Tel1/ATM and Mec1/ATR checkpoint kinases are activated by DNA double-strand breaks (DSBs). Mec1/ATR recruitment to DSBs requires the formation of RPA-coated single-stranded DNA (ssDNA), which arises from 5′-3′ nucleolytic degradation (resection) of DNA ends. Here, we show that Saccharomyces cerevisiae Mec1 regulates resection of the DSB ends. The lack of Mec1 accelerates resection and reduces the loading to DSBs of the checkpoint protein Rad9, which is known to inhibit ssDNA generation. Extensive resection is instead inhibited by the Mec1-ad mutant variant that increases the recruitment near the DSB of Rad9, which in turn blocks DSB resection by both Rad53-dependent and Rad53-independent mechanisms. The mec1-ad resection defect leads to prolonged persistence at DSBs of the MRX complex that causes unscheduled Tel1 activation, which in turn impairs checkpoint switch off. Thus, Mec1 regulates the generation of ssDNA at DSBs, and this control is important to coordinate Mec1 and Tel1 signaling activities at these breaks.

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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...

Eukaryotic cells respond to DNA double-strand breaks (DSBs) by activating a checkpoint that depends on the protein kinases Tel1/ ATM and Mec1/ATR. Mec1/ATR is activated by RPA-coated singlestranded DNA (ssDNA), which arises upon nucleolytic degradation (resection) of the DSB. Emerging evidences indicate that RNAprocessing factors play critical, yet poorly understood, roles in genomic stability. Here, we provide evidence that the Saccharomyces cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 regulate Mec1/ ATR activation by promoting generation of RPA-coated ssDNA. The lack of Xrn1 inhibits ssDNA generation at the DSB by preventing the loading of the MRX complex. By contrast, DSB resection is not affected in the absence of Rrp6 or Trf4, but their lack impairs the recruitment of RPA, and therefore of Mec1, to the DSB. Rrp6 and Trf4 inactivation affects neither Rad51/Rad52 association nor DSB repair by homologous recombination (HR), suggesting that full Mec1 activation requires higher amount of RPA-coated ssDNA than HR-mediated repair. Noteworthy, deep transcriptome analyses do not identify common misregulated gene expression that could explain the observed phenotypes. Our results provide a novel link between RNA processing and genome stability.

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) in haploid cells is generally restricted to S/G2 cell cycle phases, when DNA has been replicated and a sister chromatid is available as a repair template. This cell cycle specificity depends on cyclin-dependent protein kinases (Cdk1 in Saccharomyces cerevisiae), which initiate HR by promoting 5′–3′ nucleolytic degradation of the DSB ends. Whether Cdk1 regulates other HR steps is unknown. Here we show that yku70Δ cells, which accumulate single-stranded DNA (ssDNA) at the DSB ends independently of Cdk1 activity, are able to repair a DSB by single-strand annealing (SSA) in the G1 cell cycle phase, when Cdk1 activity is low. This ability to perform SSA depends on DSB resection, because both resection and SSA are enhanced by the lack of Rad9 in yku70Δ G1 cells. Furthermore, we found that interchromosomal noncrossover recombinants are generated in yku70Δ and yku70Δ rad9Δ G1 cells, indicating that DSB resection bypasses Cdk1 requirement also for carrying out these recombination events. By contrast, yku70Δ and yku70Δ rad9Δ cells are specifically defective in interchromosomal crossover recombination when Cdk1 activity is low. Thus, Cdk1 promotes DSB repair by single-strand annealing and noncrossover recombination by acting mostly at the resection level, whereas additional events require Cdk1-dependent regulation in order to generate crossover outcomes.