair in multiple myeloma.

Histone deacetylase inhibitors (HDI) have shown promise as candidate radiosensitizers for many types of cancers. However, the mechanisms of action are not well understood, and whether they could sensitize multiple myeloma (MM) to radiation therapy is unclear. In this study, we show that suberoylanilide hydroxamic acid (SAHA) at low concentrations has minimal cytotoxic effects, yet can significantly increase radiosensitivity of MM cells. SAHA seems to block RAD51 protein response to ionizing radiation, consistent with an inhibitory effect on the formation of RAD51 focus in irradiated MM cells. These effects of SAHA on RAD51 focus are independent of cell-cycle distribution changes. Furthermore, we show that SAHA selectively inhibits the homology-directed repair (HDR) pathway. The results of this study suggest that SAHA, a recently approved HDI in clinical trials for malignancies, at lower concentrations may act as a radiosensitizer via disruption of the RAD51-dependent HDR pathway.

The relative efficiency of homology-directed repair has distinct effects on proper anaphase chromosome separation.

Homology-directed repair (HDR) is essential to limit mutagenesis, chromosomal instability (CIN) and tumorigenesis. We have characterized the consequences of HDR deficiency on anaphase, using markers for incomplete chromosome separation: DAPI-bridges and Ultra-fine bridges (UFBs). We show that multiple HDR factors (Rad51, Brca2 and Brca1) are critical for complete chromosome separation during anaphase, while another chromosome break repair pathway, non-homologous end joining, does not affect chromosome segregation. We then examined the consequences of mild versus severe HDR disruption, using two different dominant-negative alleles of the strand exchange factor, Rad51. We show that mild HDR disruption is viable, but causes incomplete chromosome separation, as detected by DAPI-bridges and UFBs, while severe HDR disruption additionally results in multipolar anaphases and loss of clonogenic survival. We suggest that mild HDR disruption favors the proliferation of cells that are prone to CIN due to defective chromosome separation during anaphase, whereas, severe HDR deficiency leads to multipolar divisions that are prohibitive for cell proliferation.

Bcl-2 inhibits nuclear homologous recombination by localizing BRCA1 to the endomembranes.

Genetic stability requires coordination of a network of pathways including DNA repair/recombination and apoptosis. In addition to its canonical anti-apoptotic role, Bcl-2 negatively impacts genome stability. In this study, we identified the breast cancer tumor suppressor BRCA1, which plays an essential role in homologous recombination (HR), as a target for Bcl-2 in the repression of HR. Indeed, ionizing radiation-induced BRCA1 foci assembly was repressed when Bcl-2 was expressed ectopically, in human SV40 fibroblasts, or spontaneously, in lymphoma t(14:18) cells and in HeLa and H460 cancer cell lines. Moreover, we showed that the transmembrane (TM) domain of Bcl-2 was required for both inhibition of BRCA1 foci assembly and the inhibition of HR induced by a double-strand break targeted into an intrachromosomal HR substrate by the meganuclease I-SceI. Fluorescence confocal microscopy, proximity ligation assay, and electron microscopy analyses as well as Western blot analysis of subcellular fractions showed that Bcl-2 and BRCA1 colocalized to mitochondria and endoplasmic reticulum in a process requiring the TM domain of Bcl-2. Targeting BRCA1 to the endomembranes depletes BRCA1 from the nucleus and, thus, accounts for the inhibition of HR. Furthermore, our findings support an apoptosis-stimulatory role for the cytosolic form of BRCA1, suggesting a new tumor suppressor function of BRCA1. Together, our results reveal a new mode of BRCA1 regulation and for HR in the maintenance of genome stability.

Mammalian Fbh1 is important to restore normal mitotic progression following decatenation stress.

We have addressed the role of the F-box helicase 1 (Fbh1) protein during genome maintenance in mammalian cells. For this, we generated two mouse embryonic stem cell lines deficient for Fbh1: one with a homozygous deletion of the N-terminal F-box domain (Fbh1(f/f)), and the other with a homozygous disruption (Fbh1(-/-)). Consistent with previous reports of Fbh1-deficiency in vertebrate cells, we found that Fbh1(-/-) cells show a moderate increase in Rad51 localization to DNA damage, but no clear defect in chromosome break repair. In contrast, we found that Fbh1(f/f) cells show a decrease in Rad51 localization to DNA damage and increased cytoplasmic localization of Rad51. However, these Fbh1(f/f) cells show no clear defects in chromosome break repair. Since some Rad51 partners and F-box-associated proteins (Skp1-Cul1) have been implicated in progression through mitosis, we considered whether Fbh1 might play a role in this process. To test this hypothesis, we disrupted mitosis using catalytic topoisomerase II inhibitors (bisdioxopiperazines), which inhibit chromosome decatenation. We found that both Fbh1(f/f) and Fbh1(-/-) cells show hypersensitivity to topoisomerase II catalytic inhibitors, even though the degree of decatenation stress was not affected. Furthermore, following topoisomerase II catalytic inhibition, both Fbh1-deficient cell lines show substantial defects in anaphase separation of chromosomes. These results indicate that Fbh1 is important for restoration of normal mitotic progression following decatenation stress.

AKT1 inhibits homologous recombination by inducing cytoplasmic retention of BRCA1 and RAD51.

AKT1 is frequently up-regulated in sporadic breast cancer, whereas BRCA1 is frequently mutated in familial breast cancer. Because BRCA1 is involved in homologous recombination (HR), we addressed whether AKT1 also has an effect on this process. We showed that AKT1 repressed HR through cytoplasmic retention of BRCA1 and RAD51 proteins, resulting in a BRCA1-deficient-like phenotype. This process does not require direct BRCA1 phosphorylation by AKT1. The cytoplasmic retention of BRCA1 and RAD51 correlated with activated AKT1 in tumor cell lines and in biopsies from sporadic breast cancers. Under nonpathologic conditions, fibroblast growth factor, which activates AKT1 and stimulates proliferation in fibroblasts, impaired excessive HR without fully inhibiting it, promoting genome stability. Our study reveals that the regulation of BRCA1 and RAD51 is altered in a high frequency of sporadic breast cancers and highlights the role of extracellular AKT signaling-dependent regulation of HR and genome stability.