Although cancer initiation and progression are generally associated with the accumulation of somatic mutations^1,2, substantial epigenomic alterations underlie many aspects of tumorigenesis and cancer susceptibility^3–6, suggesting that genetic mechanisms might not be the only drivers of malignant transformation⁷. However, whether purely non-genetic mechanisms are sufficient to initiate tumorigenesis irrespective of mutations has been unknown. Here, we show that a transient perturbation of transcriptional silencing mediated by Polycomb group proteins is sufficient to induce an irreversible switch to a cancer cell fate in Drosophila. This is linked to the irreversible derepression of genes that can drive tumorigenesis, including members of the JAK–STAT signalling pathway and zfh1, the fly homologue of the ZEB1 oncogene, whose aberrant activation is required for Polycomb perturbation-induced tumorigenesis. These data show that a reversible depletion of Polycomb proteins can induce cancer in the absence of driver mutations, suggesting that tumours can emerge through epigenetic dysregulation leading to inheritance of altered cell fates.

The Mre11-Rad50-Xrs2 nuclease complex, together with Sae2, initiates the 5′-to-3′ resection of Double-Strand DNA Breaks (DSBs). Extended 3′ single stranded DNA filaments can be exposed from a DSB through the redundant activities of the Exo1 nuclease and the Dna2 nuclease with the Sgs1 helicase. In the absence of Sae2, Mre11 binding to a DSB is prolonged, the two DNA ends cannot be kept tethered, and the DSB is not efficiently repaired. Here we show that deletion of the yeast 53BP1-ortholog RAD9 reduces Mre11 binding to a DSB, leading to Rad52 recruitment and efficient DSB end-tethering, through an Sgs1-dependent mechanism. As a consequence, deletion of RAD9 restores DSB repair either in absence of Sae2 or in presence of a nuclease defective MRX complex. We propose that, in cells lacking Sae2, Rad9/53BP1 contributes to keep Mre11 bound to a persistent DSB, protecting it from extensive DNA end resection, which may lead to potentially deleterious DNA deletions and genome rearrangements. DNA double strand breaks (DSBs) are among the most deleterious types of damage occurring in the genome, as failure to repair these lesions through either non-homologous-end-joining (NHEJ) or homologous recombination (HR) leads to genetic instability. The 5′ strand of a DSB can be nucleolytically degraded by several nucleases and associated factors, including Mre11, CtIP/Sae2, Exo1 and Dna2 together with Bloom helicase/Sgs1, through a finely regulated process called DSB resection. Once resection is initiated, error-prone NHEJ is prevented. Several findings suggest that DSB resection is a double-edged sword, if not finely regulated, since on one hand it is needed for faithful HR, but on the other it may lead to extensive DNA deletions associated with genome instability. Both in mammals and yeast, 53BP1/Rad9 protein binds near the lesion and counteracts the resection process, limiting the formation of ssDNA. By using S. cerevisiae as a model organism, here we show that Rad9 oligomers block the removal of hypo-active Mre11 protein from a persistent DSB, thus limiting initiation of resection and the recruitment of the recombination factor Rad52, in the absence of Sae2. Altogether, these findings pinpoint a critical role of 53BP1/Rad9 in balancing HR and NHEJ repair events throughout the cell cycle.

The DNA damage checkpoint pathway is activated in response to DNA lesions and replication stress to preserve genome integrity. However, hyper-activation of this surveillance system is detrimental to the cell, because it might prevent cell cycle re-start after repair, which may also lead to senescence. Here we show that the scaffold proteins Slx4 and Rtt107 limit checkpoint signalling at a persistent double-strand DNA break (DSB) and at uncapped telomeres. We found that Slx4 is recruited within a few kilobases of an irreparable DSB, through the interaction with Rtt107 and the multi-BRCT domain scaffold Dpb11. In the absence of Slx4 or Rtt107, Rad9 binding near the irreparable DSB is increased, leading to robust checkpoint signalling and slower nucleolytic degradation of the 5′ strand. Importantly, in slx4Δ sae2Δ double mutant cells these phenotypes are exacerbated, causing a severe Rad9-dependent defect in DSB repair. Our study sheds new light on the molecular mechanism that coordinates the processing and repair of DSBs with DNA damage checkpoint signalling, preserving genome integrity.

An important but still enigmatic function of DNA:RNA hybrids is their role in DNA double-strand break (DSB) repair. Here, we show that Sen1, the budding yeast ortholog of the human helicase Senataxin, is recruited at an HO endonuclease-induced DSB and limits the local accumulation of DNA:RNA hybrids. In the absence of Sen1, hybrid accumulation proximal to the DSB promotes increased binding of the Ku70-80 (KU) complex at the break site, mutagenic non-homologous end joining (NHEJ), micro-homology-mediated end joining (MMEJ), and chromosome translocations. We also show that homology-directed recombination (HDR) by gene conversion is mostly proficient in sen1 mutants after single DSB. However, in the absence of Sen1, DNA:RNA hybrids, Mre11, and Dna2 initiate resection through a non-canonical mechanism. We propose that this resection mechanism through local DNA:RNA hybrids acts as a backup to prime HDR when canonical pathways are altered, but at the expense of genome integrity.

In all the eukaryotic cells, nucleolytic processing (resection) of a double strand DNA break (DSB) is a key step to channel the repair of the lesion toward the homologous recombination, at the expenses of the non-homologous end joining (NHEJ). The coordinated action of several nucleases and helicases generates 3' single strand (ss) DNA, which is covered by RPA and recombination factors. Molecular details of the process have been first dissected in the model organism Saccharomyces cerevisiae. When DSB ends are occupied by KU, a central component of the NHEJ, the Mre11-Rad50-Xrs2 (MRX) nuclease complex (MRN in human), aided by the associated factors Sae2 (CTIP in human), initiates the resection process, inducing a nick close to the DSB ends. Then, starting from the nick, the nucleases Mre11, Exo1, Dna2, in cooperation with Sgs1 helicase (BLM in human), degrade DNA strand in both the directions, creating the 3' ssDNA filament. Multiple levels of regulation of the break processing ensure faithful DSB repair, preventing chromosome rearrangements, and genome instability. Here we review the DSB resection process and its regulation in the context of chromatin. Particularly, we focus on proteins that limit DSB resection, acting as physical barriers toward nucleases and helicases. Moreover, we also take into consideration recent evidence regarding functional interplay between DSB repair and RNA molecules nearby the break site.

The DNA damage checkpoint (DDC) is often robustly activated during the homologous recombination (HR) repair of DNA double strand breaks (DSBs). DDC activation controls several HR repair factors by phosphorylation, preventing premature segregation of entangled chromosomes formed during HR repair. The DDC mediator 53BP1/Rad9 limits the nucleolytic processing (resection) of a DSB, controlling the formation of the 3′ single-stranded DNA (ssDNA) filament needed for recombination, from yeast to human. Here we show that Rad9 promotes stable annealing between the recombinogenic filament and the donor template in yeast, limiting strand rejection by the Sgs1 and Mph1 helicases. This regulation allows repair by long tract gene conversion, crossover recombination and break-induced replication (BIR), only after DDC activation. These findings shed light on how cells couple DDC with the choice and effectiveness of HR sub-pathways, with implications for genome instability and cancer.