Remodelling the Rad9 checkpoint complex: preparing Rad53 for action

DNA damage checkpoints are signal transduction pathways that are activated after genotoxic insults to protect genomic integrity. The Rad9 protein functions in the DNA damage checkpoint pathway in Saccharomyces cerevisiae and is essential for the Mec1-dependent activation of the effector kinase Rad53. We recently described the purification of two soluble distinct Rad9 complexes. The large 850 kDa complex consists of hypophosphorylated Rad9 and the chaperone proteins Ssa1/2. This complex is found both in undamaged cells as well as in cells treated with DNA damaging agents. The smaller 560 kDa complex contains hyperphosphorylated Rad9, Ssa1/2 and, in addition, Rad53. This complex forms only in cells with compromised DNA integrity. Once bound to the smaller complex, Rad53 can be activated by in trans autophosphorylation. Here, we propose a model in which the large Rad9 complex is remodelled after a genomic insult by chaperone activity to a smaller Rad53 activating complex.     

Rad53 phosphorylation site clusters are important for Rad53 regulation and signaling

Budding yeast Rad53 is an essential protein kinase that is phosphorylated and activated in a MEC1- and TEL1-dependent manner in response to DNA damage. We studied the role of Rad53 phosphorylation through mutation of consensus phosphorylation sites for upstream kinases Mec1 and Tel1. Alanine substitution of the Rad53 amino-terminal TQ cluster region reduced viability and impaired checkpoint functions. These substitution mutations spared the basal interaction with Asf1 and the DNA damage-induced interactions with Rad9. However, they caused a decrease in DNA damage-induced Rad53 kinase activity and an impaired interaction with the protein kinase Dun1. The Dun1 FHA (Forkhead-associated) domain recognized the amino-terminal TQ cluster of Rad53 after DNA damage or replication blockade. Thus, the phosphorylation of Rad53 by upstream kinases is important not only for Rad53 activation but also for creation of an interface between Rad53 and Dun1.     

Rad9 phosphorylation sites couple Rad53 to the Saccharomyces cerevisiae DNA damage checkpoint

Rad9 is required for the MEC1/TEL1-dependent activation of Saccharomyces cerevisiae DNA damage checkpoint pathways mediated by Rad53 and Chk1. DNA damage induces Rad9 phosphorylation, and Rad53 specifically associates with phosphorylated Rad9. We report here that multiple Mec1/Tel1 consensus [S/T]Q sites within Rad9 are phosphorylated in response to DNA damage. These Rad9 phosphorylation sites are selectively required for activation of the Rad53 branch of the checkpoint pathway. Consistent with the in vivo function in recruiting Rad53, Rad9 phosphopeptides are bound by Rad53 forkhead-associated (FHA) domains in vitro. These data suggest that functionally independent domains within Rad9 regulate Rad53 and Chk1, and support the model that FHA domain-mediated recognition of Rad9 phosphopeptides couples Rad53 to the DNA damage checkpoint pathway.     

Association of Rad9 with double-strand breaks through a Mec1-dependent mechanism

Rad9 is required for the activation of DNA damage checkpoint pathways in budding yeast. Rad9 is phosphorylated after DNA damage in a Mec1- and Tel1-dependent manner and subsequently interacts with Rad53. This Rad9-Rad53 interaction has been suggested to trigger the activation and phosphorylation of Rad53. Here we show that Mec1 controls the Rad9 accumulation at double-strand breaks (DSBs). Rad9 was phosphorylated after DSB induction and associated with DSBs. However, its phosphorylation and association with DSBs were significantly decreased in cells carrying a mec1Delta or kinase-negative mec1 mutation. Mec1 phosphorylated the S/TQ motifs of Rad9 in vitro, the same motifs that are phosphorylated after DNA damage in vivo. In addition, multiple mutations in the Rad9 S/TQ motifs resulted in its defective association with DSBs. Phosphorylation of Rad9 was partially defective in cells carrying a weak mec1 allele (mec1-81), whereas its association with DSBs occurred efficiently in the mec1-81 mutants, as found in wild-type cells. However, the Rad9-Rad53 interaction after DSB induction was significantly decreased in mec1-81 mutants, as it was in mec1Delta mutants. Deletion mutation in RAD53 did not affect the association of Rad9 with DSBs. Our results suggest that Mec1 promotes association of Rad9 with sites of DNA damage, thereby leading to full phosphorylation of Rad9 and its interaction with Rad53.     

A Tel1/MRX-dependent checkpoint inhibits the metaphase-to-anaphase transition after UV irradiation in the absence of Mec1

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 mec1Delta cells independently of Rad53 phosphorylation and activation. The inability of UV-irradiated mec1Delta 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.     

Activation of Rad53 kinase in response to DNA damage and its effect in modulating phosphorylation of the lagging strand DNA polymerase

The Saccharomyces cerevisiae Rad53 protein kinase is required for the execution of checkpoint arrest at multiple stages of the cell cycle. We found that Rad53 autophosphorylation activity depends on in trans phosphorylation mediated by Mec1 and does not require physical association with other proteins. Uncoupling in trans phosphorylation from autophosphorylation using a rad53 kinase-defective mutant results in a dominant-negative checkpoint defect. Activation of Rad53 in response to DNA damage in G(1) requires the Rad9, Mec3, Ddc1, Rad17 and Rad24 checkpoint factors, while this dependence is greatly reduced in S phase cells. Furthermore, during recovery from checkpoint activation, Rad53 activity decreases through a process that does not require protein synthesis. We also found that Rad53 modulates the lagging strand replication apparatus by controlling phosphorylation of the DNA polymerase alpha-primase complex in response to intra-S DNA damage.     

Hyperactivation of the yeast DNA damage checkpoint by TEL1 and DDC2 overexpression.

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.     

Saccharomyces cerevisiae Rad9 acts as a Mec1 adaptor to allow Rad53 activation

BACKGROUND: The DNA damage checkpoint is a protein kinase-based signaling system that detects and signals physical alterations in DNA. Despite having identified many components of this signaling cascade, the exact mechanisms by which checkpoint kinases are activated after DNA damage, as well as the role of the checkpoint mediators, remain poorly understood. RESULTS: To elucidate the mechanisms that underlie the MEC1 and RAD9-dependent activation of Rad53, the Saccharomyces cerevisiae ortholog of Chk2, we mapped and characterized in vivo phosphorylation sites present on Rad53 after DNA damage by mass spectrometry. We find that Rad53 requires for its activation multisite phosphorylation on a number of typical and atypical Mec1 phosphorylation sites, thus confirming that Rad53 is a direct target of Mec1, the mammalian ATR homolog. Moreover, by using biochemical reconstitution experiments, we demonstrate that efficient and direct phosphorylation of Rad53 by Mec1 is only observed in the presence of purified Rad9, the archetypal checkpoint mediator. We find that the stimulatory activity of Rad9 requires a phospho- and FHA-dependent interaction with Rad53, which allows Rad53 to be recognized as a substrate for Mec1. CONCLUSIONS: Our results indicate that Rad9 acts as a bona fide signaling adaptor that enables Rad53 phosphorylation by Mec1. Given the high degree of conservation of checkpoint signaling in eukaryotes, we propose that one of the critical functions of checkpoint mediators such as MDC1, 53BP1, or Brca1 is to act as PIKK adaptors during the DNA damage response.     

Activation of the checkpoint kinase Rad53 by the phosphatidyl inositol kinase-like kinase Mec1

Saccharomyces cerevisiae Rad53, the ortholog of mammalian Chk2, is an essential protein kinase in DNA damage and DNA replication checkpoint pathways. Consecutive phosphatidyl inositol kinase-like kinase (PIKK)-dependent and PIKK-independent steps in activation of Rad53 are key steps for controlling and transmitting diverse downstream responses to DNA damage. However, these activities have not been demonstrated in vitro in defined systems. Here, we have shown that enzymatically dephosphorylated purified Rad53 autoactivates in vitro through a phosphorylation-dependent mechanism. Kinetic analysis demonstrated that autophosphorylation results in a more than 9-fold increase in protein kinase activity. Autophosphorylation was Rad53 concentration-dependent, indicating that the reaction follows an intermolecular mechanism. DNA damage induced oligomerization of a subset of Rad53 molecules in vivo. At low concentrations of Rad53, preincubation of Rad53 with immune complexes containing the Mec1/Ddc2 complex can activate Rad53 kinase activity. Our findings showed that Mec1/Ddc2 complexes can directly activate Rad53 through a phosphorylation-dependent mechanism, and more generally, supported the hypothesis that PIKKs regulate Chk2 orthologs through phosphorylation. Moreover, this work has substantiated a model for PIKK-independent amplification of Rad53 activation (and by extension, activation of other Chk2 orthologs) mediated by inter-Rad53 phosphorylation.     

Mec1/ATR regulates the generation of single‐stranded DNA that attenuates Tel1/ATM signaling at DNA ends

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.     

The DNA damage checkpoint response requires histone H2B ubiquitination by Rad6-Bre1 and H3 methylation by Dot1

The cellular response to DNA lesions entails the recruitment of several checkpoint and repair factors to damaged DNA, and chromatin modifications may play a role in this process. Here we show that in Saccharomyces cerevisiae epigenetic modification of histones is required for checkpoint activity in response to a variety of genotoxic stresses. We demonstrate that ubiquitination of histone H2B on lysine 123 by the Rad6-Bre1 complex, is necessary for activation of Rad53 kinase and cell cycle arrest. We found a similar requirement for Dot1-dependent methylation of histone H3. Loss of H3-Lys(79) methylation does not affect Mec1 activation, whereas it renders cells checkpoint-defective by preventing phosphorylation of Rad9. Such results suggest that histone modifications may have a role in checkpoint function by modulating the interactions of Rad9 with chromatin and active Mec1 kinase.     

A Ddc2-Rad53 fusion protein can bypass the requirements for RAD9 and MRC1 in Rad53 activation

Activation of Rad53p by DNA damage plays an essential role in DNA damage checkpoint pathways. Rad53p activation requires coupling of Rad53p to Mec1p through a “mediator” protein, Rad9p or Mrc1p. We sought to determine whether the mediator requirement could be circumvented by making fusion proteins between the Mec1 binding partner Ddc2p and Rad53p. Ddc2-Rad53p interacted with Mec1p and other Ddc2-Rad53p molecules under basal conditions and displayed an increased oligomerization upon DNA damage. Ddc2-Rad53p was activated in a Mec1p- and Tel1p-dependent manner upon DNA damage. Expression of Ddc2-Rad53p in Δrad9 or Δrad9Δmrc1 cells increased viability on plates containing the alkylating agent methyl methane sulfonate. Ddc2-Rad53p was activated at least partially by DNA damage in Δrad9Δmrc1 cells. In addition, expression of Ddc2-Rad53p in Δrad24Δrad17Δmec3 cells increased cell survival. These results reveal minimal requirements for function of a core checkpoint signaling system.     

CDC5 Inhibits the Hyperphosphorylation of the Checkpoint Kinase Rad53, Leading to Checkpoint Adaptation

The Saccharomyces cerevisiae polo-like kinase Cdc5 promotes adaptation to the DNA damage checkpoint, in addition to its numerous roles in mitotic progression. The process of adaptation occurs when cells are presented with persistent or irreparable DNA damage and escape the cell-cycle arrest imposed by the DNA damage checkpoint. However, the precise mechanism of adaptation remains unknown. We report here that CDC5 is dose-dependent for adaptation and that its overexpression promotes faster adaptation, indicating that high levels of Cdc5 modulate the ability of the checkpoint to inhibit the downstream cell-cycle machinery. To pinpoint the step in the checkpoint pathway at which Cdc5 acts, we overexpressed CDC5 from the GAL1 promoter in damaged cells and examined key steps in checkpoint activation individually. Cdc5 overproduction appeared to have little effect on the early steps leading to Rad53 activation. The checkpoint sensors, Ddc1 (a member of the 9-1-1 complex) and Ddc2 (a member of the Ddc2/Mec1 complex), properly localized to damage sites. Mec1 appeared to be active, since the Rad9 adaptor retained its Mec1 phosphorylation. Moreover, the damage-induced interaction between phosphorylated Rad9 and Rad53 remained intact. In contrast, Rad53 hyperphosphorylation was significantly reduced, consistent with the observation that cell-cycle arrest is lost during adaptation. Thus, we conclude Cdc5 acts to attenuate the DNA damage checkpoint through loss of Rad53 hyperphosphorylation to allow cells to adapt to DNA damage. Polo-like kinase homologs have been shown to inhibit the ability of Claspin to facilitate the activation of downstream checkpoint kinases, suggesting that this function is conserved in vertebrates.     

Mec1 and Rad53 inhibit formation of single-stranded DNA at telomeres of Saccharomyces cerevisiae cdc13-1 mutants

Here we examine the roles of budding-yeast checkpoint proteins in regulating degradation of dsDNA to ssDNA at unprotected telomeres (in Cdc13 telomere-binding protein defective strains). We find that Rad17, Mec3, as well as Rad24, members of the putative checkpoint clamp loader (Rad24) and sliding clamp (Rad17, Mec3) complexes, are important for promoting degradation of dsDNA in and near telomere repeats. We find that Mec1, Rad53, as well as Rad9, have the opposite role: they inhibit degradation. Downstream checkpoint kinases Chk1 and Dun1 play no detectable role in either promoting degradation or inhibiting it. These data suggest, first, that the checkpoint sliding clamp regulates and/or recruits some nucleases for degradation, and, second, that Mec1 activates Rad9 to activate Rad53 to inhibit degradation. Further analysis shows that Rad9 inhibits ssDNA generation by both Mec1/Rad53-dependent and -independent pathways. Exo1 appears to be targeted by the Mec1/Rad53-dependent pathway. Finally, analysis of double mutants suggests a minor role for Mec1 in promoting Rad24-dependent degradation of dsDNA. Thus, checkpoint proteins orchestrate carefully ssDNA production at unprotected telomeres.     

Mdt1, a novel Rad53 FHA1 domain-interacting protein, modulates DNA damage tolerance and G(2)/M cell cycle progression in Saccharomyces cerevisiae

The Rad53 kinase plays a central role in yeast DNA damage checkpoints. Rad53 contains two FHA phosphothreonine-binding domains that are required for Rad53 activation and possibly downstream signaling. Here we show that the N-terminal Rad53 FHA1 domain interacts with the RNA recognition motif, coiled-coil, and SQ/TQ cluster domain-containing protein Mdt1 (YBl051C). The interaction of Rad53 and Mdt1 depends on the structural integrity of the FHA1 phosphothreonine-binding site as well as threonine-305 of Mdt1. Mdt1 is constitutively threonine phosphorylated and hyperphosphorylated in response to DNA damage in vivo. DNA damage-dependent Mdt1 hyperphosphorylation depends on the Mec1 and Tel1 checkpoint kinases, and Mec1 can directly phosphorylate a recombinant Mdt1 SQ/TQ domain fragment. MDT1 overexpression is synthetically lethal with a rad53 deletion, whereas mdt1 deletion partially suppresses the DNA damage hypersensitivity of checkpoint-compromised strains and generally improves DNA damage tolerance. In the absence of DNA damage, mdt1 deletion leads to delayed anaphase completion, with an elongated cell morphology reminiscent of that of G(2)/M cell cycle mutants. mdt1-dependent and DNA damage-dependent cell cycle delays are not additive, suggesting that they act in the same pathway. The data indicate that Mdt1 is involved in normal G(2)/M cell cycle progression and is a novel target of checkpoint-dependent cell cycle arrest pathways.     

Dpb11 coordinates Mec1 kinase activation with cell cycle-regulated Rad9 recruitment

Eukaryotic cells respond to DNA damage by activating checkpoint signalling pathways. Checkpoint signals are transduced by a protein kinase cascade that also requires non-kinase mediator proteins. One such mediator is the Saccharomyces cerevisiae Dpb11 protein, which binds to and activates the apical checkpoint kinase, Mec1. Here, we show that a ternary complex of Dpb11, Mec1 and another key mediator protein Rad9 is required for efficient Rad9 phosphorylation by Mec1 in vitro, and for checkpoint activation in vivo. Phosphorylation of Rad9 by cyclin-dependent kinase (CDK) on two key residues generates a binding site for tandem BRCT repeats of Dpb11, and is thereby required for Rad9 recruitment into the ternary complex. Checkpoint signalling via Dpb11, therefore, does not efficiently occur during G1 phase when CDK is inactive. Thus, Dpb11 coordinates checkpoint signal transduction both temporally and spatially, ensuring the initiator kinase is specifically activated in proximity of one of its critical substrates.     

Colocalization of Mec1 and Mrc1 is sufficient for Rad53 phosphorylation in vivo

When DNA is damaged or DNA replication goes awry, cells activate checkpoints to allow time for damage to be repaired and replication to complete. In Saccharomyces cerevisiae, the DNA damage checkpoint, which responds to lesions such as double-strand breaks, is activated when the lesion promotes the association of the sensor kinase Mec1 and its targeting subunit Ddc2 with its activators Ddc1 (a member of the 9-1-1 complex) and Dpb11. It has been more difficult to determine what role these Mec1 activators play in the replication checkpoint, which recognizes stalled replication forks, since Dpb11 has a separate role in DNA replication itself. Therefore we constructed an in vivo replication-checkpoint mimic that recapitulates Mec1-dependent phosphorylation of the effector kinase Rad53, a crucial step in checkpoint activation. In the endogenous replication checkpoint, Mec1 phosphorylation of Rad53 requires Mrc1, a replisome component. The replication-checkpoint mimic requires colocalization of Mrc1-LacI and Ddc2-LacI and is independent of both Ddc1 and Dpb11. We show that these activators are also dispensable for Mec1 activity and cell survival in the endogenous replication checkpoint but that Ddc1 is absolutely required in the absence of Mrc1. We propose that colocalization of Mrc1 and Mec1 is the minimal signal required to activate the replication checkpoint.     

Rad52 recruitment is DNA replication independent and regulated by Cdc28 and the Mec1 kinase

Recruitment of the homologous recombination machinery to sites of double‐strand breaks is a cell cycle‐regulated event requiring entry into S phase and CDK1 activity. Here, we demonstrate that the central recombination protein, Rad52, forms foci independent of DNA replication, and its recruitment requires B‐type cyclin/CDK1 activity. Induction of the intra‐S‐phase checkpoint by hydroxyurea (HU) inhibits Rad52 focus formation in response to ionizing radiation. This inhibition is dependent upon Mec1/Tel1 kinase activity, as HU‐treated cells form Rad52 foci in the presence of the PI3 kinase inhibitor caffeine. These Rad52 foci colocalize with foci formed by the replication clamp PCNA. These results indicate that Mec1 activity inhibits the recruitment of Rad52 to both sites of DNA damage and stalled replication forks during the intra‐S‐phase checkpoint. We propose that B‐type cyclins promote the recruitment of Rad52 to sites of DNA damage, whereas Mec1 inhibits spurious recombination at stalled replication forks.     

Temperature-sensitive defects of the GSP1gene, yeast Ran homologue, activate the Tel1-dependent pathway

RanGTPase is involved in many cellular processes. It functions in nuclear-cytosolic transport and centrosome formation. Ran also localizes to chromatin as RCC1 does, its guanine nucleotide exchange factor, but Ran's function on chromatin is not known. We found that gsp1, a temperature-sensitive mutant of GSP1, a Saccharomyces cerevisiae Ran homologue, suppressed the hydroxyurea (HU) and ultra violet (UV) sensitivities of the mec1 mutant. In UV-irradiated mec1 gsp1 cells, Rad53 was phosphorylated despite the lack of Mec1. This suppression depended on the TEL1 gene, given that the triple mutant, mec1 gsp1 tel1, was unable to grow. The gsp1 mutations also suppressed the HU sensitivity of the rad9 mutant in a Tel1-dependent manner, but not the HU sensitivity of the rad53 mutant. These results indicated that Rad53 was activated by the Tel1 pathway in mec1 gsp1 cells, suggesting that Gsp1 helps regulate the role switching the ATM family kinases Mec1 and Tel1.     

Double-strand breaks trigger MRX- and Mec1-dependent, but Tel1-independent, checkpoint activation

Together with the Tel1 PI3 kinase, the Mre11/Rad50/Xrs2 (MRX) complex is involved in checkpoint activation in response to double-strand breaks (DSBs), a function also conserved in human cells by Mre11/Rad50/Nbs1 acting with ATM. It has been proposed that the yeast Tel1/MRX pathway is activated in the presence of DSBs that cannot be resected. The Mec1 PI3 kinase, by contrast, would be involved in detecting breaks that can be processed. The significance of a Mec1/MRX DSB-activated DNA damage checkpoint has yet to be reported. To understand whether the MRX complex works specifically with Tel1 or Mec1, we investigated MRX function in checkpoint activation in response to endonuclease-induced DSBs in synchronized cells. We found that the expression of EcoRI activated the G1 and intra-S phase checkpoints in a MRX- and Mec1-dependent, but Tel1-independent manner. The pathways identified here are therefore different from the Tel1/MRX pathway that was previously reported. Thus, our results demonstrate that MRX can function in concert with both Mec1 and Tel1 PI3K-like kinases to trigger checkpoint activation in response to DSBs. Importantly, we also describe a novel MRX-independent checkpoint that is activated in late S-phase when cells replicate their DNA in the presence of DSBs. The existence of this novel mode of checkpoint activation explains why several previous studies had reported that mutations in the MRX complex did not abrogate DSB-induced checkpoint activation in asynchronous cells.