In response to chromosomal double-strand breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint, which is orchestrated by the PI3 kinase-like protein kinases ATR and ATM (Mec1 and Tel1 in budding yeast). Following DSB formation, Mec1 and Tel1 phosphorylate histone H2A on serine 129 (known as -H2AX). We used caffeine to inhibit the checkpoint kinases after DSB induction. We show that prolonged phosphorylation of H2A-S129 does not require continuous Mec1 and Tel1 activity. Un-expectedly, caffeine treatment impaired homologous recombination by inhibiting 5 ′ to 3 ′ end resection, in-dependent of Mec1 and Tel1 inhibition. Caffeine treat-ment led to the rapid loss, by proteasomal degrada-tion, of both Sae2, a nuclease that plays a role in early steps of resection, and Dna2, a nuclease that facilitates one of two extensive resection pathways. Sae2’s instability is evident in the absence of DNA damage. A similar loss is seen when protein synthe-sis is inhibited by cycloheximide. Caffeine treatment had similar effects on irradiated HeLa cells, blocking the formation of RPA and Rad51 foci that depend on 5 ′ to 3 ′ resection of broken chromosome ends. Our findings provide insight toward the use of caffeine as a DNA damage-sensitizing agent in cancer cells.

The DNA damage checkpoint pathway is acti-vated in response to DNA lesions and replication stress to preserve genome integrity. However, hyper-activation of this surveillance system is detrimental to the cell, because it might prevent cell cycle re-start after repair, which may also lead to senescence. Here we show that the scaffold proteins Slx4 and Rtt107 limit checkpoint signalling at a persistent double-strand DNA break (DSB) and at uncapped telomeres. We found that Slx4 is recruited within a few kilobases of an irreparable DSB, through the interaction with Rtt107 and the multi-BRCT domain scaffold Dpb11. In the absence of Slx4 or Rtt107, Rad9 binding near the irreparable DSB is increased, leading to robust checkpoint signalling and slower nucleolytic degra-dation of the 5 ′ strand. Importantly, in slx4 sae2 double mutant cells these phenotypes are exacer-bated, causing a severe Rad9-dependent defect in DSB repair. Our study sheds new light on the molec-ular mechanism that coordinates the processing and repair of DSBs with DNA damage checkpoint sig-nalling, preserving genome integrity.