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DNA 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.

The evolutionarily conserved yeast Mec1 and Tel1 protein kinases, as well as the Mec1-interacting protein Ddc2, are involved in the DNA damage checkpoint response. We show that regulation of Tel1 and Ddc2-Mec1 activities is important to modulate both activation and termination of checkpoint-mediated cell cycle arrest. In fact, overproduction of either Tel1 or Ddc2 causes a prolonged cell cycle arrest and cell death in response to DNA damage, impairing the ability of cells to recover from checkpoint activation. This cell cycle arrest is independent of Mec1 in UV-irradiated Tel1-overproducing cells, while it is strictly Mec1 dependent in similarly treated DDC2-overexpressing cells. The Rad53 checkpoint kinase is instead required in both cases for cell cycle arrest, which correlates with its enhanced and persistent phosphorylation, suggesting that unscheduled Rad53 phosphorylation might prevent cells from re-entering the cell cycle after checkpoint activation. In addition, Tel1 overproduction results in transient nuclear division arrest and concomitant Rad53 phosphorylation in the absence of exogenous DNA damage independently of Mec1 and Ddc1.

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.