MEC1-dependent redistribution of the Sir3 silencing protein from telomeres to DNA double-strand breaks.

The yeast Sir2/3/4p complex is found in abundance at telomeres, where it participates in the formation of silent heterochromatin and telomere maintenance. Here, we show that Sir3p is released from telomeres in response to DNA double-strand breaks (DSBs), binds to DSBs, and mediates their repair, independent of cell mating type. Sir3p relocalization is S phase specific and, importantly, requires the DNA damage checkpoint genes MEC1 and RAD9. MEC1 is a homolog of ATM, mutations in which cause ataxia telangiectasia (A-T), a disease characterized by various neurologic and immunologic abnormalities, a predisposition for cancer, and a cellular defect in repair of DSBs. This novel mode by which preformed DNA repair machinery is mobilized by DNA damage sensors may have implications for human diseases resulting from defective DSB repair.     

Understanding the limitations of radiation-induced cell cycle checkpoints

The DNA damage response pathways involve processes of double-strand break (DSB) repair and cell cycle checkpoint control to prevent or limit entry into S phase or mitosis in the presence of unrepaired damage. Checkpoints can function to permanently remove damaged cells from the actively proliferating population but can also halt the cell cycle temporarily to provide time for the repair of DSBs. Although efficient in their ability to limit genomic instability, checkpoints are not foolproof but carry inherent limitations. Recent work has demonstrated that the G1/S checkpoint is slowly activated and allows cells to enter S phase in the presence of unrepaired DSBs for about 4-6?h post irradiation. During this time, only a slowing but not abolition of S-phase entry is observed. The G2/M checkpoint, in contrast, is quickly activated but only responds to a level of 10-20 DSBs such that cells with a low number of DSBs do not initiate the checkpoint or terminate arrest before repair is complete. Here, we discuss the limitations of these checkpoints in the context of the current knowledge of the factors involved. We suggest that the time needed to fully activate G1/S arrest reflects the existence of a restriction point in G1-phase progression. This point has previously been defined as the point when mitogen starvation fails to prevent cells from entering S phase. However, cells that passed the restriction point can respond to DSBs, albeit with reduced efficiency.     

Regulation of Saccharomyces Rad53 checkpoint kinase during adaptation from DNA damage-induced G2/M arrest

Saccharomyces cells with one unrepaired double-strand break (DSB) adapt after checkpoint-mediated G2/M arrest. Adaptation is accompanied by loss of Rad53p checkpoint kinase activity and Chk1p phosphorylation. Rad53p kinase remains elevated in yku70delta and cdc5-ad cells that fail to adapt. Permanent G2/M arrest in cells with increased single-stranded DNA is suppressed by the rfa1-t11 mutation, but this RPA mutation does not suppress permanent arrest in cdc5-ad cells. Checkpoint kinase activation and inactivation can be followed in G2-arrested cells, but there is no kinase activation in G1-arrested cells. We conclude that activation of the checkpoint kinases in response to a single DNA break is cell cycle regulated and that adaptation is an active process by which these kinases are inactivated.     

Tid1/Rdh54 translocase is phosphorylated through a Mec1- and Rad53-dependent manner in the presence of DSB lesions in budding yeast

Saccharomyces cerevisiae cells with a single double-strand break (DSB) activate the ATR/Mec1-dependent checkpoint response as a consequence of extensive ssDNA accumulation. The recombination factor Tid1/Rdh54, a member of the Swi2-like family proteins, has an ATPase activity and may contribute to the remodelling of nucleosomes on DNA. Tid1 dislocates Rad51 recombinase from dsDNA, can unwind and supercoil DNA filaments, and has been implicated in checkpoint adaptation from a G2/M arrest induced by an unrepaired DSB.Here we show that both ATR/Mec1 and Chk2/Rad53 kinases are implicated in the phosphorylation of Tid1 in the presence of DNA damage, indicating that the protein is regulated during the DNA damage response. We show that Tid1 ATPase activity is dispensable for its phosphorylation and for its recruitment near a DSB, but it is required to switch off Rad53 activation and for checkpoint adaptation. Mec1 and Rad53 kinases, together with Rad51 recombinase, are also implicated in the hyper-phosphorylation of the ATPase defective Tid1-K318R variant and in the efficient binding of the protein to the DSB site.In summary, Tid1 is a novel target of the DNA damage checkpoint pathway that is also involved in checkpoint adaptation.     

Radiation-induced double-strand breaks require ATM but not Artemis for homologous recombination during S-phase

Double-strand breaks (DSBs) are repaired by two distinct pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). The endonuclease Artemis and the PIK kinase Ataxia-Telangiectasia Mutated (ATM), mutated in prominent human radiosensitivity syndromes, are essential for repairing a subset of DSBs via NHEJ in G1 and HR in G2. Both proteins have been implicated in DNA end resection, a mandatory step preceding homology search and strand pairing in HR. Here, we show that during S-phase Artemis but not ATM is dispensable for HR of radiation-induced DSBs. In replicating AT cells, numerous Rad51 foci form gradually, indicating a Rad51 recruitment process that is independent of ATM-mediated end resection. Those DSBs decorated with Rad51 persisted through S- and G2-phase indicating incomplete HR resulting in unrepaired DSBs and a pronounced G2 arrest. We demonstrate that in AT cells loading of Rad51 depends on functional ATR/Chk1. The ATR-dependent checkpoint response is most likely activated when the replication fork encounters radiation-induced single-strand breaks leading to generation of long stretches of single-stranded DNA. Together, these results provide new insight into the role of ATM for initiation and completion of HR during S- and G2-phase. The DSB repair defect during S-phase significantly contributes to the radiosensitivity of AT cells.     

Mechanisms of checkpoint kinase Rad53 inactivation after a double-strand break in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, double-strand breaks (DSBs) activate DNA checkpoint pathways that trigger several responses including a strong G(2)/M arrest. We have previously provided evidence that the phosphatases Ptc2 and Ptc3 of the protein phosphatase 2C type are required for DNA checkpoint inactivation after a DSB and probably dephosphorylate the checkpoint kinase Rad53. In this article we have investigated further the interactions between Ptc2 and Rad53. We showed that forkhead-associated domain 1 (FHA1) of Rad53 interacts with a specific threonine of Ptc2, T376, located outside its catalytic domain in a TXXD motif which constitutes an optimal FHA1 binding sequence in vitro. Mutating T376 abolishes Ptc2 interaction with the Rad53 FHA1 domain and results in adaptation and recovery defects following a DSB. We found that Ckb1 and Ckb2, the regulatory subunits of the protein kinase CK2, are necessary for the in vivo interaction between Ptc2 and the Rad53 FHA1 domain, that Ckb1 binds Ptc2 in vitro and that ckb1Delta and ckb2Delta mutants are defective in adaptation and recovery after a DSB. Our data thus strongly suggest that CK2 is the kinase responsible for the in vivo phosphorylation of Ptc2 T376.     

Role of Dot1-dependent histone H3 methylation in G1 and S phase DNA damage checkpoint functions of Rad9

We screened radiation-sensitive yeast mutants for DNA damage checkpoint defects and identified Dot1, the conserved histone H3 Lys 79 methyltransferase. DOT1 deletion mutants (dot1Delta) are G1 and intra-S phase checkpoint defective after ionizing radiation but remain competent for G2/M arrest. Mutations that affect Dot1 function such as Rad6-Bre1/Paf1 pathway gene deletions or mutation of H2B Lys 123 or H3 Lys 79 share dot1Delta checkpoint defects. Whereas dot1Delta alone confers minimal DNA damage sensitivity, combining dot1Delta with histone methyltransferase mutations set1Delta and set2Delta markedly enhances lethality. Interestingly, set1Delta and set2Delta mutants remain G1 checkpoint competent, but set1Delta displays a mild S phase checkpoint defect. In human cells, H3 Lys 79 methylation by hDOT1L likely mediates recruitment of the signaling protein 53BP1 via its paired tudor domains to double-strand breaks (DSBs). Consistent with this paradigm, loss of Dot1 prevents activation of the yeast 53BP1 ortholog Rad9 or Chk2 homolog Rad53 and decreases binding of Rad9 to DSBs after DNA damage. Mutation of Rad9 to alter tudor domain binding to methylated Lys 79 phenocopies the dot1Delta checkpoint defect and blocks Rad53 phosphorylation. These results indicate a key role for chromatin and methylation of histone H3 Lys 79 in yeast DNA damage signaling.     

Role of the Saccharomyces cerevisiae Rad53 checkpoint kinase in signaling double-strand breaks during the meiotic cell cycle

DNA double-strand breaks (DSBs) can arise at unpredictable locations after DNA damage or in a programmed manner during meiosis. DNA damage checkpoint response to accidental DSBs during mitosis requires the Rad53 effector kinase, whereas the meiosis-specific Mek1 kinase, together with Red1 and Hop1, mediates the recombination checkpoint in response to programmed meiotic DSBs. Here we provide evidence that exogenous DSBs lead to Rad53 phosphorylation during the meiotic cell cycle, whereas programmed meiotic DSBs do not. However, the latter can trigger phosphorylation of a protein fusion between Rad53 and the Mec1-interacting protein Ddc2, suggesting that the inability of Rad53 to transduce the meiosis-specific DSB signals might be due to its failure to access the meiotic recombination sites. Rad53 phosphorylation/activation is elicited when unrepaired meiosis-specific DSBs escape the recombination checkpoint. This activation requires homologous chromosome segregation and delays the second meiotic division. Altogether, these data indicate that Rad53 prevents sister chromatid segregation in the presence of unrepaired programmed meiotic DSBs, thus providing a salvage mechanism ensuring genetic integrity in the gametes even in the absence of the recombination checkpoint.     

DNA resection proteins Sgs1 and Exo1 are required for G1 checkpoint activation in budding yeast

Double-strand breaks (DSBs) in budding yeast trigger activation of DNA damage checkpoints, allowing repair to occur. Although resection is necessary for initiating damage-induced cell cycle arrest in G2, no role has been assigned to it in the activation of G1 checkpoint. Here we demonstrate for the first time that the resection proteins Sgs1 and Exo1 are required for efficient G1 checkpoint activation. We find in G1 arrested cells that histone H2A phosphorylation in response to ionizing radiation is independent of Sgs1 and Exo1. In contrast, these proteins are required for damage-induced recruitment of Rfa1 to the DSB sites, phosphorylation of the Rad53 effector kinase, cell cycle arrest and RNR3 expression. Checkpoint activation in G1 requires the catalytic activity of Sgs1, suggesting that it is DNA resection mediated by Sgs1 that stimulates the damage response pathway rather than protein–protein interactions with other DDR proteins. Together, these results implicate DNA resection, which is thought to be minimal in G1, as necessary for activation of the G1 checkpoint.     

Requirement for the SRS2 DNA helicase gene in non-homologous end joining in yeast

Mitotic cells experience double-strand breaks (DSBs) from both exogenous and endogenous sources. Since unrepaired DSBs can result in genome rearrangements or cell death, cells mobilize multiple pathways to repair the DNA damage. In the yeast Saccharomyces cerevisiae, mitotic cells preferentially use a homologous recombination repair pathway. However, when no significant homology to the DSB ends is available, cells utilize a repair process called non-homologous end joining (NHEJ), which can join ends with no homology through resection to uncover microhomologies of a few nucleotides. Although components of the homologous recombination repair system are also involved in NHEJ, the rejoining does not involve all of the homologous recombination repair genes. The SRS2 DNA helicase has been shown to be required for DSB repair when the homologous single-stranded regions are short. Here it is shown that SRS2 is also required for NHEJ, regardless of the cell mating type. Efficient NHEJ of sticky ends requires the Ku70 and Ku80 proteins and the silencing genes SIR2, SIR3 and SIR4. However, NHEJ of blunt ends, while very inefficient, is not further reduced by mutations in YKU70, SIR2, SIR3, SIR4 or SRS2, suggesting that this rejoining process occurs by a different mechanism.     

Analysis of human syndromes with disordered chromatin reveals the impact of heterochromatin on the efficacy of ATM-dependent G2/M checkpoint arrest

Heterochromatin (HC) poses a barrier to γH2AX focus expansion and DNA double-strand break (DSB) repair, the latter being relieved by ATM-dependent KAP-1 phosphorylation. Using high-resolution imaging, we show here that the HC superstructure markedly restricts ATM signaling to cell cycle checkpoint proteins. The impact of HC is greater than anticipated from the percentage of HC-DNA and, in distinction to DSB repair, ATM only partly overcomes the constraints posed by HC. Importantly, we examine ATM signaling in human syndromes with disordered HC. After depletion of MeCP2 and DNMT3B, proteins defective in the Rett and immunodeficiency with centromere instability and facial anomalies (ICF) syndromes, respectively, we demonstrate enhanced γH2AX signal expansion at HC-chromocenters in mouse NIH 3T3 cells, which have visible HC-chromocenters. Previous studies have shown that the G(2)/M checkpoint is inefficient requiring multiple DSBs to initiate arrest. MeCP2 and DNMT3B depletion leads to hypersensitive radiation-induced G(2)/M checkpoint arrest despite normal DSB repair. Cell lines from Rett, ICF, and Hutchinson-Guildford progeria syndrome patients similarly showed hyperactivated ATM signaling and hypersensitive and prolonged G(2)/M checkpoint arrest. Collectively, these findings reveal that heterochromatin contributes to the previously described inefficient G(2)/M checkpoint arrest and demonstrate how the signaling response can be uncoupled from DSB repair.     

The Saccharomyces cerevisiae Sae2 protein negatively regulates DNA damage checkpoint signalling

Double-strand breaks (DSBs) elicit a DNA damage response, resulting in checkpoint-mediated cell-cycle delay and DNA repair. The Saccharomyces cerevisiae Sae2 protein is known to act together with the MRX complex in meiotic DSB processing, as well as in DNA damage response during the mitotic cell cycle. Here, we report that cells lacking Sae2 fail to turn off both Mec1- and Tel1-dependent checkpoints activated by a single irreparable DSB, and delay Mre11 foci disassembly at DNA breaks, indicating that Sae2 may negatively regulate checkpoint signalling by modulating MRX association at damaged DNA. Consistently, high levels of Sae2 prevent checkpoint activation and impair MRX foci formation in response to unrepaired DSBs. Mec1- and Tel1-dependent Sae2 phosphorylation is necessary for these Sae2 functions, suggesting that the two kinases, once activated, may regulate checkpoint switch off through Sae2-mediated inhibition of MRX signalling.     

An Imperfect G2M Checkpoint Contributes to Chromosome Instability Following Irradiation of S and G2 Phase Cells

DNA double strand break (DSB) repair and checkpoint control represent two major mechanisms that function to reduce chromosomal instability following ionising irradiation (IR). Ataxia telangiectasia (A-T) cells have long been known to have defective checkpoint responses. Recent studies have shown that they also have a DSB repair defect following IR raising the issue of how ATM’s repair and checkpoint functions interplay to maintain chromosomal stability. A-T and Artemis cells manifest an identical and epistatic repair defect throughout the cell cycle demonstrating that ATM’s major repair defect following IR represents Artemis-dependent end-processing. Artemis cells show efficient G2/M checkpoint induction and a prolonged arrest relative to normal cells. Following irradiation of G2 cells, this checkpoint is dependent on ATM and A-T cells fail to show checkpoint arrest. In contrast, cells irradiated during S phase initiate a G2/M checkpoint which is independent of ATM and, significantly, both Artemis and A-T cells show a prolonged arrest at the G2/M checkpoint likely reflecting their repair defect. Strikingly, the G2/M checkpoint is released before the completion of repair when approximately 10-20 DSBs remain both for S phase and G2 phase irradiated cells. This defined sensitivity level of the G2/M checkpoint explains the prolonged arrest in repair-deficient relative to normal cells and provides a conceptual framework for the co-operative phenotype between checkpoint and repair functions in maintaining chromosomal stability.     

Pch2 links chromatin silencing to meiotic checkpoint control.

The PCH2 gene of Saccharomyces cerevisiae is required for the meiotic checkpoint that prevents chromosome segregation when recombination and chromosome synapsis are defective. Mutation of PCH2 relieves the checkpoint-induced pachytene arrest of the zip1, zip2, and dmc1 mutants, resulting in chromosome missegregation and low spore viability. Most of the Pch2 protein localizes to the nucleolus, where it represses meiotic interhomolog recombination in the ribosomal DNA, apparently by excluding the meiosis-specific Hop1 protein. Nucleolar localization of Pch2 depends on the silencing factor Sir2, and mutation of SIR2 also bypasses the zip1 pachytene arrest. Under certain circumstances, Sir3-dependent localization of Pch2 to telomeres also provides checkpoint function. These unexpected findings link the nucleolus, chromatin silencing, and the pachytene checkpoint.     

SIR–nucleosome interactions: Structure–function relationships in yeast silent chromatin

Discrete regions of the eukaryotic genome assume a heritable chromatin structure that is refractory to gene expression, referred to as heterochromatin or “silent” chromatin. Constitutively silent chromatin is found in subtelomeric domains in a number of species, ranging from yeast to man. In addition, chromatin-dependent repression of mating type loci occurs in both budding and fission yeasts, to enable sexual reproduction. The silencing of chromatin in budding yeast is characterized by an assembly of Silent Information Regulatory (SIR) proteins—Sir2, Sir3 and Sir4—with unmodified nucleosomes. Silencing requires the lysine deacetylase activity of Sir2, extensive contacts between Sir3 and the nucleosome, as well as interactions among the SIR proteins, to generate the Sir2–3–4 or SIR complex. Results from recent structural and reconstitution studies suggest an updated model for the ordered assembly and organization of SIR-dependent silent chromatin in yeast. Moreover, studies of subtelomeric gene expression reveal the importance of subtelomeric silent chromatin in the regulation of genes other than the silent mating type loci. This review covers recent advances in this field.     

Break dosage, cell cycle stage and DNA replication influence DNA double strand break response

DNA double strand breaks (DSBs) can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HR). HR requires nucleolytic degradation of 5' DNA ends to generate tracts of single-stranded DNA (ssDNA), which are also important for the activation of DNA damage checkpoints. Here we describe a quantitative analysis of DSB processing in the budding yeast Saccharomyces cerevisiae. We show that resection of an HO endonuclease-induced DSB is less extensive than previously estimated and provide evidence for significant instability of the 3' ssDNA tails. We show that both DSB resection and checkpoint activation are dose-dependent, especially during the G1 phase of the cell cycle. During G1, processing near the break is inhibited by competition with NHEJ, but extensive resection is regulated by an NHEJ-independent mechanism. DSB processing and checkpoint activation are more efficient in G2/M than in G1 phase, but are most efficient at breaks encountered by DNA replication forks during S phase. Our findings identify unexpected complexity of DSB processing and its regulation, and provide a framework for further mechanistic insights.     

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.     

Evidence for DNA-PK-dependent and -independent DNA double-strand break repair pathways in mammalian cells as a function of the cell cycle.

Mice homozygous for the scid (severe combined immune deficiency) mutation are defective in the repair of DNA double-strand breaks (DSBs) and are consequently very X-ray sensitive and defective in the lymphoid V(D)J recombination process. Recently, a strong candidate for the scid gene has been identified as the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) complex. Here, we show that the activity of the DNA-PK complex is regulated in a cell cycle-dependent manner, with peaks of activity found at the G1/early S phase and again at the G2 phase in wild-type cells. Interestingly, only the deficit of the G1/early S phase DNA-PK activity correlated with an increased hypersensitivity to X-irradiation and a DNA DSB repair deficit in synchronized scid pre-B cells. Finally, we demonstrate that the DNA-PK activity found at the G2 phase may be required for exit from a DNA damage-induced G2 checkpoint arrest. These observations suggest the presence of two pathways (DNA-PK-dependent and -independent) of illegitimate mammalian DNA DSB repair and two distinct roles (DNA DSB repair and G2 checkpoint traversal) for DNA-PK in the cellular response to ionizing radiation.