Telomere Length Determines TERRA and R-Loop Regulation through the Cell Cycle.

Maintenance of a minimal telomere length is essential to prevent cellular senescence. When critically short telomeres arise in the absence of telomerase, they can be repaired by homology-directed repair (HDR) to prevent premature senescence onset. It is unclear why specifically the shortest telomeres are targeted for HDR. We demonstrate that the non-coding RNA TERRA accumulates as HDR-promoting RNA-DNA hybrids (R-loops) preferentially at very short telomeres. The increased level of TERRA and R-loops, exclusively at short telomeres, is due to a local defect in RNA degradation by the Rat1 and RNase H2 nucleases, respectively. Consequently, the coordination of TERRA degradation with telomere replication is altered at shortened telomeres. R-loop persistence at short telomeres contributes to activation of the DNA damage response (DDR) and promotes recruitment of the Rad51 recombinase. Thus, the telomere length-dependent regulation of TERRA and TERRA R-loops is a critical determinant of the rate of replicative senescence.

DNA binding modes influence Rap1 activity in the regulation of telomere length and MRX functions at DNA ends.

The cellular response to DNA double-strand breaks (DSBs) is initiated by the Mre11-Rad50-Xrs2 (MRX) complex that has structural and catalytic functions. MRX association at DSBs is counteracted by Rif2, which is known to interact with Rap1 that binds telomeric DNA through two tandem Myb-like domains. Whether and how Rap1 acts at DSBs is unknown. Here we show that Rif2 inhibits MRX association to DSBs in a manner dependent on Rap1, which binds to DSBs and promotes Rif2 association to them. Rap1 in turn can negatively regulate MRX function at DNA ends also independently of Rif2. In fact, a characterization of Rap1 mutant variants shows that Rap1 binding to DNA through both Myb-like domains results in formation of Rap1-DNA complexes that control MRX functions at both DSBs and telomeres primarily through Rif2. By contrast, Rap1 binding to DNA through a single Myb-like domain results in formation of high stoichiometry complexes that act at DNA ends mostly in a Rif2-independent manner. Altogether these findings indicate that the DNA binding modes of Rap1 influence its functional properties, thus highlighting the structural plasticity of this protein.

Rad9/53BP1 protects stalled replication forks from degradation in Mec1/ATR-defective cells.

Nucleolytic processing by nucleases can be a relevant mechanism to allow repair/restart of stalled replication forks. However, nuclease action needs to be controlled to prevent overprocessing of damaged replication forks that can be detrimental to genome stability. The checkpoint protein Rad9/53BP1 is known to limit nucleolytic degradation (resection) of DNA double-strand breaks (DSBs) in both yeast and mammals. Here, we show that loss of the inhibition that Rad9 exerts on resection exacerbates the sensitivity to replication stress of Mec1/ATR-defective yeast cells by exposing stalled replication forks to Dna2-dependent degradation. This Rad9 protective function is independent of checkpoint activation and relies mainly on Rad9-Dpb11 interaction. We propose that Rad9/53BP1 supports cell viability by protecting stalled replication forks from extensive resection when the intra-S checkpoint is not fully functional.

Regulation of telomere metabolism by the RNA processing protein Xrn1.

Telomeric DNA consists of repetitive G-rich sequences that terminate with a 3΄-ended single stranded overhang (G-tail), which is important for telomere extension by telomerase. Several proteins, including the CST complex, are necessary to maintain telomere structure and length in both yeast and mammals. Emerging evidence indicates that RNA processing factors play critical, yet poorly understood, roles in telomere metabolism. Here, we show that the lack of the RNA processing proteins Xrn1 or Rrp6 partially bypasses the requirement for the CST component Cdc13 in telomere protection by attenuating the activation of the DNA damage checkpoint. Xrn1 is necessary for checkpoint activation upon telomere uncapping because it promotes the generation of single-stranded DNA. Moreover, Xrn1 maintains telomere length by promoting the association of Cdc13 to telomeres independently of ssDNA generation and exerts this function by downregulating the transcript encoding the telomerase inhibitor Rif1. These findings reveal novel roles for RNA processing proteins in the regulation of telomere metabolism with implications for genome stability in eukaryotes.

Increased TERRA levels and RNase H sensitivity are conserved hallmarks of post-senescent survivors in budding yeast.

Cancer cells activate telomere maintenance mechanisms (TMMs) to bypass replicative senescence and achieve immortality by either upregulating telomerase or promoting homology-directed repair (HDR) at chromosome ends to maintain telomere length, the latter being referred to as ALT (Alternative Lengthening of Telomeres). In yeast telomerase mutants, the HDR-based repair of telomeres leads to the generation of 'survivors' that escape senescence and divide indefinitely. So far, yeast has proven to provide an accurate model to study the generation and maintenance of telomeres via HDR. Recently, it has been established that up-regulation of the lncRNA, TERRA (telomeric repeat-containing RNA), is a novel hallmark of ALT cells. Moreover, RNA-DNA hybrids are thought to trigger HDR at telomeres in ALT cells to maintain telomere length and function. Here we show that, also in established yeast type II survivors, TERRA levels are increased in an analogous manner to human ALT cells. The elevated TERRA levels are independent of yeast-specific subtelomeric structures, i.e. the presence or absence of Y' repetitive elements. Furthermore, we show that RNase H1 overexpression, which degrades the RNA moiety in RNA-DNA hybrids, impairs the growth of yeast survivors. We suggest that even in terms of TERRA regulation, yeast survivors serve as an accurate model that recapitulates many key features of human ALT cells.

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.

Escape of Sgs1 from Rad9 inhibition reduces the requirement for Sae2 and functional MRX in DNA end resection.

Homologous recombination requires nucleolytic degradation (resection) of DNA double-strand break (DSB) ends. In Saccharomyces cerevisiae, the MRX complex and Sae2 are involved in the onset of DSB resection, whereas extensive resection requires Exo1 and the concerted action of Dna2 and Sgs1. Here, we show that the checkpoint protein Rad9 limits the action of Sgs1/Dna2 in DSB resection by inhibiting Sgs1 binding/persistence at the DSB ends. When inhibition by Rad9 is abolished by the Sgs1-ss mutant variant or by deletion of RAD9, the requirement for Sae2 and functional MRX in DSB resection is reduced. These results provide new insights into how early and long-range resection is coordinated.

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.

Coupling end resection with the checkpoint response at DNA double-strand breaks.

DNA double-strand breaks (DSBs) are a nasty form of damage that needs to be repaired to ensure genome stability. The DSB ends can undergo a strand-biased nucleolytic processing (resection) to generate 3'-ended single-stranded DNA (ssDNA) that channels DSB repair into homologous recombination. Generation of ssDNA also triggers the activation of the DNA damage checkpoint, which couples cell cycle progression with DSB repair. The checkpoint response is intimately linked to DSB resection, as some checkpoint proteins regulate the resection process. The present review will highlight recent works on the mechanism and regulation of DSB resection and its interplays with checkpoint activation/inactivation in budding yeast.

Saccharomyces cerevisiae Rif1 cooperates with MRX-Sae2 in promoting DNA-end resection.

Diverse roles in DNA metabolism have been envisaged for budding yeast and mammalian Rif1. In particular, yeast Rif1 is involved in telomere homeostasis, while its mammalian counterpart participates in the cellular response to DNA double-strand breaks (DSBs). Here, we show that Saccharomyces cerevisiae Rif1 supports cell survival to DNA lesions in the absence of MRX or Sae2. Furthermore, it contributes to the nucleolytic processing (resection) of DSBs. This Rif1-dependent control of DSB resection becomes important for DSB repair by homologous recombination when resection activities are suboptimal.

Telomere-end processing: mechanisms and regulation.

Telomeres are specialized nucleoprotein complexes that provide protection to the ends of eukaryotic chromosomes. Telomeric DNA consists of tandemly repeated G-rich sequences that terminate with a 3' single-stranded overhang, which is important for telomere extension by the telomerase enzyme. This structure, as well as most of the proteins that specifically bind double and single-stranded telomeric DNA, are conserved from yeast to humans, suggesting that the mechanisms underlying telomere identity are based on common principles. The telomeric 3' overhang is generated by different events depending on whether the newly synthesized strand is the product of leading- or lagging-strand synthesis. Here, we review the mechanisms that regulate these processes at Saccharomyces cerevisiae and mammalian telomeres.

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.

Rif1 supports the function of the CST complex in yeast telomere capping.

Telomere integrity in budding yeast depends on the CST (Cdc13-Stn1-Ten1) and shelterin-like (Rap1-Rif1-Rif2) complexes, which are thought to act independently from each other. Here we show that a specific functional interaction indeed exists among components of the two complexes. In particular, unlike RIF2 deletion, the lack of Rif1 is lethal for stn1ΔC cells and causes a dramatic reduction in viability of cdc13-1 and cdc13-5 mutants. This synthetic interaction between Rif1 and the CST complex occurs independently of rif1Δ-induced alterations in telomere length. Both cdc13-1 rif1Δ and cdc13-5 rif1Δ cells display very high amounts of telomeric single-stranded DNA and DNA damage checkpoint activation, indicating that severe defects in telomere integrity cause their loss of viability. In agreement with this hypothesis, both DNA damage checkpoint activation and lethality in cdc13 rif1Δ cells are partially counteracted by the lack of the Exo1 nuclease, which is involved in telomeric single-stranded DNA generation. The functional interaction between Rif1 and the CST complex is specific, because RIF1 deletion does not enhance checkpoint activation in case of CST-independent telomere capping deficiencies, such as those caused by the absence of Yku or telomerase. Thus, these data highlight a novel role for Rif1 in assisting the essential telomere protection function of the CST complex.

Proteasome-mediated degradation of long-range nucleases negatively regulates resection of DNA double-strand breaks.

Homologous recombination is initiated by the nucleolytic degradation (resection) of DNA double-strand breaks (DSBs). DSB resection is a two-step process. In the short-range step, the MRX (Mre11-Rad50-Xrs2) complex, together with Sae2, incises the 5'-terminated strand at the DSB end and resects back toward the DNA end. Then, the long-range resection nucleases Exo1 and Dna2 further elongate the resected DNA tracts. We found that mutations lowering proteasome functionality bypass the need for Sae2 in DSB resection. In particular, the dysfunction of the proteasome subunit Rpn11 leads to hyper-resection and increases the levels of both Exo1 and Dna2 to such an extent that it allows the bypass of the requirement for either Exo1 or Dna2, but not for both. These observations, along with the finding that Exo1 and Dna2 are ubiquitylated, indicate a role of the proteasome in restraining DSB resection by negatively controlling the abundance of the long-range resection nucleases.

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.

The multistep path to replicative senescence onset: zooming on triggering and inhibitory events at telomeric DNA.

Replicative senescence is an essential cellular process playing important physiological functions, but it is better known for its implications in aging, cancer, and other pathologies. One of the main triggers of replicative senescence is telomere shortening and/or its dysfunction and, therefore, a deep understanding of the molecular determinants is crucial. However, replicative senescence is a heterogeneous and hard to study process, especially in mammalian cells, and some important questions still need an answer. These questions concern i) the exact molecular causes triggering replicative senescence, ii) the role of DNA repair mechanisms and iii) the importance of R-loops at telomeres in regulating senescence onset, and iv) the mechanisms underlying the bypass of replicative senescence. In this review, we will report and discuss recent findings about these mechanisms both in mammalian cells and in the model organism Saccharomyces cerevisiae.

To Fix or Not to Fix: Maintenance of Chromosome Ends Versus Repair of DNA Double-Strand Breaks.

Early work by Muller and McClintock discovered that the physical ends of linear chromosomes, named telomeres, possess an inherent ability to escape unwarranted fusions. Since then, extensive research has shown that this special feature relies on specialized proteins and structural properties that confer identity to the chromosome ends, thus allowing cells to distinguish them from intrachromosomal DNA double-strand breaks. Due to the inability of conventional DNA replication to fully replicate the chromosome ends and the downregulation of telomerase in most somatic human tissues, telomeres shorten as cells divide and lose this protective capacity. Telomere attrition causes the activation of the DNA damage checkpoint that leads to a cell-cycle arrest and the entering of cells into a nondividing state, called replicative senescence, that acts as a barrier against tumorigenesis. However, downregulation of the checkpoint overcomes this barrier and leads to further genomic instability that, if coupled with re-stabilization of telomeres, can drive tumorigenesis. This review focuses on the key experiments that have been performed in the model organism Saccharomyces cerevisiae to uncover the mechanisms that protect the chromosome ends from eliciting a DNA damage response, the conservation of these pathways in mammals, as well as the consequences of their loss in human cancer.

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.

MRX-dependent DNA damage response to short telomeres.

Telomere structure allows cells to distinguish the natural chromosome ends from double-strand breaks (DSBs). However, DNA damage response proteins are intimately involved in telomere metabolism, suggesting that functional telomeres may be recognized as DNA damage during a time window. Here we show by two different systems that short telomeres are recognized as DSBs during the time of their replication, because they induce a transient MRX-dependent DNA damage checkpoint response during their prolonged elongation. The MRX complex, which is recruited at telomeres under these conditions, dissociates from telomeres concomitantly with checkpoint switch off when telomeres reach a new equilibrium length. We also show that MRX recruitment to telomeres is sufficient to activate the checkpoint independently of telomere elongation. We propose that MRX can signal checkpoint activation by binding to short telomeres only when they become competent for elongation. Because full-length telomeres are refractory to MRX binding and the shortest telomeres are elongated of only a few base pairs per generation, this limitation may prevent unscheduled checkpoint activation during an unperturbed S phase.

Sensing R-Loop-Associated DNA Damage to Safeguard Genome Stability.

DNA transcription and replication are two essential physiological processes that can turn into a threat for genome integrity when they compete for the same DNA substrate. During transcription, the nascent RNA strongly binds the template DNA strand, leading to the formation of a peculiar RNA-DNA hybrid structure that displaces the non-template single-stranded DNA. This three-stranded nucleic acid transition is called R-loop. Although a programed formation of R-loops plays important physiological functions, these structures can turn into sources of DNA damage and genome instability when their homeostasis is altered. Indeed, both R-loop level and distribution in the genome are tightly controlled, and the list of factors involved in these regulatory mechanisms is continuously growing. Over the last years, our knowledge of R-loop homeostasis regulation (formation, stabilization, and resolution) has definitely increased. However, how R-loops affect genome stability and how the cellular response to their unscheduled formation is orchestrated are still not fully understood. In this review, we will report and discuss recent findings about these questions and we will focus on the role of ATM- and Rad3-related (ATR) and Ataxia-telangiectasia-mutated (ATM) kinases in the activation of an R-loop-dependent DNA damage response.