The DNA damage checkpoint senses the presence of DNA lesions and controls the cellular response thereto. A crucial DNA damage signal is single-stranded DNA (ssDNA), which is frequently found at sites of DNA damage and recruits the sensor checkpoint kinase Mec1-Ddc2. However, how this signal – and therefore the cells’ DNA damage load – is quantified, is poorly understood. Here, we use genetic manipulation of DNA end resection at a site-specific DNA double-strand break (DSB) in budding yeast to generate quantitatively different DNA damage (ssDNA) signals. Interestingly, two major targets of the Mec1-Ddc2 kinase – Rad53 and γH2A – differ in their response to the ssDNA signal, indicating distinct signalling circuits within the checkpoint. The local checkpoint signalling circuit leading to γH2A phosphorylation is non-quantitative and unresponsive to increased amounts of damage-associated Mec1-Ddc2 kinase. In contrast, the global checkpoint signalling circuit, which triggers Rad53 activation, integrates the ssDNA signal quantitatively. We find that not only Mec1-Ddc2 association, but also loading of the 9-1-1 co-sensor complex is enhanced during ongoing resection. Moreover, we can uncouple global checkpoint activation from the amount of Mec1-Ddc2 kinase at the lesion by using mutant conditions that hyper-activate the 9-1-1 signalling axis and at the same time show reduced amounts of damage-associated Mec1-Ddc2 kinase. We therefore propose that a key function of the 9-1-1 complex and the downstream checkpoint mediators is to generate a checkpoint response, which is quantitative and proportional to the cellular DNA damage load. The DNA damage checkpoint senses the presence of DNA lesions and controls the cellular response thereto. A crucial DNA damage signal is single-stranded DNA (ssDNA), which is frequently found at sites of DNA damage and recruits the sensor checkpoint kinase Mec1-Ddc2. However, how this signal – and therefore the cells’ DNA damage load – is quantified, is poorly understood. Here, we use genetic manipulation of DNA end resection at a site-specific DNA double-strand break (DSB) in budding yeast to generate quantitatively different DNA damage (ssDNA) signals. Interestingly, two major targets of the Mec1-Ddc2 kinase – Rad53 and γH2A – differ in their response to the ssDNA signal, indicating distinct signalling circuits within the checkpoint. The local checkpoint signalling circuit leading to γH2A phosphorylation is non-quantitative and unresponsive to increased amounts of damage-associated Mec1-Ddc2 kinase. In contrast, the global checkpoint signalling circuit, which triggers Rad53 activation, integrates the ssDNA signal quantitatively. We find that not only Mec1-Ddc2 association, but also loading of the 9-1-1 co-sensor complex is enhanced during ongoing resection. Moreover, we can uncouple global checkpoint activation from the amount of Mec1-Ddc2 kinase at the lesion by using mutant conditions that hyper-activate the 9-1-1 signalling axis and at the same time show reduced amounts of damage-associated Mec1-Ddc2 kinase. We therefore propose that a key function of the 9-1-1 complex and the downstream checkpoint mediators is to generate a checkpoint response, which is quantitative and proportional to the cellular DNA damage load.
Overall design: In this experiment, RPA and gammaH2A enrichments around two permanent HO-induced DNA double-strand break are analyzed using ChIP-seq before and after 4 hours of DSB induction in M phase-arrested cells. WT cells were compared to resection-defective exo1∆ sgs1∆ cells.
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