The 9-1-1 checkpoint clamp coordinates resection at DNA double strand breaks.

DNA-end resection, the generation of single-stranded DNA at DNA double strand break (DSB) ends, is critical for controlling the many cellular responses to breaks. Here we show that the conserved DNA damage checkpoint sliding clamp (the 9-1-1 complex) plays two opposing roles coordinating DSB resection in budding yeast. We show that the major effect of 9-1-1 is to inhibit resection by promoting the recruitment of Rad9(53BP1) near DSBs. However, 9-1-1 also stimulates resection by Exo1- and Dna2-Sgs1-dependent nuclease/helicase activities, and this can be observed in the absence of Rad9(53BP1). Our new data resolve the controversy in the literature about the effect of the 9-1-1 complex on DSB resection. Interestingly, the inhibitory role of 9-1-1 on resection is not observed near uncapped telomeres because less Rad9(53BP1) is recruited near uncapped telomeres. Thus, 9-1-1 both stimulates and inhibits resection and the effects of 9-1-1 are modulated by different regions of the genome. Our experiments illustrate the central role of the 9-1-1 checkpoint sliding clamp in the DNA damage response network that coordinates the response to broken DNA ends. Our results have implications in all eukaryotic cells.

The 9-1-1 checkpoint clamp stimulates DNA resection by Dna2-Sgs1 and Exo1.

Single-stranded DNA (ssDNA) at DNA ends is an important regulator of the DNA damage response. Resection, the generation of ssDNA, affects DNA damage checkpoint activation, DNA repair pathway choice, ssDNA-associated mutation and replication fork stability. In eukaryotes, extensive DNA resection requires the nuclease Exo1 and nuclease/helicase pair: Dna2 and Sgs1(BLM). How Exo1 and Dna2-Sgs1(BLM) coordinate during resection remains poorly understood. The DNA damage checkpoint clamp (the 9-1-1 complex) has been reported to play an important role in stimulating resection but the exact mechanism remains unclear. Here we show that the human 9-1-1 complex enhances the cleavage of DNA by both DNA2 and EXO1 in vitro, showing that the resection-stimulatory role of the 9-1-1 complex is direct. We also show that in Saccharomyces cerevisiae, the 9-1-1 complex promotes both Dna2-Sgs1 and Exo1-dependent resection in response to uncapped telomeres. Our results suggest that the 9-1-1 complex facilitates resection by recruiting both Dna2-Sgs1 and Exo1 to sites of resection. This activity of the 9-1-1 complex in supporting resection is strongly inhibited by the checkpoint adaptor Rad9(53BP1). Our results provide important mechanistic insights into how DNA resection is regulated by checkpoint proteins and have implications for genome stability in eukaryotes.

BCL-3 loss sensitises colorectal cancer cells to DNA damage by targeting homologous recombination.

The proto-oncogene BCL-3 is upregulated in a subset of colorectal cancers (CRC), where it has been shown to enhance tumour cell survival. However, although increased expression correlates with poor patient prognosis, the role of BCL-3 in determining therapeutic response remains largely unknown. In this study, we use combined approaches in multiple cell lines and pre-clinical mouse models to investigate the function of BCL-3 in the DNA damage response. We show that suppression of BCL-3 increases γH2AX foci formation and decreases homologous recombination in CRC cells, resulting in reduced RAD51 foci number and increased sensitivity to PARP inhibition. Importantly, a similar phenotype is seen in Bcl3-/- mice, where Bcl3-/- mouse crypts also exhibit sensitivity to DNA damage with increased γH2AX foci compared to wild type mice. Additionally, Apc.Kras-mutant x Bcl3-/- mice are more sensitive to cisplatin chemotherapy compared to wild type mice. Taken together, our results identify BCL-3 as a regulator of the cellular response to DNA damage and suggests that elevated BCL-3 expression, as observed in CRC, could increase resistance of tumour cells to DNA damaging agents including radiotherapy. These findings offer a rationale for targeting BCL-3 in CRC as an adjunct to conventional therapies and suggest that BCL-3 expression in tumours could be a useful biomarker in stratification of rectal cancer patients for neo-adjuvant chemoradiotherapy.

UPF1 promotes the formation of R loops to stimulate DNA double-strand break repair.

DNA-RNA hybrid structures have been detected at the vicinity of DNA double-strand breaks (DSBs) occurring within transcriptional active regions of the genome. The induction of DNA-RNA hybrids strongly affects the repair of these DSBs, but the nature of these structures and how they are formed remain poorly understood. Here we provide evidence that R loops, three-stranded structures containing DNA-RNA hybrids and the displaced single-stranded DNA (ssDNA) can form at sub-telomeric DSBs. These R loops are generated independently of DNA resection but are induced alongside two-stranded DNA-RNA hybrids that form on ssDNA generated by DNA resection. We further identified UPF1, an RNA/DNA helicase, as a crucial factor that drives the formation of these R loops and DNA-RNA hybrids to stimulate DNA resection, homologous recombination, microhomology-mediated end joining and DNA damage checkpoint activation. Our data show that R loops and DNA-RNA hybrids are actively generated at DSBs to facilitate DNA repair.

PARP inhibition prevents escape from a telomere-driven crisis and inhibits cell immortalisation.

Telomeric crisis is the final replicative barrier to cell immortalisation; it is characterised by genome instability and cell death and is triggered when telomeres become critically short and are subjected to fusion. Pre-cancerous lesions, or early stage cancers, often show signs of a telomere crisis, suggesting that escape from telomere crisis is a prerequisite for disease progression. Telomeric crisis therefore represents an attractive, and as yet unexplored, opportunity for therapeutic intervention. Here, we show that two clinically approved PARP inhibitors, selectively eliminate human cells undergoing a telomere-driven crisis. Clonal populations of a colorectal cancer cell line (HCT116), or the plasma cell leukaemia cell line (JJN-3), expressing a dominant-negative telomerase, entered a telomere-driven crisis at defined population doubling points and telomere lengths. The addition of the PARP inhibitors, olaparib or rucaparib prevented these cells from escaping crisis. PARP inhibition did not alter cellular proliferation prior to crisis, rates of telomere erosion or the telomere length at which crisis was initiated, but affected repair of eroded telomeres, resulting in an increased in intra-chromosomal telomere fusion. This was accompanied by enhanced DNA damage checkpoint activation and elevated levels of apoptosis. We propose that PARP inhibitors impair the repair of dysfunctional telomeres and/or induce replicative stress at telomeres to inhibit escape from a telomere crisis. This is the first demonstration that a drug can selectively kill cells experiencing telomeric crisis. We propose that this type of drug, which we term 'crisolytic', has the potential to eliminate pre-cancerous lesions and tumours exhibiting short dysfunctional telomeres.

RadB acts in homologous recombination in the archaeon Haloferax volcanii, consistent with a role as recombination mediator.

Homologous recombination plays a central role in the repair of double-strand DNA breaks, the restart of stalled replication forks and the generation of genetic diversity. Regulation of recombination is essential since defects can lead to genome instability and chromosomal rearrangements. Strand exchange is a key step of recombination - it is catalysed by RecA in bacteria, Rad51/Dmc1 in eukaryotes and RadA in archaea. RadB, a paralogue of RadA, is present in many archaeal species. RadB has previously been proposed to function as a recombination mediator, assisting in RadA-mediated strand exchange. In this study, we use the archaeon Haloferax volcanii to provide evidence to support this hypothesis. We show that RadB is required for efficient recombination and survival following treatment with DNA-damaging agents, and we identify two point mutations in radA that suppress the ΔradB phenotype. Analysis of these point mutations leads us to propose that the role of RadB is to act as a recombination mediator, which it does by inducing a conformational change in RadA and thereby promoting its polymerisation on DNA.

Systematic Analysis of the DNA Damage Response Network in Telomere Defective Budding Yeast.

Functional telomeres are critically important to eukaryotic genetic stability. Scores of proteins and pathways are known to affect telomere function. Here, we report a series of related genome-wide genetic interaction screens performed on budding yeast cells with acute or chronic telomere defects. Genetic interactions were examined in cells defective in Cdc13 and Stn1, affecting two components of CST, a single stranded DNA (ssDNA) binding complex that binds telomeric DNA. For comparison, genetic interactions were also examined in cells with defects in Rfa3, affecting the major ssDNA binding protein, RPA, which has overlapping functions with CST at telomeres. In more complex experiments, genetic interactions were measured in cells lacking EXO1 or RAD9, affecting different aspects of the DNA damage response, and containing a cdc13-1 induced telomere defect. Comparing fitness profiles across these data sets helps build a picture of the specific responses to different types of dysfunctional telomeres. The experiments show that each context reveals different genetic interactions, consistent with the idea that each genetic defect causes distinct molecular defects. To help others engage with the large volumes of data, the data are made available via two interactive web-based tools: Profilyzer and DIXY. One particularly striking genetic interaction observed was that the chk1∆ mutation improved fitness of cdc13-1 exo1∆ cells more than other checkpoint mutations (ddc1∆, rad9∆, rad17∆, and rad24∆), whereas, in cdc13-1 cells, the effects of all checkpoint mutations were similar. We show that this can be explained by Chk1 stimulating resection-a new function for Chk1 in the eukaryotic DNA damage response network.