MRX protects telomeric DNA at uncapped telomeres of budding yeast cdc13-1 mutants

MRX, an evolutionally conserved DNA damage response complex composed of Mre11, Rad50 and Xrs2, is involved in DNA double strand break (DSB) repair, checkpoint activation and telomere maintenance. At DSBs, MRX plays a role in generating single stranded DNA (ssDNA) and signalling cell cycle arrest. Here we investigated whether MRX also contributes to generating ssDNA or signalling cell cycle arrest at uncapped telomeres. To investigate the role of MRX, we generated a conditionally degradable Rad50 protein and combined this with cdc13-1, a temperature sensitive mutation in the Cdc13 telomere capping protein. We show that Rad50 does not contribute to ssDNA generation or cell cycle arrest in response to cdcl3-1 uncapped telomeres. Instead, we find that Rad50 inhibits ssDNA accumulation and promotes cdc13-1 cell viability, consistent with a major role for MRX in telomere capping.     

Telomeres and DNA damage checkpoints

In all eukaryotic organisms, interruptions in duplex DNA molecules elicit a DNA damage response, which includes activation of DNA repair machineries and surveillance mechanisms, known as DNA damage checkpoints. Telomeres and double-strand breaks (DSBs) share the common feature of being physical ends of chromosomes. However, unlike DSBs, telomeres do not activate the DNA damage checkpoints and are usually protected from end-to-end fusions and other processing events that normally promote repair of DNA breaks. This indicates that they are shielded from being recognized and processed as DSBs. On the other hand, chromosome ends resemble damaged DNA, as several factors required for DNA repair and checkpoint networks play important roles in telomere length maintenance. Due to the critical role of both DNA damage checkpoints and telomere homeostasis in maintaining genetic stability and in counteracting cancer development, the knowledge of their interconnections is essential for our understanding of these key cellular controls.     

Histone methyltransferase Dot1 and Rad9 inhibit single-stranded DNA accumulation at DSBs and uncapped telomeres

Cells respond to DNA double-strand breaks (DSBs) and uncapped telomeres by recruiting checkpoint and repair factors to the site of lesions. Single-stranded DNA (ssDNA) is an important intermediate in the repair of DSBs and is produced also at uncapped telomeres. Here, we provide evidence that binding of the checkpoint protein Rad9, through its Tudor domain, to methylated histone H3-K79 inhibits resection at DSBs and uncapped telomeres. Loss of DOT1 or mutations in RAD9 influence a Rad50-dependent nuclease, leading to more rapid accumulation of ssDNA, and faster activation of the critical checkpoint kinase, Mec1. Moreover, deletion of RAD9 or DOT1 partially bypasses the requirement for CDK1 in DSB resection. Interestingly, Dot1 contributes to checkpoint activation in response to low levels of telomere uncapping but is not essential with high levels of uncapping. We suggest that both Rad9 and histone H3 methylation allow transmission of the damage signal to checkpoint kinases, and keep resection of damaged DNA under control influencing, both positively and negatively, checkpoint cascades and contributing to a tightly controlled response to DNA damage.     

Dissection of Rad9 BRCT domain function in the mitotic checkpoint response to telomere uncapping

In Saccharomyces cerevisiae, destabilizing telomeres, via inactivation of telomeric repeat binding factor Cdc13, induces a cell cycle checkpoint that arrests cells at the metaphase to anaphase transition—much like the response to an unrepaired DNA double strand break (DSB). Throughout the cell cycle, the multi-domain adaptor protein Rad9 is required for the activation of checkpoint effector kinase Rad53 in response to DSBs and is similarly necessary for checkpoint signaling in response to telomere uncapping. Rad53 activation in G1 and S phase depends on Rad9 association with modified chromatin adjacent to DSBs, which is mediated by Tudor domains binding histone H3 di-methylated at K79 and BRCT domains to histone H2A phosphorylated at S129. Nonetheless, Rad9 Tudor or BRCT mutants can initiate a checkpoint response to DNA damage in nocodazole-treated cells. Mutations affecting di-methylation of H3 K79, or its recognition by Rad9 enhance 5′ strand resection upon telomere uncapping, and potentially implicate Rad9 chromatin binding in the checkpoint response to telomere uncapping. Indeed, we report that Rad9 binds to sub-telomeric chromatin, upon telomere uncapping, up to 10 kb from the telomere. Rad9 binding occurred within 30 min after inactivating Cdc13, preceding Rad53 phosphorylation. In turn, Rad9 Tudor and BRCT domain mutations blocked chromatin binding and led to attenuated checkpoint signaling as evidenced by decreased Rad53 phosphorylation and impaired cell cycle arrest. Our work identifies a role for Rad9 chromatin association, during mitosis, in the DNA damage checkpoint response to telomere uncapping, suggesting that chromatin binding may be an initiating event for checkpoints throughout the cell cycle.     

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.     

Pif1- and Exo1-dependent nucleases coordinate checkpoint activation following telomere uncapping

Essential telomere 'capping' proteins act as a safeguard against ageing and cancer by inhibiting the DNA damage response (DDR) and regulating telomerase recruitment, thus distinguishing telomeres from double-strand breaks (DSBs). Uncapped telomeres and unrepaired DSBs can both stimulate a potent DDR, leading to cell cycle arrest and cell death. Using the cdc13-1 mutation to conditionally 'uncap' telomeres in budding yeast, we show that the telomere capping protein Cdc13 protects telomeres from the activity of the helicase Pif1 and the exonuclease Exo1. Our data support a two-stage model for the DDR at uncapped telomeres; Pif1 and Exo1 resect telomeric DNA <5 kb from the chromosome end, stimulating weak checkpoint activation; resection is extended >5 kb by Exo1 and full checkpoint activation occurs. Cdc13 is also crucial for telomerase recruitment. However, cells lacking Cdc13, Pif1 and Exo1, do not senesce and maintain their telomeres in a manner dependent upon telomerase, Ku and homologous recombination. Thus, attenuation of the DDR at uncapped telomeres can circumvent the need for otherwise-essential telomere capping proteins.     

Telomeres and the DNA damage response: why the fox is guarding the henhouse

DNA double strand breaks (DSBs) are repaired by an extensive network of proteins that recognize damaged DNA and catalyze its repair. By virtue of their similarity, the normal ends of linear chromosomes and internal DNA DSBs are both potential substrates for DSB repair enzymes. Thus, telomeres, specialized nucleo-protein complexes that cap chromosomal ends, serve a critical function to differentiate themselves from internal DNA strand breaks, and as a result prevent genomic instability that can result from their inappropriate involvement in repair reactions. Telomeres that become critically short due to failure of telomere maintenance mechanisms, or which become dysfunctional by loss of telomere binding proteins, elicit extensive checkpoint responses that in normal cells blocks proliferation. In this situation, the DNA DSB repair machinery plays a major role in responding to these "damaged" telomeres - creating chromosome fusions or capturing telomeres from other chromosomes in an effort to rid the cell of the perceived damage. However, a surprising aspect of telomere maintenance is that many of the same proteins that facilitate this repair of damaged telomeres are also necessary for their proper integrity. Here, we review recent work defining the roles for DSB repair machinery in telomere maintenance and in response to telomere dysfunction.     

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.     

Subtelomere-binding protein Tbf1 and telomere-binding protein Rap1 collaborate to inhibit localization of the Mre11 complex to DNA ends in budding yeast

Chromosome ends, known as telomeres, have to be distinguished from DNA double-strand breaks that activate DNA damage checkpoints. In budding yeast, the Mre11-Rad50-Xrs2 (MRX) complex associates with DNA ends and promotes checkpoint activation. Rap1 binds to double-stranded telomeric regions and recruits Rif1 and Rif2 to telomeres. Rap1 collaborates with Rif1 and Rif2 and inhibits MRX localization to DNA ends. This Rap1-Rif1-Rif2 function becomes attenuated at shortened telomeres. Here we show that Rap1 acts together with the subtelomere-binding protein Tbf1 and inhibits MRX localization to DNA ends. The placement of a subtelomeric sequence or TTAGGG repeats together with a short telomeric TG repeat sequence inhibits MRX accumulation at nearby DNA ends in a Tbf1-dependent manner. Moreover, tethering of both Tbf1 and Rap1 proteins decreases MRX and Tel1 accumulation at nearby DNA ends. This Tbf1- and Rap1-dependent pathway operates independently of Rif1 or Rif2 function. Depletion of Tbf1 protein stimulates checkpoint activation in cells containing short telomeres but not in cells containing normal-length telomeres. These data support a model in which Tbf1 and Rap1 collaborate to maintain genomic stability of short telomeres.     

Sudden telomere lengthening triggers a Rad53-dependent checkpoint in Saccharomyces cerevisiae

Telomeres are specialized functional complexes that ensure chromosome stability by protecting chromosome ends from fusions and degradation and avoiding chromosomal termini from being sensed as DNA breaks. Budding yeast Tel1 is required both for telomere metabolism and for a Rad53-dependent checkpoint responding to unprocessed double-strand breaks. We show that overexpression of a GAL1-TEL1 fusion causes transient telomere lengthening and activation of a Rad53-dependent G2/M checkpoint in cells whose telomeres are short due to the lack of either Tel1 or Yku70. Sudden telomere elongation and checkpoint-mediated cell cycle arrest are also triggered in wild-type cells by overproducing a protein fusion between the telomeric binding protein Cdc13 and the telomerase-associated protein Est1. Checkpoint activation by GAL1-TEL1 requires ongoing telomere elongation. In fact, it is turned off concomitantly with telomeres reaching a new stable length and is partially suppressed by deletion of the telomerase EST2 gene. Moreover, both telomere length rebalancing and checkpoint inactivation under galactose-induced conditions are accelerated by high levels of either the Sae2 protein, involved in double-strand breaks processing, or the negative telomere length regulator Rif2. These data suggest that sudden telomere lengthening elicits a checkpoint response that inhibits the G2/M transition.     

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.     

Telomere biology: integrating chromosomal end protection with DNA damage response

Telomeres play the key protective role at chromosomes. Many studies indicate that loss of telomere function causes activation of DNA damage response. Here, we review evidence supporting interdependence between telomere maintenance and DNA damage response and present a model in which these two pathways are combined into a single mechanism for protecting chromosomal integrity. Proteins directly involved in telomere maintenance and DNA damage response include Ku, DNA-PKcs, RAD51D, PARP-2, WRN and RAD50/MRE11/NBS1 complex. Since most of these proteins participate in the repair of DNA double-strand breaks (DSBs), this was perceived by many authors as a paradox, given that telomeres function to conceal natural DNA ends from mechanisms that detect and repair DSBs. However, we argue here that the key function of one particular DSB protein, Ku, is to prevent or control access of telomerase, the enzyme that synthesises telomeric sequences, to both internal DSBs and natural chromosomal ends. This view is supported by observations that Ku has a high affinity for DNA ends; it acts as a negative regulator of telomerase and that telomerase itself can target internal DSBs. Ku then directs other DSB repair/telomere maintenance proteins to either repair DSBs at internal chromosomal sites or prevent uncontrolled elongation of telomeres by telomerase. This model eliminates the above paradox and provides a testable scenario in which the role of DSB repair proteins is to protect chromosomal integrity by balancing repair activities and telomere maintenance. In our model, a close association between telomeres and different DNA damage response factors is not an unexpected event, but rather a logical result of chromosomal integrity maintenance activities. Review related to the 15th International Chromosome Conference (ICC XV), held in September 2004, Brunel University, London, UK     

Origin-dependent initiation of DNA replication within telomeric sequences

Replication of telomeres requires the action of telomerase, the semi-conservative replication machinery and the stabilization of the replication fork during passage through telomeric DNA. Whether vertebrate telomeres support initiation of replication has not been experimentally addressed. Using Xenopus cell free extracts we established a system to study replication initiation within linear telomeric DNA substrates. We show binding of TRF2 to telomeric DNA, indicating that exogenous DNA exclusively composed of telomeric repeats is recognized by shelterin components. Interaction with telomere binding proteins is not sufficient to prevent a DNA damage response. Notably, we observe regulated assembly of the pre-replicative complex proteins ORC2, MCM6 and Cdc6 to telomeric DNA. Most importantly, we detect origin-dependent replication of telomeric substrates under conditions that inhibit checkpoint activation. These results indicate that pre-replicative complexes assemble within telomeric DNA and can be converted into functional origins.     

Dual role for Saccharomyces cerevisiae Tel1 in the checkpoint response to double-strand breaks

The main responder to DNA double-strand breaks (DSBs) in mammals is ataxia telangiectasia mutated (ATM), whereas DSB-induced checkpoint activation in budding yeast seems to depend primarily on the ATM and Rad-3-related (ATR) orthologue Mec1. Here, we show that Saccharomyces cerevisiae Tel1, the ATM orthologue, has two functions in checkpoint response to DSBs. First, Tel1 participates, together with the MRX complex, in Mec1-dependent DSB-induced checkpoint activation by increasing the efficiency of single-stranded DNA accumulation at the ends of DSBs, and this checkpoint function can be overcome by overproducing the exonuclease Exo1. Second, Tel1 can activate the checkpoint response to DSBs independently of Mec1, although its signalling activity only becomes apparent when several DSBs are generated. Furthermore, we provide evidence that the kinetics of DSB resection can influence Tel1 activation, indicating that processing of the DSB termini might influence the transition from Tel1/ATM- to Mec1/ATR-dependent checkpoint.     

Xrs2p regulates Mre11p translocation to the nucleus and plays a role in telomere elongation and meiotic recombination

The Mre11-Rad50-Xrs2 (MRX) protein complex plays pivotal roles in meiotic recombination, repair of damaged DNA, telomere elongation, and cell cycle checkpoint control. Xrs2p is known to be essential for all the functions of the complex, but its role in the complex has not been clearly elucidated. A 32-amino acid region near the C terminus of Xrs2p was identified as an Mre11p-binding site. No more function of Xrs2p than translocation of Mre11p from the cytoplasm to the nucleus is necessary for response to DNA damage. However, domains in Xrs2p located both 49 amino acids upstream and 104 amino acids downstream of the Mre11p binding site are required for meiotic recombination and telomere elongation, respectively, in addition to the 32-amino acid region. These findings demonstrate that Xrs2p acts as a specificity factor that allows the MRX complex to function in meiotic recombination and in telomere elongation.     

Exonuclease activity is required for sequence addition and Cdc13p loading at a de novo telomere.

The Saccharomyces cerevisiae Mre11p/Rad50p/Xrs2p (MRX) complex is evolutionarily conserved and functions in DNA repair and at telomeres [1-3]. In vivo, MRX is required for a 5' --> 3' exonuclease activity that mediates DNA recombination at double-strand breaks (DSBs). Paradoxically, abolition of this exonuclease activity in MRX mutants results in shortened telomeric DNA tracts. To further explore the role of MRX at telomeres, we analyzed MRX mutants in a de novo telomere addition assay in yeast cells [4]. We found that the MRX genes were absolutely required for telomerase-mediated addition in this assay. Furthermore, we found that Cdc13p, a single-stranded telomeric DNA binding protein essential for telomere DNA synthesis and protection [5], was unable to bind to the de novo telomeric DNA substrate in cells lacking Rad50p. Based on the results from this model system, we propose that the MRX complex helps to prepare telomeric DNA for the loading of Cdc13p, which then protects the chromosome from further degradation and recruits telomerase and other DNA replication components to synthesize telomeric DNA.     

细胞DNA损伤检控系统在维持端粒稳定中的功能

真核生物的DNA损伤检控系统是维持细胞基因组稳定的一个重要机制,该系统能检测细胞在生命活动过程中出现的DNA损伤并引发细胞周期阻滞,对DNA损伤进行修复,以维持细胞遗传的稳定性。端粒是位于真核细胞染色体末端由重复DNA序列和蛋白质组成的复合物,具有保护染色体、介导染色体复制、引导减数分裂时的同源染色体配对和调节细胞衰老等作用。虽然端粒与DNA双链断裂都具有作为线性染色体末端的共同特点,但正常端粒并不像DNA双链断裂那样激活DNA损伤检控系统。另一方面,端粒又与DNA损伤相似,因为多种DNA损伤检控蛋白在端粒长度稳定中起重要作用。因此DNA损伤检控系统既参与了维持正常端粒的完整性,又可对端粒损伤作出应答。现就DNA损伤检控系统在维持端粒稳定中的作用及其对功能缺陷端粒的应答作一简要综述。     

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.     

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.