Eukaryotic chromosomes are replicated from multiple origins that initiate throughout the S-phase of the cell cycle. Why all origins do not fire simultaneously at the beginning of S-phase is not known, but two kinase activities, cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), are continually required throughout the S-phase for all replication initiation events. Here, we show that the two CDK substrates Sld3 and Sld2 and their binding partner Dpb11, together with the DDK subunit Dbf4 are in low abundance in the budding yeast, Saccharomyces cerevisiae. Over-expression of these factors is sufficient to allow late firing origins of replication to initiate early and together with deletion of the histone deacetylase RPD3, promotes the firing of heterochromatic, dormant origins. We demonstrate that the normal programme of origin firing prevents inappropriate checkpoint activation and controls S-phase length in budding yeast. These results explain how the competition for limiting DDK kinase and CDK targets at origins regulates replication initiation kinetics during S-phase and establishes a unique system with which to investigate the biological roles of the temporal programme of origin firing.

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.

The main responder to DNA double-strand breaks (DSBs) in mammals is ataxia telangiectasia mutated (ATM), whereas DSBinduced checkpoint activation in budding yeast seems to depend primarily on the ATM and Rad-3-related (ATR) orthologue Mec1. Here, we show that Saccharomyces cerevisiae Tel1, the ATM orthologue, has two functions in checkpoint response to DSBs. First, Tel1 participates, together with the MRX complex, in Mec1-dependent DSB-induced checkpoint activation by increasing the efficiency of single-stranded DNA accumulation at the ends of DSBs, and this checkpoint function can be overcome by overproducing the exonuclease Exo1. Second, Tel1 can activate the checkpoint response to DSBs independently of Mec1, although its signalling activity only becomes apparent when several DSBs are generated. Furthermore, we provide evidence that the kinetics of DSB resection can influence Tel1 activation, indicating that processing of the DSB termini might influence the transition from Tel1/ATM-to Mec1/ATR-dependent checkpoint.

Replication fork stalling caused by deoxynucleotide depletion triggers Rad53 phosphorylation and subsequent checkpoint activation, which in turn play a crucial role in maintaining functional DNA replication forks. How cells regulate checkpoint deactivation after inhibition of DNA replication is poorly understood. Here, we show that the budding yeast protein phosphatase Glc7/protein phosphatase 1 (PP1) promotes disappearance of phosphorylated Rad53 and recovery from replication fork stalling caused by the deoxynucleoside triphosphate (dNTP) synthesis inhibitor hydroxyurea (HU). Glc7 is also required for recovery from a double-strand break-induced checkpoint, while it is dispensable for checkpoint inactivation during methylmethane sulfonate exposure, which instead requires the protein phosphatases Pph3, Ptc2, and Ptc3. Furthermore, Glc7 counteracts in vivo histone H2A phosphorylation on serine 129 (␥H2A) and dephosphorylates ␥H2A in vitro. Finally, the replication recovery defects of HU-treated glc7 mutants are partially rescued by Rad53 inactivation or lack of ␥H2A formation, and the latter also counteracts hyperphosphorylated Rad53 accumulation. We therefore propose that Glc7 activity promotes recovery from replication fork stalling caused by dNTP depletion and that ␥H2A dephosphorylation is a critical Glc7 function in this process.Eukaryotic cells require specialized surveillance mechanisms called checkpoints to preserve genome integrity in the presence of genotoxic insults. An efficient checkpoint response is also important during S phase, where it inhibits late origin firing, prevents stalled replication fork breakdown, and promotes the restart of replication (6,22,23,33,34). Checkpoint activation requires protein phosphorylation cascades that in Saccharomyces cerevisiae are initiated by the two protein kinases Mec1 (ATR in humans), which functions in a complex with Ddc2 (27), and Tel1 (ATM in humans) (reviewed in reference 20).Mec1 and Tel1 phosphorylate the central effector kinases Rad53 and Chk1, which transfer the arrest signal to a myriad of downstream proteins (reviewed in reference 20). Rad53 and Chk1 activation is not governed by their simple interaction with Mec1 or Tel1 but rather requires a stepwise process. Once recruited to the double-strand break (DSB) ends, Mec1 phosphorylates Rad9, which promotes the recruitment of inactive Rad53 in a forkhead-associated domain (FHA)-dependent manner, thus allowing its activating phosphorylation by Mec1 (31), as well as Rad53 in trans autophosphorylation, by increasing the local concentration of Rad53 molecules (14). Active Rad53 kinase molecules are then released from the complex and can phosphorylate downstream targets to arrest mitotic cell cycle progression. Mec1 activation is supported by independent loading onto DNA of the Ddc1-Rad17-Mec3 complex by Rad24-RFC, which enhances Mec1 ability to transmit and amplify the DNA damage signals (24).Mec1 and Tel1 also phosphorylate histone H2A on serine 129 (␥H2A) in response to DNA DSBs (12, 28, 30) and inhibition...

In Saccharomyces cerevisiae, Mec1/ATR plays a primary role in sensing and transducing checkpoint signals in response to different types of DNA lesions, while the role of the Tel1/ATM kinase in DNA damage checkpoints is not as well defined. We found that UV irradiation in G 1 in the absence of Mec1 activates a Tel1/MRX-dependent checkpoint, which specifically inhibits the metaphase-to-anaphase transition. Activation of this checkpoint leads to phosphorylation of the downstream checkpoint kinases Rad53 and Chk1, which are required for Tel1-dependent cell cycle arrest, and their adaptor Rad9. The spindle assembly checkpoint protein Mad2 also partially contributes to the G 2 /M arrest of UV-irradiated mec1⌬ cells independently of Rad53 phosphorylation and activation. The inability of UV-irradiated mec1⌬ cells to undergo anaphase can be relieved by eliminating the anaphase inhibitor Pds1, whose phosphorylation and stabilization in these cells depend on Tel1, suggesting that Pds1 persistence may be responsible for the inability to undergo anaphase. Moreover, while UV irradiation can trigger Mec1-dependent Rad53 phosphorylation and activation in G 1 -and G 2 -arrested cells, Tel1-dependent checkpoint activation requires entry into S phase independently of the cell cycle phase at which cells are UV irradiated, and it is decreased when single-stranded DNA signaling is affected by the rfa1-t11 allele. This indicates that UV-damaged DNA molecules need to undergo structural changes in order to activate the Tel1-dependent checkpoint. Active Clb-cyclin-dependent kinase 1 (CDK1) complexes also participate in triggering this checkpoint and are required to maintain both Mec1-and Tel1-dependent Rad53 phosphorylation, suggesting that they may provide critical phosphorylation events in the DNA damage checkpoint cascade.Eukaryotic cells have developed sophisticated surveillance mechanisms called checkpoints to ensure proper response to the presence of damaged or incompletely replicated DNA molecules (reviewed in references 49 and 72) and to alterations in the mitotic apparatus (reviewed in references 46 and 64). The failure of checkpoints causes accumulation of genetic changes and chromosome instability that may lead to cancer in multicellular eukaryotes (reviewed in reference 87).DNA damage checkpoints are specialized in detecting abnormal DNA structures, serving at least two primary purposes: (i) to arrest the cell cycle in response to DNA damage, thereby coordinating cell cycle progression with DNA repair capacity (109); and (ii) to regulate transcription of DNA damage response genes, as well as to regulate activation and recruitment of various repair and recombination proteins that help cells survive genotoxic stress to sites of damage (reviewed in references 72 and 90). Therefore, DNA damage checkpoints are considered one of the main lines of defense against genomic instability. In fact, similar to mutations in recombination, replication, and repair genes, defective S-phase checkpoint genes increase the rate of gross chromosome...

SummaryDNA double-strand breaks (DSBs) are among the most deleterious types of damage that can occur in the genome of eukaryotic cells because failure to repair them can lead to loss of genetic information and chromosome rearrangements. DSBs can arise by failures in DNA replication and by exposure to environmental factors, such as ionizing radiations and radiomimetic chemicals. Moreover, they might arise when telomeres undergo extensive erosion, leading to the activation of the DNA damage response pathways and the onset of apoptosis and/or senescence. Importantly, DSBs can also form in a programmed manner during development. For example, meiotic recombination and rearrangement of the immunoglobulin genes in lymphocytes require the generation of site-or region-specific DSBs through the action of specific endonucleases. Efficient DSB repair is crucial in safeguarding genome integrity, whose maintenance in the face of DSBs involves branched signalling networks that switch on DNA damage checkpoints, activate DNA repair, induce chromatin reorganization and modulate numerous cellular processes. Not surprisingly, defects in these networks result in a variety of diseases ranging from severe genetic disorders to cancer predisposition and accelerated ageing.