Transcription inhibition by DRB potentiates recombinational repair of UV lesions in mammalian cells.

Homologous recombination (HR) is intricately associated with replication, transcription and DNA repair in all organisms studied. However, the interplay between all these processes occurring simultaneously on the same DNA molecule is still poorly understood. Here, we study the interplay between transcription and HR during ultraviolet light (UV)-induced DNA damage in mammalian cells. Our results show that inhibition of transcription with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) increases the number of UV-induced DNA lesions (γH2AX, 53BP1 foci formation), which correlates with a decrease in the survival of wild type or nucleotide excision repair defective cells. Furthermore, we observe an increase in RAD51 foci formation, suggesting HR is triggered in response to an increase in UV-induced DSBs, while inhibiting transcription. Unexpectedly, we observe that DRB fails to sensitise HR defective cells to UV treatment. Thus, increased RAD51 foci formation correlates with increased cell death, suggesting the existence of a futile HR repair of UV-induced DSBs which is linked to transcription inhibition.

miR-182-mediated downregulation of BRCA1 impacts DNA repair and sensitivity to PARP inhibitors.

Expression of BRCA1 is commonly decreased in sporadic breast tumors, and this correlates with poor prognosis of breast cancer patients. Here we show that BRCA1 transcripts are selectively enriched in the Argonaute/miR-182 complex and miR-182 downregulates BRCA1 expression. Antagonizing miR-182 enhances BRCA1 protein levels and protects them from IR-induced cell death, while overexpressing miR-182 reduces BRCA1 protein, impairs homologous recombination-mediated repair, and render cells hypersensitive to IR. The impaired DNA repair phenotype induced by miR-182 overexpression can be fully rescued by overexpressing miR-182-insensitive BRCA1. Consistent with a BRCA1-deficiency phenotype, miR-182-overexpressing breast tumor cells are hypersensitive to inhibitors of poly (ADP-ribose) polymerase 1 (PARP1). Conversely, antagonizing miR-182 enhances BRCA1 levels and induces resistance to PARP1 inhibitor. Finally, a clinical-grade PARP1 inhibitor impacts outgrowth of miR-182-expressing tumors in animal models. Together these results suggest that miR-182-mediated downregulation of BRCA1 impedes DNA repair and may impact breast cancer therapy.

Transcription inhibition by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) causes DNA damage and triggers homologous recombination repair in mammalian cells.

Transcription, replication and homologous recombination are intrinsically connected and it is well established that an increase of transcription is associated with an increase in homologous recombination. Here, we have studied how homologous recombination is affected during transcription inhibition by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a compound that prevents activating phosphorylations of the RNA Pol II C-terminal domain. We identify that DRB triggers an increase in homologous recombination within the hprt gene as well as increasing RAD51 foci formation in mammalian cells. Furthermore, we find that DRB-induced transcriptional stress is associated with formation of the nuclear foci of the phosphorylated form of H2AX (γH2AX). We accounted that about 72% of RAD51 foci co-localized with the observed γH2AX foci. Interestingly, we find that XRCC3 mutated, homologous recombination defective cells are hypersensitive to the toxic effect of DRB and fail to form RAD51 foci. In conclusion, we show that DRB-induced transcription inhibition is associated with the formation of a lesion that triggers RAD51-dependent homologous recombination repair, required for survival under transcriptional stress.

Contextual synthetic lethality of cancer cell kill based on the tumor microenvironment.

Acute and chronic hypoxia exists within the three-dimensional microenvironment of solid tumors and drives therapy resistance, genetic instability, and metastasis. Replicating cells exposed to either severe acute hypoxia (16 hours with 0.02% O(2)) followed by reoxygenation or moderate chronic hypoxia (72 hours with 0.2% O(2)) treatments have decreased homologous recombination (HR) protein expression and function. As HR defects are synthetically lethal with poly(ADP-ribose) polymerase 1 (PARP1) inhibition, we evaluated the sensitivity of repair-defective hypoxic cells to PARP inhibition. Although PARP inhibition itself did not affect HR expression or function, we observed increased clonogenic killing in HR-deficient hypoxic cells following chemical inhibition of PARP1. This effect was partially reversible by RAD51 overexpression. PARP1(-/-) murine embryonic fibroblasts (MEF) showed a proliferative disadvantage under hypoxic gassing when compared with PARP1(+/+) MEFs. PARP-inhibited hypoxic cells accumulated γH2AX and 53BP1 foci as a consequence of altered DNA replication firing during S phase-specific cell killing. In support of this proposed mode of action, PARP inhibitor-treated xenografts displayed increased γH2AX and cleaved caspase-3 expression in RAD51-deficient hypoxic subregions in vivo, which was associated with decreased ex vivo clonogenic survival following experimental radiotherapy. This is the first report of selective cell killing of HR-defective hypoxic cells in vivo as a consequence of microenvironment-mediated "contextual synthetic lethality." As all solid tumors contain aggressive hypoxic cells, this may broaden the clinical utility of PARP and DNA repair inhibition, either alone or in combination with radiotherapy and chemotherapy, even in tumor cells lacking synthetically lethal, genetic mutations.

6-thioguanine selectively kills BRCA2-defective tumors and overcomes PARP inhibitor resistance.

Familial breast and ovarian cancers are often defective in homologous recombination (HR) due to mutations in the BRCA1 or BRCA2 genes. Cisplatin chemotherapy or poly(ADP-ribose) polymerase (PARP) inhibitors were tested for these tumors in clinical trials. In a screen for novel drugs that selectively kill BRCA2-defective cells, we identified 6-thioguanine (6TG), which induces DNA double-strand breaks (DSB) that are repaired by HR. Furthermore, we show that 6TG is as efficient as a PARP inhibitor in selectively killing BRCA2-defective tumors in a xenograft model. Spontaneous BRCA1-defective mammary tumors gain resistance to PARP inhibitors through increased P-glycoprotein expression. Here, we show that 6TG efficiently kills such BRCA1-defective PARP inhibitor-resistant tumors. We also show that 6TG could kill cells and tumors that have gained resistance to PARP inhibitors or cisplatin through genetic reversion of the BRCA2 gene. Although HR is reactivated in PARP inhibitor-resistant BRCA2-defective cells, it is not fully restored for the repair of 6TG-induced lesions. This is likely to be due to several recombinogenic lesions being formed after 6TG. We show that BRCA2 is also required for survival from mismatch repair-independent lesions formed by 6TG, which do not include DSBs. This suggests that HR is involved in the repair of 6TG-induced DSBs as well as mismatch repair-independent 6TG-induced DNA lesion. Altogether, our data show that 6TG efficiently kills BRCA2-defective tumors and suggest that 6TG may be effective in the treatment of advanced tumors that have developed resistance to PARP inhibitors or platinum-based chemotherapy.

Poly(ADP-ribose) polymerase is hyperactivated in homologous recombination-defective cells.

Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) is activated by DNA single-strand breaks (SSB) or at stalled replication forks to facilitate DNA repair. Inhibitors of PARP efficiently kill breast, ovarian, or prostate tumors in patients carrying hereditary mutations in the homologous recombination (HR) genes BRCA1 or BRCA2 through synthetic lethality. Here, we surprisingly show that PARP1 is hyperactivated in replicating BRCA2-defective cells. PARP1 hyperactivation is explained by the defect in HR as shRNA depletion of RAD54, RAD52, BLM, WRN, and XRCC3 proteins, which we here show are all essential for efficient HR and also caused PARP hyperactivation and correlated with an increased sensitivity to PARP inhibitors. BRCA2-defective cells were not found to have increased levels of SSBs, and PAR polymers formed in HR-defective cells do not colocalize to replication protein A or gammaH2AX, excluding the possibility that PARP hyperactivity is due to increased SSB repair or PARP induced at damaged replication forks. Resistance to PARP inhibitors can occur through genetic reversion in the BRCA2 gene. Here, we report that PARP inhibitor-resistant BRCA2-mutant cells revert back to normal levels of PARP activity. We speculate that the reason for the sensitivity of HR-defective cells to PARP inhibitors is related to the hyperactivated PARP1 in these cells. Furthermore, the presence of PAR polymers can be used to identify HR-defective cells that are sensitive to PARP inhibitors, which may be potential biomarkers.

Transcription-associated recombination in eukaryotes: link between transcription, replication and recombination.

Homologous recombination (HR) is an important DNA repair pathway and is essential for cellular survival. It plays a major role in repairing replication-associated lesions and is functionally connected to replication. Transcription is another cellular process, which has emerged to have a connection with HR. Transcription enhances HR, which is a ubiquitous phenomenon referred to as transcription-associated recombination (TAR). Recent evidence suggests that TAR plays a role in inducing genetic instability, for example in the THO mutants (Tho2, Hpr1, Mft1 and Thp2) in yeast or during the development of the immune system leading to genetic diversity in mammals. On the other hand, evidence also suggests that TAR may play a role in preventing genetic instability in many different ways, one of which is by rescuing replication during transcription. Hence, TAR is a double-edged sword and plays a role in both preventing and inducing genetic instability. In spite of the interesting nature of TAR, the mechanism behind TAR has remained elusive. Recent advances in the area, however, suggest a link between TAR and replication and show specific genetic requirements for TAR that differ from regular HR. In this review, we aim to present the available evidence for TAR in both lower and higher eukaryotes and discuss its possible mechanisms, with emphasis on its connection with replication.

Transcription-associated recombination is dependent on replication in Mammalian cells.

Transcription can enhance recombination; this is a ubiquitous phenomenon from prokaryotes to higher eukaryotes. However, the mechanism of transcription-associated recombination in mammalian cells is poorly understood. Here we have developed a construct with a recombination substrate in which levels of recombination can be studied in the presence or absence of transcription. We observed a direct enhancement in recombination when transcription levels through the substrate were increased. This increase in homologous recombination following transcription is locus specific, since homologous recombination at the unrelated hprt gene is unaffected. In addition, we have shown that transcription-associated recombination involves both short-tract and long-tract gene conversions in mammalian cells, which are different from double-strand-break-induced recombination events caused by endonucleases. Transcription fails to enhance recombination in cells that are not in the S phase of the cell cycle. Furthermore, inhibition of transcription suppresses induction of recombination at stalled replication forks, suggesting that recombination may be involved in bypassing transcription during replication.