The Chromatin Landscape around DNA Double-Strand Breaks in Yeast and Its Influence on DNA Repair Pathway Choice.

DNA double-strand breaks (DSBs) are harmful DNA lesions, which elicit catastrophic consequences for genome stability if not properly repaired. DSBs can be repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR). The choice between these two pathways depends on which proteins bind to the DSB ends and how their action is regulated. NHEJ initiates with the binding of the Ku complex to the DNA ends, while HR is initiated by the nucleolytic degradation of the 5'-ended DNA strands, which requires several DNA nucleases/helicases and generates single-stranded DNA overhangs. DSB repair occurs within a precisely organized chromatin environment, where the DNA is wrapped around histone octamers to form the nucleosomes. Nucleosomes impose a barrier to the DNA end processing and repair machinery. Chromatin organization around a DSB is modified to allow proper DSB repair either by the removal of entire nucleosomes, thanks to the action of chromatin remodeling factors, or by post-translational modifications of histones, thus increasing chromatin flexibility and the accessibility of repair enzymes to the DNA. Here, we review histone post-translational modifications occurring around a DSB in the yeast Saccharomyces cerevisiae and their role in DSB repair, with particular attention to DSB repair pathway choice.

The regulation of the DNA damage response at telomeres: focus on kinases.

The natural ends of linear chromosomes resemble those of accidental double-strand breaks (DSBs). DSBs induce a multifaceted cellular response that promotes the repair of lesions and slows down cell cycle progression. This response is not elicited at chromosome ends, which are organized in nucleoprotein structures called telomeres. Besides counteracting DSB response through specialized telomere-binding proteins, telomeres also prevent chromosome shortening. Despite of the different fate of telomeres and DSBs, many proteins involved in the DSB response also localize at telomeres and participate in telomere homeostasis. In particular, the DSB master regulators Tel1/ATM and Mec1/ATR contribute to telomere length maintenance and arrest cell cycle progression when chromosome ends shorten, thus promoting a tumor-suppressive process known as replicative senescence. During senescence, the actions of both these apical kinases and telomere-binding proteins allow checkpoint activation while bulk DNA repair activities at telomeres are still inhibited. Checkpoint-mediated cell cycle arrest also prevents further telomere erosion and deprotection that would favor chromosome rearrangements, which are known to increase cancer-associated genome instability. This review summarizes recent insights into functions and regulation of Tel1/ATM and Mec1/ATR at telomeres both in the presence and in the absence of telomerase, focusing mainly on discoveries in budding yeast.

Tel1/ATM prevents degradation of replication forks that reverse after topoisomerase poisoning.

In both yeast and mammals, the topoisomerase poison camptothecin (CPT) induces fork reversal, which has been proposed to stabilize replication forks, thus providing time for the repair of CPT-induced lesions and supporting replication restart. We show that Tel1, the Saccharomyces cerevisiae orthologue of human ATM kinase, stabilizes CPT-induced reversed forks by counteracting their nucleolytic degradation by the MRX complex. Tel1-lacking cells are hypersensitive to CPT specifically and show less reversed forks in the presence of CPT The lack of Mre11 nuclease activity restores wild-type levels of reversed forks in CPT-treated tel1Δ cells without affecting fork reversal in wild-type cells. Moreover, Mrc1 inactivation prevents fork reversal in wild-type, tel1Δ, and mre11 nuclease-deficient cells and relieves the hypersensitivity of tel1Δ cells to CPT Altogether, our data indicate that Tel1 counteracts Mre11 nucleolytic activity at replication forks that undergo Mrc1-mediated reversal in the presence of CPT.

Tel1 and Rif2 Regulate MRX Functions in End-Tethering and Repair of DNA Double-Strand Breaks.

The cellular response to DNA double-strand breaks (DSBs) is initiated by the MRX/MRN complex (Mre11-Rad50-Xrs2 in yeast; Mre11-Rad50-Nbs1 in mammals), which recruits the checkpoint kinase Tel1/ATM to DSBs. In Saccharomyces cerevisiae, the role of Tel1 at DSBs remains enigmatic, as tel1Δ cells do not show obvious hypersensitivity to DSB-inducing agents. By performing a synthetic phenotype screen, we isolated a rad50-V1269M allele that sensitizes tel1Δ cells to genotoxic agents. The MRV1269MX complex associates poorly to DNA ends, and its retention at DSBs is further reduced by the lack of Tel1. As a consequence, tel1Δ rad50-V1269M cells are severely defective both in keeping the DSB ends tethered to each other and in repairing a DSB by either homologous recombination (HR) or nonhomologous end joining (NHEJ). These data indicate that Tel1 promotes MRX retention to DSBs and this function is important to allow proper MRX-DNA binding that is needed for end-tethering and DSB repair. The role of Tel1 in promoting MRX accumulation to DSBs is counteracted by Rif2, which is recruited to DSBs. We also found that Rif2 enhances ATP hydrolysis by MRX and attenuates MRX function in end-tethering, suggesting that Rif2 can regulate MRX activity at DSBs by modulating ATP-dependent conformational changes of Rad50.

The RNA binding protein Npl3 promotes resection of DNA double-strand breaks by regulating the levels of Exo1.

Eukaryotic cells preserve genome integrity upon DNA damage by activating a signaling network that promotes DNA repair and controls cell cycle progression. One of the most severe DNA damage is the DNA double-strand break (DSB), whose 5΄ ends can be nucleolitically resected by multiple nucleases to create 3΄-ended single-stranded DNA tails that trigger DSB repair by homologous recombination. Here, we identify the Saccharomyces cerevisiae RNA binding protein Npl3 as a new player in DSB resection. Npl3 is related to both the metazoan serine-arginine-rich and the heterogeneous nuclear ribonucleo-proteins. NPL3 deletion impairs the generation of long ssDNA tails at the DSB ends, whereas it does not exacerbate the resection defect of exo1Δ cells. Furthermore, either the lack of Npl3 or the inactivation of its RNA-binding domains causes decrease of the exonuclease Exo1 protein levels as well as generation of unusual and extended EXO1 RNA species. These findings, together with the observation that EXO1 overexpression partially suppresses the resection defect of npl3Δ cells, indicate that Npl3 participates in DSB resection by promoting the proper biogenesis of EXO1 mRNA.

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.

Sae2 Function at DNA Double-Strand Breaks Is Bypassed by Dampening Tel1 or Rad53 Activity.

The MRX complex together with Sae2 initiates resection of DNA double-strand breaks (DSBs) to generate single-stranded DNA (ssDNA) that triggers homologous recombination. The absence of Sae2 not only impairs DSB resection, but also causes prolonged MRX binding at the DSBs that leads to persistent Tel1- and Rad53-dependent DNA damage checkpoint activation and cell cycle arrest. Whether this enhanced checkpoint signaling contributes to the DNA damage sensitivity and/or the resection defect of sae2Δ cells is not known. By performing a genetic screen, we identify rad53 and tel1 mutant alleles that suppress both the DNA damage hypersensitivity and the resection defect of sae2Δ cells through an Sgs1-Dna2-dependent mechanism. These suppression events do not involve escaping the checkpoint-mediated cell cycle arrest. Rather, defective Rad53 or Tel1 signaling bypasses Sae2 function at DSBs by decreasing the amount of Rad9 bound at DSBs. As a consequence, reduced Rad9 association to DNA ends relieves inhibition of Sgs1-Dna2 activity, which can then compensate for the lack of Sae2 in DSB resection and DNA damage resistance. We propose that persistent Tel1 and Rad53 checkpoint signaling in cells lacking Sae2 increases the association of Rad9 at DSBs, which in turn inhibits DSB resection by limiting the activity of the Sgs1-Dna2 resection machinery.

Tbf1 and Vid22 promote resection and non-homologous end joining of DNA double-strand break ends.

The repair of DNA double-strand breaks (DSBs) is crucial for maintaining genome stability. The Saccharomyces cerevisiae protein Tbf1, which is characterized by a Myb domain and is related to mammalian TRF1 and TRF2, has been proposed to act as a transcriptional activator. Here, we show that Tbf1 and its interacting protein Vid22 are new players in the response to DSBs. Inactivation of either TBF1 or VID22 causes hypersensitivity to DSB-inducing agents and shows strong negative interactions with mutations affecting homologous recombination. Furthermore, Tbf1 and Vid22 are recruited to an HO-induced DSB, where they promote both resection of DNA ends and repair by non-homologous end joining. Finally, inactivation of either Tbf1 or Vid22 impairs nucleosome eviction around the DSB, suggesting that these proteins promote efficient repair of the break by influencing chromatin identity in its surroundings.

Multiple pathways regulate 3' overhang generation at S. cerevisiae telomeres.

Generation of 3' G strand overhangs at telomere ends may play a role in regulating telomerase action and occurs by still unclear mechanisms. We show by an inducible short telomere assay that Sae2 and the Sgs1 RecQ helicase control two distinct but partially complementary pathways for nucleolytic processing of S. cerevisiae telomeres, with Sae2 function requiring its serine 267 phosphorylation. No processing activity is detectable in sae2Delta sgs1Delta cells, while the Exo1 exonuclease contributes to telomere end processing and elongation in both sae2Delta and sgs1Delta cells, suggesting that Exo1 telomeric function requires either Sgs1 or Sae2 action. Moreover, Dna2 might also support Sgs1 activity, as it acts redundantly with Exo1, but not with Sgs1. Finally, both length maintenance and G strand overhang generation at native telomeres are affected in sae2Delta sgs1Delta cells, further supporting the notion that Sae2 and Sgs1 combined activities control telomere length by regulating telomere processing.

Mechanisms and regulation of DNA end resection.

DNA double-strand breaks (DSBs) are highly hazardous for genome integrity, because failure to repair these lesions can lead to genomic instability. DSBs can arise accidentally at unpredictable locations into the genome, but they are also normal intermediates in meiotic recombination. Moreover, the natural ends of linear chromosomes resemble DSBs. Although intrachromosomal DNA breaks are potent stimulators of the DNA damage response, the natural ends of linear chromosomes are packaged into protective structures called telomeres that suppress DNA repair/recombination activities. Although DSBs and telomeres are functionally different, they both undergo 5'-3' nucleolytic degradation of DNA ends, a process known as resection. The resulting 3'-single-stranded DNA overhangs enable repair of DSBs by homologous recombination (HR), whereas they allow the action of telomerase at telomeres. The molecular activities required for DSB and telomere end resection are similar, indicating that the initial steps of HR and telomerase-mediated elongation are related. Resection of both DSBs and telomeres must be tightly regulated in time and space to ensure genome stability and cell survival.

Distinct Cdk1 requirements during single-strand annealing, noncrossover, and crossover recombination.

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.

Shelterin-like proteins and Yku inhibit nucleolytic processing of Saccharomyces cerevisiae telomeres.

Eukaryotic cells distinguish their chromosome ends from accidental DNA double-strand breaks (DSBs) by packaging them into protective structures called telomeres that prevent DNA repair/recombination activities. Here we investigate the role of key telomeric proteins in protecting budding yeast telomeres from degradation. We show that the Saccharomyces cerevisiae shelterin-like proteins Rif1, Rif2, and Rap1 inhibit nucleolytic processing at both de novo and native telomeres during G1 and G2 cell cycle phases, with Rif2 and Rap1 showing the strongest effects. Also Yku prevents telomere resection in G1, independently of its role in non-homologous end joining. Yku and the shelterin-like proteins have additive effects in inhibiting DNA degradation at G1 de novo telomeres, where Yku plays the major role in preventing initiation, whereas Rif1, Rif2, and Rap1 act primarily by limiting extensive resection. In fact, exonucleolytic degradation of a de novo telomere is more efficient in yku70Delta than in rif2Delta G1 cells, but generation of ssDNA in Yku-lacking cells is limited to DNA regions close to the telomere tip. This limited processing is due to the inhibitory action of Rap1, Rif1, and Rif2, as their inactivation allows extensive telomere resection not only in wild-type but also in yku70Delta G1 cells. Finally, Rap1 and Rif2 prevent telomere degradation by inhibiting MRX access to telomeres, which are also protected from the Exo1 nuclease by Yku. Thus, chromosome end degradation is controlled by telomeric proteins that specifically inhibit the action of different nucleases.

Elevated levels of the polo kinase Cdc5 override the Mec1/ATR checkpoint in budding yeast by acting at different steps of the signaling pathway.

Checkpoints are surveillance mechanisms that constitute a barrier to oncogenesis by preserving genome integrity. Loss of checkpoint function is an early event in tumorigenesis. Polo kinases (Plks) are fundamental regulators of cell cycle progression in all eukaryotes and are frequently overexpressed in tumors. Through their polo box domain, Plks target multiple substrates previously phosphorylated by CDKs and MAPKs. In response to DNA damage, Plks are temporally inhibited in order to maintain the checkpoint-dependent cell cycle block while their activity is required to silence the checkpoint response and resume cell cycle progression. Here, we report that, in budding yeast, overproduction of the Cdc5 polo kinase overrides the checkpoint signaling induced by double strand DNA breaks (DSBs), preventing the phosphorylation of several Mec1/ATR targets, including Ddc2/ATRIP, the checkpoint mediator Rad9, and the transducer kinase Rad53/CHK2. We also show that high levels of Cdc5 slow down DSB processing in a Rad9-dependent manner, but do not prevent the binding of checkpoint factors to a single DSB. Finally, we provide evidence that Sae2, the functional ortholog of human CtIP, which regulates DSB processing and inhibits checkpoint signaling, is regulated by Cdc5. We propose that Cdc5 interferes with the checkpoint response to DSBs acting at multiple levels in the signal transduction pathway and at an early step required to resect DSB ends.

The PP2A phosphatase counteracts the function of the 9-1-1 axis in checkpoint activation.

DNA damage elicits a checkpoint response depending on the Mec1/ATR kinase, which detects the presence of single-stranded DNA and activates the effector kinase Rad53/CHK2. In Saccharomyces cerevisiae, one of the signaling circuits leading to Rad53 activation involves the evolutionarily conserved 9-1-1 complex, which acts as a platform for the binding of Dpb11 and Rad9 (referred to as the 9-1-1 axis) to generate a protein complex that allows Mec1 activation. By examining the effects of both loss-of-function and hypermorphic mutations, here, we show that the Cdc55 and Tpd3 subunits of the PP2A phosphatase counteract activation of the 9-1-1 axis. The lack of this inhibitory function results in DNA-damage sensitivity, sustained checkpoint-mediated cell-cycle arrest, and impaired resection of DNA double-strand breaks. This PP2A anti-checkpoint role depends on the capacity of Cdc55 to interact with Ddc1 and to counteract Ddc1-Dpb11 complex formation by preventing Dpb11 recognition of Ddc1 phosphorylated on Thr602.

Interplay between Sae2 and Rif2 in the regulation of Mre11-Rad50 activities at DNA ends.

DNA double-strand breaks (DSBs) can be repaired by non-homologous end-joining (NHEJ) or homologous recombination (HR). HR is initiated by nucleolytic degradation of the DSB ends in a process termed resection. The Mre11-Rad50-Xrs2/NBS1 (MRX/N) complex is a multifunctional enzyme that, aided by the Sae2/CtIP protein, promotes DSB resection and maintains the DSB ends tethered to each other to facilitate their re-ligation. Furthermore, it activates the protein kinase Tel1/ATM, which initiates DSB signaling. In Saccharomyces cerevisiae, these MRX functions are inhibited by the Rif2 protein, which is enriched at telomeres and protects telomeric DNA from being sensed and processed as a DSB. The present review focuses on recent data showing that Sae2 and Rif2 regulate MRX functions in opposite manners by interacting with Rad50 and influencing ATP-dependent Mre11-Rad50 conformational changes. As Sae2 is enriched at DSBs whereas Rif2 is predominantly present at telomeres, the relative abundance of these two MRX regulators can provide an effective mechanism to activate or inactivate MRX depending on the nature of chromosome ends.

Resection of a DNA Double-Strand Break by Alkaline Gel Electrophoresis and Southern Blotting.

Generation of 3' single-stranded DNA (ssDNA) at the ends of a double-strand break (DSB) is essential to initiate repair by homology-directed mechanisms. Here we describe a Southern blot-based method to visualize the generation of ssDNA at the ends of site-specific DSBs generated in the Saccharomyces cerevisiae genome.

Dpb4 promotes resection of DNA double-strand breaks and checkpoint activation by acting in two different protein complexes.

Budding yeast Dpb4 (POLE3/CHRAC17 in mammals) is a highly conserved histone fold protein that is shared by two protein complexes: the chromatin remodeler ISW2/hCHRAC and the DNA polymerase ε (Pol ε) holoenzyme. In Saccharomyces cerevisiae, Dpb4 forms histone-like dimers with Dls1 in the ISW2 complex and with Dpb3 in the Pol ε complex. Here, we show that Dpb4 plays two functions in sensing and processing DNA double-strand breaks (DSBs). Dpb4 promotes histone removal and DSB resection by interacting with Dls1 to facilitate the association of the Isw2 ATPase to DSBs. Furthermore, it promotes checkpoint activation by interacting with Dpb3 to facilitate the association of the checkpoint protein Rad9 to DSBs. Persistence of both Isw2 and Rad9 at DSBs is enhanced by the A62S mutation that is located in the Dpb4 histone fold domain and increases Dpb4 association at DSBs. Thus, Dpb4 exerts two distinct functions at DSBs depending on its interactors.

The Yku70-Yku80 complex contributes to regulate double-strand break processing and checkpoint activation during the cell cycle.

DNA double-strand breaks (DSBs) are repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). HR requires 5' DSB end degradation that occurs in the presence of cyclin-dependent kinase (CDK) activity. Here, we show that a lack of any of the NHEJ proteins Yku (Yku70-Yku80), Lif1 or DNA ligase IV (Dnl4) increases 5' DSB end degradation in G1 phase, with ykuDelta cells showing the strongest effect. This increase depends on MRX, the recruitment of which at DSBs is enhanced in ykuDelta G1 cells. DSB processing in G2 is not influenced by the absence of Yku, but it is delayed by Yku overproduction, which also decreases MRX loading on DSBs. Moreover, DSB resection in ykuDelta cells occurs independently of CDK activity, suggesting that it might be promoted by CDK-dependent inhibition of Yku.

Tel1/ATM Signaling to the Checkpoint Contributes to Replicative Senescence in the Absence of Telomerase.

Telomeres progressively shorten at every round of DNA replication in the absence of telomerase. When they become critically short, telomeres trigger replicative senescence by activating a DNA damage response that is governed by the Mec1/ATR and Tel1/ATM protein kinases. While Mec1/ATR is known to block cell division when extended single-stranded DNA (ssDNA) accumulates at eroded telomeres, the molecular mechanism by which Tel1/ATM promotes senescence is still unclear. By characterizing a Tel1-hy184 mutant variant that compensates for the lack of Mec1 functions, we provide evidence that Tel1 promotes senescence by signaling to a Rad9-dependent checkpoint. Tel1-hy184 anticipates senescence onset in telomerase-negative cells, while the lack of Tel1 or the expression of a kinase-defective (kd) Tel1 variant delays it. Both Tel1-hy184 and Tel1-kd do not alter ssDNA generation at telomeric DNA ends. Furthermore, Rad9 and (only partially) Mec1 are responsible for the precocious senescence promoted by Tel1-hy184. This precocious senescence is mainly caused by the F1751I, D1985N, and E2133K amino acid substitutions, which are located in the FRAP-ATM-TRAPP domain of Tel1 and also increase Tel1 binding to DNA ends. Altogether, these results indicate that Tel1 induces replicative senescence by directly signaling dysfunctional telomeres to the checkpoint machinery.

Uncoupling Sae2 Functions in Downregulation of Tel1 and Rad53 Signaling Activities.

The Mre11-Rad50-Xrs2 (MRX) complex acts together with the Sae2 protein to initiate resection of DNA double-strand breaks (DSBs) and to regulate a checkpoint response that couples cell cycle progression with DSB repair. Sae2 supports resistance to DNA damage and downregulates the signaling activities of MRX, Tel1, and Rad53 checkpoint proteins at the sites of damage. How these functions are connected to each other is not known. Here, we describe the separation-of-function sae2-ms mutant that, similar to SAE2 deletion, upregulates MRX and Tel1 signaling activities at DSBs by reducing Mre11 endonuclease activity. However, unlike SAE2 deletion, Sae2-ms causes neither DNA damage sensitivity nor enhanced Rad53 activation, indicating that DNA damage resistance depends mainly on Sae2-mediated Rad53 inhibition. The lack of Sae2, but not the presence of Sae2-ms, impairs long-range resection and increases both Rad9 accumulation at DSBs and Rad53-Rad9 interaction independently of Mre11 nuclease activity. Altogether, these data lead to a model whereby Sae2 plays distinct functions in limiting MRX-Tel1 and Rad9 abundance at DSBs, with the control on Rad9 association playing the major role in supporting DNA damage resistance and in regulating long-range resection and checkpoint activation.