The DNA damage response (DDR) arrests cell-cycle progression until damage is removed. DNA damage-induced cellular senescence is associated with persistent DDR. The molecular bases that distinguish transient from persistent DDR are unknown. Here we show that a large fraction of exogenously-induced persistent DDR markers are associated with telomeric DNA in cultured cells and mammalian tissues. In yeast, a chromosomal DNA double-strand break (DSB) next to telomeric sequences resists repair and impairs DNA ligase 4 recruitment. In mammalian cells, ectopic localization of telomeric factor TRF2 next to a DSB induces persistent DNA damage and DDR. Linear telomeric DNA, but not circular or scrambled DNA, induces a prolonged checkpoint in normal cells. In terminally-differentiated tissues of old primates, DDR markers accumulate at telomeres which are not critically short. We propose that linear genomes are not uniformly reparable and telomeric DNA tracts, if damaged, are irreparable and trigger persistent DDR and cellular senescence.

When eukaryotic chromosomes undergo double strand breaks (DSBs), several evolutionarily conserved proteins, among which the MRX complex, are recruited to the break site, leading to checkpoint activation and DNA repair. The function of the Saccharomyces cerevisiae Sae2 protein, which is known to work together with the MRX complex in meiotic DSB processing and in specific mitotic DSB repair events, is only beginning to be elucidated. Here we provide new insights into the role of Sae2 in mitotic DSB repair. We show that repair by single strand annealing of a single DSB, which is generated by the HO endonuclease between direct repeats, is defective both in the absence of Sae2 and in the presence of the hypomorphic rad50s allele altering the Rad50 subunit of MRX. Moreover, SAE2 overexpression partially suppresses the rad50s single strand annealing repair defects, suggesting that the latter might arise from defective MRX-Sae2 interactions. Finally, SAE2 deletion slows down resection of an HO-induced DSB and impairs DSB end bridging. Thus, Sae2 participates in DSB single strand annealing repair by ensuring both resection and intrachromosomal association of the broken ends.DNA double strand breaks (DSB 3 (s)) are a particularly dangerous form of DNA damage, because failure to repair these lesions can lead to loss of genetic information by deletions, duplications, translocations, and missegregation of large chromosome fragments (1). DSBs can arise by failures in DNA replication and by exposure to environmental factors, such as ionizing radiations and genotoxic drugs. However, they also play an important role as intermediates in meiotic and mitotic crossing over, V(D)J recombination and yeast mating type switching.When DSBs occur, many proteins are recruited to the break sites and serve both to promote a checkpoint response and to initiate DNA repair that can occur through non-homologous end joining or homologous recombination (HR) (2). Whereas non-homologous end joining implies recombination between sequences with little or no homology, HR involves exchange of genetic information between homologous DNA sequences and is the major DSB repair process in Saccharomyces cerevisiae.HR initiates with a DSB (3, 4), whose 5Ј-ends resection leaves 3Ј-ended single-stranded DNA (ssDNA) tails. Then, depending on the position of the homologous partner, on the initiation event and on the length of the homology region in the recombinant molecules, HR may occur by different mechanisms, including double strand break repair, synthesis-dependent strand annealing, and break-induced replication (2, 5). Moreover, when a DSB occurs between direct repeats, its repair is primarily achieved by a particular kind of HR named single strand annealing (SSA). SSA requires DSB resection to generate long 3Ј-ended single-stranded tails that can anneal with each other when resection is sufficient to uncover the duplicated sequences. Single-stranded tails are then removed by nucleases, and the resulting gaps/nicks are filled in by DNA repair synthesis and lig...

DNA double-strand breaks (DSBs) are repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). HR requires 5 0 DSB end degradation that occurs in the presence of cyclin-dependent kinase (CDK) activity. Here, we show that a lack of any of the NHEJ proteins Yku (Yku70-Yku80), Lif1 or DNA ligase IV (Dnl4) increases 5 0 DSB end degradation in G1 phase, with ykuD cells showing the strongest effect. This increase depends on MRX, the recruitment of which at DSBs is enhanced in ykuD G1 cells. DSB processing in G2 is not influenced by the absence of Yku, but it is delayed by Yku overproduction, which also decreases MRX loading on DSBs. Moreover, DSB resection in ykuD cells occurs independently of CDK activity, suggesting that it might be promoted by CDK-dependent inhibition of Yku.

Double‐strand breaks (DSBs) elicit a DNA damage response, resulting in checkpoint‐mediated cell‐cycle delay and DNA repair. The Saccharomyces cerevisiae Sae2 protein is known to act together with the MRX complex in meiotic DSB processing, as well as in DNA damage response during the mitotic cell cycle. Here, we report that cells lacking Sae2 fail to turn off both Mec1‐ and Tel1‐dependent checkpoints activated by a single irreparable DSB, and delay Mre11 foci disassembly at DNA breaks, indicating that Sae2 may negatively regulate checkpoint signalling by modulating MRX association at damaged DNA. Consistently, high levels of Sae2 prevent checkpoint activation and impair MRX foci formation in response to unrepaired DSBs. Mec1‐ and Tel1‐dependent Sae2 phosphorylation is necessary for these Sae2 functions, suggesting that the two kinases, once activated, may regulate checkpoint switch off through Sae2‐mediated inhibition of MRX signalling.

Generation of 3 0 G strand overhangs at telomere ends may play a role in regulating telomerase action and occurs by still unclear mechanisms. We show by an inducible short telomere assay that Sae2 and the Sgs1 RecQ helicase control two distinct but partially complementary pathways for nucleolytic processing of S. cerevisiae telomeres, with Sae2 function requiring its serine 267 phosphorylation. No processing activity is detectable in sae2D sgs1D cells, while the Exo1 exonuclease contributes to telomere end processing and elongation in both sae2D and sgs1D cells, suggesting that Exo1 telomeric function requires either Sgs1 or Sae2 action. Moreover, Dna2 might also support Sgs1 activity, as it acts redundantly with Exo1, but not with Sgs1. Finally, both length maintenance and G strand overhang generation at native telomeres are affected in sae2D sgs1D cells, further supporting the notion that Sae2 and Sgs1 combined activities control telomere length by regulating telomere processing. Molecular CellSae2, Sgs1, and 3 0 Overhangs at Telomeres