RPA and POT1: Friends or foes at telomeres?

Telomere maintenance in cycling cells relies on both DNA replication and capping by the protein complex shelterin. Two single-stranded DNA (ssDNA)-binding proteins, replication protein A (RPA) and protection of telomere 1 (POT1) play critical roles in DNA replication and telomere capping, respectively. While RPA binds to ssDNA in a non-sequence-specific manner, POT1 specifically recognizes singlestranded TTAGGG telomeric repeats. Loss of POT1 leads to aberrant accumulation of RPA at telomeres and activation of the ataxia telangiectasia and Rad3-related kinase (ATR)-mediated checkpoint response, suggesting that POT1 antagonizes RPA binding to telomeric ssDNA. The requirement for both POT1 and RPA in telomere maintenance and the antagonism between the two proteins raises the important question of how they function in concert on telomeric ssDNA. Two interesting models were proposed by recent studies to explain the regulation of POT1 and RPA at telomeres. Here, we discuss how these models help unravel the coordination, and also the antagonism, between POT1 and RPA during the cell cycle.Key words: RPA, POT1, telomere, ATR, checkpointTelomeres, the natural ends of chromosomes, are composed of repetitive DNA sequences and “capped” by both specific proteins and non-coding RNAs.^1–3 One of the critical functions of telomeres is to prevent chromosomal ends from recognition by the DNA damage response machinery. Critically short or improperly capped telomeres lead to telomere dysfunction and are a major source of genomic instability.⁴ While telomeres need to be properly capped to remain stable, they also need to be duplicated during each cell division by the DNA replication machinery. The requirement of these two seemingly competing processes for telomere maintenance suggests that the cell must coordinate DNA replication and capping of telomeres to ensure faithful telomere duplication yet avoid an inappropriate DNA damage response.Telomeric DNA is unique in several ways. The bulk of each human telomere is comprised of double-stranded TTA GGG repeats. At the very end of each telomere, a stretch of single-stranded TTAGGG repeats exists as a 3′ overhang. The TTA GGG repeats in the telomeric single-stranded DNA (ssDNA) allow it to loop back and invade telomeric double-stranded DNA (dsDNA), forming a structure called the t-loop.⁵ At the base of the t-loop, the TTAGGG strand of the telomeric dsDNA is displaced by the invading single-stranded 3′ overhang to form a single-stranded D-loop. Thus, the unique DNA sequence and structures of telomeres confer the ability to bind proteins in both sequence- and structure-specific manners, providing the basis for additional regulations.In human cells, telomere capping is orchestrated by the protein complex shelterin, which contains TRF1, TRF2, RAP1, TIN2, TPP1 and POT1.³ Among these shelterin components, TRF1 and TRF2 interact with telomeric dsDNA in a sequence-specific manner, whereas POT1, in a complex with TPP1, binds to telomeric ssDNA in a sequence-specific manner.^6–8 While the human genome contains only one POT1 gene, the mouse genome contains two POT1-related genes, POT1a and POT1b.^9–11 TIN2 functions to stabilize TRF1 and TRF2 DNA binding and also tethers the POT1-TPP1 heterodimer to the rest of the shelterin complex on telomeric dsDNA.^12,13Unlike the properly capped telomeres, double-stranded DNA breaks (DSBs) with ssDNA overhangs are known to activate the ATR checkpoint kinase.^14,15 In a complex with its functional partner ATRIP, ATR is recruited to ssDNA by RPA, a non-sequence-specific ssDNA-binding protein complex.¹⁶ In addition to the ATR-ATRIP kinase complex, several other checkpoint proteins involved in ATR activation are also recruited in the presence of RPA-ssDNA.¹⁵ The structural resemblance between DSBs and telomeres and the presence of ssDNA at telomeres raise the important question as to how ATR activation is repressed at telomeres.     

Telomere dysfunction puts the brakes on oncogene-induced cancers

EMBO J 31 13, 2839–2851 (2012); published online May082012Senescence represents a major tumour suppressor checkpoint activated by telomere dysfunction or cellular stress factors such as oncogene activation. In this issue of The EMBO Journal, Suram et al (2012) reveal a surprising interconnection between oncogene activation and telomere dysfunction induced senescence. The study supports an alternative model of tumour suppression, indicating that oncogene-induced accumulation of telomeric DNA damage contributes to the induction of senescence in telomerase-negative tumours.Telomere shortening limits the proliferative capacity of primary human cells after 50–70 cell divisions by induction of replicative senescence activated by critically short, dysfunctional telomeres. Different mechanisms were thought to initiate senescence in response to oncogene activation, which occurs abruptly within a few cell doublings (Serrano et al, 1997). Oncogene-induced senescence (OIS) involves an activation of DNA damage signals at stalled replication forks induced by DNA replication stress (Bartkova et al, 2006; Di Micco et al, 2006). Replication fork stalling in response to oncogene activation preferentially affects common fragile sites of the DNA (Tsantoulis et al, 2008). The ends of eukaryotic chromosomes—the telomeres–represent common fragile sites that are sensitive to replication fork stalling (Sfeir et al, 2009). These data made it tempting to speculate whether replication fork stalling at telomeres was causatively involved in OIS. Studies on replicative senescence in human fibroblast also supported this possibility showing that mitogenic signals amplify DNA damage responses in senescent cells (Satyanarayana et al, 2004).Multiple studies revealed experimental evidences that senescence suppresses tumour progression in mouse models and early human tumours (for review see Collado and Serrano, 2010). The relative contribution of OIS and telomere dysfunction induced senescence (TDIS) to tumour suppression and possible interconnections between the two pathways at the level of checkpoint induction were not investigated in previous studies. In this issue of The EMBO Journal, Suram et al (2012) describe the presence of TDIS in human precursor lesions but not in the corresponding malignant tumours. Mechanistically, the study shows that oncogenic signals cause replication fork stalling, resulting in telomeric DNA damage accumulation and activation of DNA damage checkpoints reminiscent to TDIS. Telomerase expression does not rescue replication fork stalling but prevents the accumulation of DNA damage at telomeres allowing a bypass of OIS.The study has several important implications for molecular pathways and therapeutic approaches in cancer that need to be further explored (Figure 1):Open in a separate windowFigure 1Traditional and new models of senescence in tumour suppression. (A) Traditional model of replicative senescence: Telomerase-negative tumour cell clones experience telomere shortening as a consequence of cell division. After a lack period depending on the initial telomere length, tumour cells accumulate telomere dysfunction and activation of senescence impairs tumour growth. Telomerase activation represents a late event allowing tumour progression. (B) New model of oncogene induced, telomere-dependent senescence: Oncogene activation leads to abrupt accumulation of DNA damage at telomeres resulting in senescence and tumour suppression. Telomerase-positive stem cells could be resistant to OIS and may be selected as the cell type of origin of tumour development.(i) Telomere length independent roles of telomeres in tumour suppressionThe classical model of telomere-dependent tumour suppression indicates that proliferation-dependent telomere shortening leads to telomere dysfunction, activation of DNA damage checkpoints, and induction of senescence suppressing the growth of telomerase-negative tumour clones. Studies on mouse models supported this concept showing that telomere shortening impairs the progression of initiated tumours in a telomere length-dependent manner (Feldser and Greider, 2007). The new data from Suram et al (2012) indicate that oncogene-induced replication fork stalling activates a telomere-dependent senescence checkpoint, which is independent of telomere length. The study shows that replication forks stall in response to oncogene activation throughout the genome. However, stalled replication forks are resolved in non-telomeric regions, whereas fork stalling inside telomeres leads to un-repairable DNA damage in telomerase-negative cells. These findings are in line with recent publication showing accumulation of un-repairable DNA damage in telomeric DNA in response to aging and stress-induced DNA damage (Fumagalli et al, 2012).(ii) Telomere length independent roles of telomerase in tumour progressionFollowing the classical model telomeres in tumour suppression (Figure 1A), telomerase re-activation is required for tumour progression by limiting telomere dysfunction and the induction of DNA damage checkpoints in response to telomere shortening. The new data from Suram et al (2012) indicate that telomerase has an additional telomere length independent role in tumour progression. The study shows that catalytically active telomerase prevents the activation of DNA damage signals originating from stalled replication forks inside telomeres in response to oncogene activation (Figure 1B). The exact mechanisms of telomerase-dependent healing of stalled replication forks at telomeres remain to be elucidated. It is also unclear whether telomerase activity can prevent any type of DNA damage at telomeres as an over-expression of TERT could not suppress irradiation-induced cellular senescence or the persistence of telomeric DDR following irradiation, H₂O₂, or chemotherapy induced DNA damage (Hewitt et al, 2012).The data could provide a plausible explanation for the increased tumorigenesis in telomerase transgenic mice—a finding which is difficult to explain by telomere length dependent effects of telomerase given the long telomere reserves in mouse tissues (Gonzalez-Suarez et al, 2001). According to the findings of Suram et al (2012), anti-telomerase therapies could have immediate anti-cancer effects in tumours depending on telomerase-mediated healing of stalled replication forks at telomeres. Specific markers for this dependency could be of clinical value. In addition, the data support the concept that somatic stem cells could represent the cell type of origin of cancers. In contrast to differentiated somatic cells, tissues stem cells are often telomerase-positive, indicating that stem cells might be less sensitive to OIS.     

Telomere Protection by TPP1 Is Mediated by POT1a and POT1b

Mammalian telomeres are protected by the shelterin complex, which contains single-stranded telomeric DNA binding proteins (POT1a and POT1b in rodents, POT1 in other mammals). Mouse POT1a prevents the activation of the ATR kinase and contributes to the repression of the nonhomologous end-joining pathway (NHEJ) at newly replicated telomeres. POT1b represses unscheduled resection of the 5′-ended telomeric DNA strand, resulting in long 3′ overhangs in POT1b KO cells. Both POT1 proteins bind TPP1, forming heterodimers that bind to other proteins in shelterin. Short hairpin RNA (shRNA)-mediated depletion had previously demonstrated that TPP1 contributes to the normal function of POT1a and POT1b. However, these experiments did not establish whether TPP1 has additional functions in shelterin. Here we report on the phenotypes of the conditional deletion of TPP1 from mouse embryo fibroblasts. TPP1 deletion resulted in the release of POT1a and POT1b from chromatin and loss of these proteins from telomeres, indicating that TPP1 is required for the telomere association of POT1a and POT1b but not for their stability. The telomere dysfunction phenotypes associated with deletion of TPP1 were identical to those of POT1a/POT1b DKO cells. No additional telomere dysfunction phenotypes were observed, establishing that the main role of TPP1 is to allow POT1a and POT1b to protect chromosome ends.Mammalian cells solve the chromosome end protection problem through the binding of shelterin to the telomeric TTAGGG repeat arrays at chromosome ends (5). Shelterin contains two double-stranded telomeric DNA binding proteins, TRF1 and TRF2, which both interact with the shelterin subunit TIN2. These three shelterin components, as well as the TRF2 interacting factor Rap1, are abundant, potentially covering the majority of the TTAGGG repeat sequences at chromosome ends (30). TIN2 interacts with the less abundant TPP1/POT1 heterodimers and is thought to facilitate the recruitment of the single-stranded telomeric DNA binding proteins to telomeres (15, 21, 35).Shelterin represses the four major pathways that threaten mammalian telomeres (6). It prevents activation of the ATM and ATR kinases, which can induce cell cycle arrest in response to double-strand breaks (DSBs). Shelterin also blocks the two major repair pathways that act on DSBs: nonhomologous end joining (NHEJ) and homology-directed repair (HDR). Removal of individual components of shelterin leads to highly specific telomere dysfunction phenotypes, allowing assignment of shelterin functions to each of its components.The POT1 proteins are critical for the repression of ATR signaling (20). Concurrent deletion of POT1a and POT1b from mouse embryo fibroblasts (POT1a/b DKO cells [12]) activates the ATR kinase at most telomeres, presumably because the single-stranded telomeric DNA is exposed to RPA. POT1a/b DKO cells also have a defect in the structure of the telomere terminus, showing extended 3′ overhangs that are thought to be due to excessive resection of the 5′-ended strand in the absence of POT1b (11-13). The combination of these two phenotypes, activation of the ATR kinase and excess single-stranded telomeric DNA, is not observed when either TRF1 or TRF2 is deleted.In contrast to the activation of ATR signaling in POT1a/b DKO cells, TRF2 deletion results in activation of the ATM kinase at telomeres (3, 16, 20). In addition, TRF2-deficient cells show widespread NHEJ-mediated telomere-telomere fusions (3, 31). This phenotype is readily distinguished from the consequences of POT1a/b loss. POT1a/b DKO cells have a minor telomere fusion phenotype that primarily manifests after DNA replication, resulting in the fusion of sister telomeres (12). In TRF2-deficient cells, most telomere fusions take place in G₁ (18), resulting in chromosome-type telomere fusions in the subsequent metaphase. Chromosome-type fusions also occur in the POT1a/b DKO setting, but they are matched in frequency by sister telomere fusions.The type of telomere dysfunction induced by TRF1 loss is also distinct. Deletion of TRF1 gives rise to DNA replication problems at telomeres that activate the ATR kinase in S phase and leads to aberrant telomere structures in metaphase (referred to as “fragile telomeres”) (28). This fragile telomere phenotype is not observed upon deletion of POT1a and POT1b, and the activation of the ATR kinase at telomeres in POT1a/b DKO cells is not dependent on the progression through S phase (Y. Gong and T. de Lange, unpublished data). Furthermore, deletion of TRF1 does not induce excess single-stranded DNA.These phenotypic distinctions bear witness to the separation of functions within shelterin and also serve as a guide to understanding the contribution of the other shelterin proteins, including TPP1. TPP1 is an oligonucleotide/oligosaccharide-binding fold (OB fold) protein in shelterin that forms a heterodimer with POT1 (32). TPP1 and POT1 are distantly related to the TEBPα/β heterodimer, which is bound to telomeric termini of certain ciliates (2, 32, 33). Several lines of evidence indicate that TPP1 mediates the recruitment of POT1 to telomeres. Mammalian TPP1 was discovered based on its interaction with TIN2, and diminished TPP1 levels affect the ability of POT1 to bind to telomeres and protect chromosome ends (14, 15, 21, 26, 33, 35). Since TPP1 enhances the in vitro DNA binding activity of POT1 (32), it might mediate the recruitment of POT1 through improving its interaction with the single-stranded telomeric DNA. However, POT1 does not require its DNA binding domain for telomere recruitment, although this domain is critical for telomere protection (23, 26). Thus, it is more likely that the TPP1-TIN2 interaction mediates the binding of POT1 to telomeres. However, POT1 has also been shown to bind to TRF2 in vitro, and this interaction has been suggested to constitute a second mechanism for the recruitment of POT1 to telomeres (1, 34).TPP1 has been suggested to have additional functions at telomeres. Biochemical data showed that TPP1 promotes the interaction between TIN2, TRF1, and TRF2 (4, 25). Therefore, it was suggested that TPP1 plays an essential organizing function in shelterin, predicting that its deletion would affect TRF1 and TRF2 (25). Furthermore, cytogenetic data on cells from the adrenocortical dysplasia (Acd) mouse strain, which carries a hypomorphic mutation for TPP1 (14), revealed complex chromosomal rearrangements in addition to telomere fusions, leading to the suggestion that TPP1 might have additional telomeric or nontelomeric functions (9).In order to determine the role of TPP1 at telomeres and possibly elsewhere in the genome, we generated a conditional knockout setting in mouse embryo fibroblasts. The results indicate that the main function of TPP1 is to ensure the protection of telomeres by POT1 proteins. Each of the phenotypes of TPP1 loss was also observed in the POT1a/b DKO cells. No evidence was found for a role of TPP1 in stabilizing or promoting the function of other components of shelterin. Furthermore, the results argue against a TPP1-independent mode of telomeric recruitment of POT1.     

Cell Cycle-Dependent Role of MRN at Dysfunctional Telomeres: ATM Signaling-Dependent Induction of Nonhomologous End Joining (NHEJ) in G1 and Resection-Mediated Inhibition of NHEJ in G2

Here, we address the role of the MRN (Mre11/Rad50/Nbs1) complex in the response to telomeres rendered dysfunctional by deletion of the shelterin component TRF2. Using conditional NBS1/TRF2 double-knockout MEFs, we show that MRN is required for ATM signaling in response to telomere dysfunction. This establishes that MRN is the only sensor for the ATM kinase and suggests that TRF2 might block ATM signaling by interfering with MRN binding to the telomere terminus, possibly by sequestering the telomere end in the t-loop structure. We also examined the role of the MRN/ATM pathway in nonhomologous end joining (NHEJ) of damaged telomeres. NBS1 deficiency abrogated the telomere fusions that occur in G₁, consistent with the requirement for ATM and its target 53BP1 in this setting. Interestingly, NBS1 and ATM, but not H2AX, repressed NHEJ at dysfunctional telomeres in G₂, specifically at telomeres generated by leading-strand DNA synthesis. Leading-strand telomere ends were not prone to fuse in the absence of either TRF2 or MRN/ATM, indicating redundancy in their protection. We propose that MRN represses NHEJ by promoting the generation of a 3′ overhang after completion of leading-strand DNA synthesis. TRF2 may ensure overhang formation by recruiting MRN (and other nucleases) to newly generated telomere ends. The activation of the MRN/ATM pathway by the dysfunctional telomeres is proposed to induce resection that protects the leading-strand ends from NHEJ when TRF2 is absent. Thus, the role of MRN at dysfunctional telomeres is multifaceted, involving both repression of NHEJ in G₂ through end resection and induction of NHEJ in G₁ through ATM-dependent signaling.Mammalian telomeres solve the end protection problem through their association with shelterin. The shelterin factor TRF2 (telomere repeat-binding factor 2) protects chromosome ends from inappropriate DNA repair events that threaten the integrity of the genome (reviewed in reference 32). When TRF2 is removed by Cre-mediated deletion from conditional knockout mouse embryo fibroblasts (TRF2^F/− MEFs), telomeres activate the ATM kinase pathway and are processed by the canonical nonhomologous end-joining (NHEJ) pathway to generate chromosome end-to-end fusions (10, 11).The repair of telomeres in TRF2-deficient cells is readily monitored in metaphase spreads. Over the course of four or five cell divisions, the majority of chromosome ends become fused, resulting in metaphase spreads displaying the typical pattern of long trains of joined chromosomes (10). The reproducible pace and the efficiency of telomere NHEJ have allowed the study of factors involved in its execution and regulation. In addition to depending on the NHEJ factors Ku70 and DNA ligase IV (10, 11), telomere fusions are facilitated by the ATM kinase (26). This aspect of telomere NHEJ is mediated through the ATM kinase target 53BP1. 53BP1 accumulates at telomeres in TRF2-depleted cells and stimulates chromatin mobility, thereby promoting the juxtaposition of distantly positioned chromosome ends prior to their fusion (18). Telomere NHEJ is also accelerated by the ATM phosphorylation target MDC1, which is required for the prolonged association of 53BP1 at sites of DNA damage (19).Although loss of TRF2 leads to telomere deprotection at all stages of the cell cycle, NHEJ of uncapped telomeres takes place primarily before their replication in G₁ (25). Postreplicative (G₂) telomere fusions can occur at a low frequency upon TRF2 deletion, but only when cyclin-dependent kinase activity is inhibited with roscovitine (25). The target of Cdk1 in this setting is not known.Here, we dissect the role of the MRN (Mre11/Rad50/Nbs1) complex and H2AX at telomeres rendered dysfunctional through deletion of TRF2. The highly conserved MRN complex has been proposed to function as the double-stranded break (DSB) sensor in the ATM pathway (reviewed in references 34 and 35). In support of this model, Mre11 interacts directly with DNA ends via two carboxy-terminal DNA binding domains (13, 14); the recruitment of MRN to sites of damage is independent of ATM signaling, as it occurs in the presence of the phosphoinositide-3-kinase-related protein kinase inhibitor caffeine (29, 44); in vitro analysis has demonstrated that MRN is required for activation of ATM by linear DNAs (27); a mutant form of Rad50 (Rad50S) can induce ATM signaling in the absence of DNA damage (31); and phosphorylation of ATM targets in response to ionizing radiation is completely abrogated upon deletion of NBS1 from MEFs (17). These data and the striking similarities between syndromes caused by mutations in ATM, Nbs1, and Mre11 (ataxia telangiectasia, Nijmegen breakage syndrome, and ataxia telangiectasia-like disease, respectively) are consistent with a sensor function for MRN.MRN has also been implicated in several aspects of DNA repair. Potentially relevant to DNA repair events, Mre11 dimers can bridge and align the two DNA ends in vitro (49) and Rad50 may promote long-range tethering of sister chromatids (24, 50). In addition, a binding partner of the MRN complex, CtIP, has been implicated in end resection of DNA ends during homology-directed repair (39, 45). The role of MRN in NHEJ has been much less clear. MRX, the yeast orthologue of MRN, functions during NHEJ in Saccharomyces cerevisiae but not in Schizosaccharomyces pombe (28, 30). In mammalian cells, MRN is not recruited to I-SceI-induced DSBs in G₁, whereas Ku70 is, and MRN does not appear to be required for NHEJ-mediated repair of these DSBs (38, 54). On the other hand, MRN promotes class switch recombination (37) and has been implicated in accurate NHEJ repair during V(D)J recombination (22).The involvement of MRN in ATM signaling and DNA repair pathways has been intriguing from the perspective of telomere biology. While several of the attributes of MRN might be considered a threat to telomere integrity, MRN is known to associate with mammalian telomeres, most likely through an interaction with the TRF2 complex (48, 51, 57). MRN has been implicated in the generation of the telomeric overhang (12), the telomerase pathway (36, 52), the ALT pathway (55), and the protection of telomeres from stochastic deletion events (1). It has also been speculated that MRN may contribute to formation of the t-loop structure (16). t-loops, the lariats formed through the strand invasion of the telomere terminus into the duplex telomeric DNA (21), are thought to contribute to telomere protection by effectively shielding the chromosome end from DNA damage response factors that interact with DNA ends, including nucleases, and the Ku heterodimer (15).H2AX has been studied extensively in the context of chromosome-internal DSBs. When a DSB is formed, ATM acts near the lesion to phosphorylate a conserved carboxy-terminal serine of H2AX, a histone variant present throughout the genome (7). Phosphorylated H2AX (referred to as γ-H2AX) promotes the spreading of DNA damage factors over several megabases along the damaged chromatin and mediates the amplification of the DNA damage signal (43). The signal amplification is accomplished through a sequence of phospho-specific interactions among γ-H2AX, MDC1, NBS1, RNF8, and RNF168, which results in the additional binding of ATM and additional phosphorylation of H2AX in adjacent chromatin (reviewed in reference 33). The formation of these large domains of altered chromatin, referred to as irradiation-induced foci at DSBs and telomere dysfunction-induced foci (TIFs) at dysfunctional telomeres (44), promotes the binding of several factors implicated in DNA repair, including the BRCA1 A complex and 53BP1 (33).In agreement with a role for H2AX in DNA repair, H2AX-deficient cells exhibit elevated levels of irradiation-induced chromosome abnormalities (5, 9). In addition, H2AX-null B cells are prone to chromosome breaks and translocations in the immunoglobulin locus, indicative of impaired class switch recombination, a process that involves the repair of DSBs through the NHEJ pathway (9, 20). Since H2AX is dispensable for the activation of irradiation-induced checkpoints (8), these data argue that H2AX contributes directly to DNA repair. However, a different set of studies has concluded that H2AX is not required for NHEJ during V(D)J recombination (5, 9) but that it plays a role in homology-directed repair (53). In this study, we have further queried the contribution of H2AX to NHEJ in the context of dysfunctional telomeres.Our aim was to dissect the contribution of MRN and H2AX to DNA damage signaling and NHEJ-mediated repair in response to telomere dysfunction elicited by deletion of TRF2. Importantly, since ATM is the only kinase activated in this setting, deletion of TRF2 can illuminate the specific contribution of these factors in the absence of the confounding effects of ATR signaling (26). This approach revealed a dual role for MRN at telomeres, involving both its function as a sensor in the ATM pathway and its ability to protect telomeres from NHEJ under certain circumstances.     

On the chromatin structure of eukaryotic telomeres

Telomeres prevent chromosome fusions and degradation by exonucleases and are implicated in DNA repair, homologous recombination, chromosome pairing and segregation. All these functions of telomeres require the integrity of their chromatin structure, which has been traditionally considered as heterochromatic. In agreement with this idea, different studies have reported that telomeres associate with heterochromatic marks. However, these studies addressed simultaneously the chromatin structures of telomeres and subtelomeric regions or the chromatin structure of telomeres and Interstitial Telomeric Sequences (ITSs). The independent analysis of Arabidopsis telomeres, subtelomeric regions and ITSs has allowed the discovery of euchromatic telomeres. In Arabidopsis, whereas subtelomeric regions and ITSs associate with heterochromatic marks, telomeres exhibit euchromatic features. We think that this scenario could be found in other model systems if the chromatin organizations of telomeres, subtelomeric regions and ITSs are independently analyzed.Key words: telomeres, subtelomeres, euchromatin, heterochromatin, ChIP, immunolocalizationTelomeric DNA usually contains tandem repeats of a short GC rich motif. The number of repeats and, therefore, the length of telomeres is subject to regulation and influences relevant biological processes like aging and cancer.^1–3 In situ hybridization studies have revealed that telomeric repeats are also present at interstitial chromosomal loci.^4,5 An analysis of the genome sequence from different eukaryotes indicates that ITSs have a widespread distribution in different model systems including zebrafish, chicken, opossum, mouse, dog, cattle, horse, human, rice, poplar or Arabidopsis (see Fig. 1 for an example; www.ncbi.nlm.nih.gov/mapview). These ITSs have been related to chromosomal aberrations, fragile sites, hot spots for recombination and diseases caused by genomic instability, although their functions remain unknown.⁶Open in a separate windowFigure 1Distribution of the main telomeric repeat arrays in the genome of several model organisms. These representations have been performed by using the megaBLAST program and the all assemblies genomic databases at NCBI (www.ncbi.nlm.nih.gov/mapview). Searches for homology with 100 tandem telomeric repeats were done using the default parameters except that the expected threshold was set to 10 and the filters were turned off. Chromosomes are represented as vertical bars and numbered at the bottom. The horizontal bars represent the telomeric repeat arrays. Colors indicate the BLAST scores (red ≥200; pink 80–200; green 50–80).Telomeres and ITSs have probably cross talk through evolution. In some instances, ITSs could have been generated by telomeric fusions. Pioneering studies performed by Hermann J. Muller in Drosophila and Barbara McClintock in maize showed that newly formed chromosome ends tend to fuse giving rise to the so-called breakage-fusion-bridge cycle.^7,8 This cycle can lead to stable chromosomal reorganizations after healing of the broken ends. In addition, Muller and McClintock found that, unlike these newly formed broken chromosome ends, natural chromosomal ends are quite stable and do not tend to fuse.⁹ It is currently known that telomere dysfunction due to mutations that cause telomeric shortening or abolish the expression of certain telomeric proteins can lead to telomeric fusions, anaphase bridges and genome reorganizations.^1–3,10,11 Therefore, telomeric shortening or alterations of telomeric chromatin structure might be expected to generate ITSs through evolution by promoting telomeric fusions.¹² ITSs might also originate through the activity of telomerase during the repair process of double strand breaks or by recombination.^13–16 In addition, telomerase activity might lead to the formation of new telomeres by healing of chromosome breaks within internal telomeric repeats and even within other sequences.^17–19 This process of healing involves the acquisition of telomeric chromatin structure.DNA folds into two major chromatin organizations inside the cell nucleus: heterochromatin and euchromatin. Heterochromatin is highly condensed in interphase nuclei and is usually associated with repetitive and silent DNA. By contrast, euchromatin has an open conformation and is often related to the capacity to be transcribed. Both kinds of chromatin exhibit defined epigenetic modifications that influence their biochemical behavior. Thus, the study of these epigenetic marks is an issue of major interest.The chromatin structures of telomeres and ITSs might be different. Therefore, they should be studied independently. Chromatin structure analyses are usually performed by immunocytolocalization or by chromatin immunoprecipitation (ChIP).^20–23 Special care should be taken when the epigenetic status of telomeres is analyzed by immunocytolocalization. This technique does not allow differentiating between telomeres and subtelomeric regions. Since subtelomeric regions are known to be heterochromatic in many eukaryotic organisms, heterochromatic marks should be immunolocalized at the chromosome ends of these organisms. However, these marks could correspond to subtelomeric regions and not to telomeres.The ChIP technique implies the immunoprecipitation of chromatin with specific antibodies and the further analysis of the immunoprecipitated DNA. DNA sequences immunoprecipitated by a specific antibody are thought to associate in vivo with the feature recognized by this antibody. Whereas the enrichment of single copy sequences in the immunoprecipitated DNA has been usually analyzed by quantitative PCR, the analyses of repetitive DNA sequences have been often performed by hybridization. Thus, multiple telomeric chromatin structure analyses have been performed by hybridizing immunoprecipitated DNA with a telomeric probe. However, these analyses displayed simultaneously the chromatin structures of telomeres and ITSs. High throughput sequencing analyses of the immunoprecipitated DNA might help overcome this problem. Nevertheless, since the reads obtained with these techniques at present are short, it is still difficult to ascertain whether the enrichment of immunoprecipitated telomeric sequences corresponds to telomeres or to ITSs. Third-generation long-read accurate technologies and new algorithms that discriminate between telomeres and ITSs should solve the problem.In principle, the combination of immunocytolocalization and ChIP experiments should help to differentiate between telomeres and ITSs. However, since subtelomeric regions are known to influence telomere function and contain degenerated ITSs, at least in some organisms like humans or Arabidopsis, this may not be necessarily true.⁶ A specific epigenetic mark might be required for telomere function, found associated with telomeric repeats by ChIP and with the end of chromosomes by immunocytolocalization and still not associate with true telomeres but with subtelomeric regions and ITSs or just with subtelomeric ITSs.An alternative way to analyze the chromatin structure of telomeres by ChIP involves the use of frequently cutting restriction enzymes. The chromatin structures of Arabidopsis telomeres and ITSs have been independently studied by using Tru9I, a restriction enzyme that recognizes the sequence TTAA.²⁴ Since telomeres in Arabidopsis and in other model systems are composed of perfect telomeric repeat arrays, they remain uncut after digestion with Tru9I.²⁵ In contrast, Arabidopsis ITSs are frequently cut because they are composed of short arrays of perfect telomeric repeats interspersed with degenerated repeats.^25–28 Thus, when Arabidopsis genomic DNA is digested with Tru9I and hybridized with a telomeric probe, most of the signals corresponding to ITSs disappear.²⁵ The use of Tru9I has made possible to discover that Arabidopsis telomeres exhibit euchromatic features. In contrast, Arabidopsis ITSs and subtelomeric regions are heterochromatic.²⁴ In Arabidopsis, heterochromatin is characterized by cytosine methylation, which can be targeted at CpG, CpNpG or CpNpN residues (where N is any nucleotide), and by H3K9me1,2, H3K27me1,2 and H4K20me1. In turn, Arabidopsis euchromatin is characterized by H3K4me1,2,3, H3K36me1,2,3, H4K20me2,3 and by histones acetylation.²⁹ ChIP experiments processed with Tru9I have revealed that Arabidopsis telomeres have high levels of euchromatic marks (H3K4me2, H3K9 and H4K16 acetylation) and low levels of heterochromatic marks (H3K9me2, H3K27me1 and DNA methylation).²⁴ Therefore, Arabidopsis telomeres exhibit epigenetic modifications characteristic of euchromatin.Different studies in mice, humans or Arabidopsis have reported that telomeres are heterochromatic based on the existence of siRNAs containing telomeric sequences, on the association of telomeric sequences with telomeric and with heterochromatin proteins, on the methylation of telomeric sequences or on the histones modifications associated with telomeric sequences.^30–34 However, the experiments presented in those studies addressed simultaneously the chromatin organizations of telomeres and subtelomeric regions or of telomeres and ITSs. Telomeres have also been reported to be heterochromatic based on the existence of the so-called TElomeric Repeat containing RNAs (TERRA), which are present in different eukaryotes.³⁵ At telomeric regions, TERRA are transcribed from subtelomeric promoters towards chromosome ends. Since human subtelomeric TERRA are mostly composed of subtelomeric sequences, with only about 200 bp of telomeric sequences at their 3′ ends, they might be related to subtelomeric heterochromatin formation rather than to the formation of telomeric chromatin. Nevertheless, TERRA interact with human telomeric proteins and influence telomere function. In addition, TERRA might also be related to ITSs heterochromatinization.^34,35We believe that the scenario found in Arabidopsis could also be found in other model systems if the chromatin structures of telomeres, subtelomeric regions and ITSs are independently analyzed. Several reports have described the presence of histone H3.3 at mice telomeres.^36–39 Since this histone variant has been previously associated with active chromatin, these studies are compatible with a euchromatic organization of telomeres. However, again in these reports, the experiments shown addressed simultaneously the chromatin organization of telomeres and subtelomeric regions or of telomeres and ITSs. In general terms, we believe that a clear distinction between telomeres and ITSs should be established when future ChIP experiments are analyzed. The use of third generation high throughput sequencing technologies or of frequently cutting restriction enzymes might help in this task.As mentioned above, the epigenetic modifications associated with telomeric regions are known to be important for telomere function. These modifications are required to provide genome stability.³³ In this context, it will be relevant to ascertain how the function of Arabidopsis telomeres is influenced by their euchromatic marks and by the presence of heterochromatin at subtelomeric regions.     

Setting the stage for cohesion establishment by the replication fork

Comment on: Rudra S, et al. Cell Cycle 2012; 2114-21The complex process of semi-conservative DNA replication involves a mechanism whereby the leading and lagging strands with opposite polarity serve as templates for concerted synthesis of complementary base pairs.¹ Lagging-strand synthesis creates discontinuous Okazaki fragments that require timely processing of the 5′ flaps, so that adjacent nascent DNA strands are ligated together to insure genomic stability. While the genetic and molecular requirements of Okazaki fragment maturation have been studied in much detail, the precise temporal and spatial relationship of lagging-strand processing to sister chromatid cohesion remains unclear.² The newly replicated daughter duplex DNA molecules (i.e., the sister chromatids) become tethered during DNA replication and remain paired in order to permit proper segregation of the chromosomes to respective poles during mitosis and nuclear division. Elegant genetic studies in yeast have implicated posttranslational modification of cohesins (specialized protein complexes responsible for tethering sister pairs) by Ctf7/Eco1 acetylase as a key regulatory step in the process, enabling cohesins to perform their function in capturing the newly synthesized sister chromatids. Previous work suggested that genetic and physical interactions among the yeast acetyltransferase Ctf7/Eco1, helicase Chl1, Flap Endonuclease (Fen1) and accessory replication factors [e.g., RFC (clamp loader) and PCNA (clamp)] play an integral role in cohesion establishment. Based on these pieces of evidence, several models to explain the relationship between replication fork dynamics and sister chromatid cohesion have been proposed; however, our understanding of the precise timing of cohesin acetylation and the passage of the replication fork machinery has remained murky at best. Given the importance of proper chromosome segregation for chromosomal stability and the suppression of developmental disorders and tumorigenesis, a comprehensive understanding of the molecular acrobatics involved in sister chromatid cohesion is highly important.In a recent study, the temporal relationship between sister chromatid establishment and lagging-strand synthesis was illuminated.³ The authors have elucidated the link between the catalytic functions of DNA unwinding, flap processing and acetylation, which supports a model of cohesion deposition and establishment that occurs after the passage of the replication fork, similar to how genomic DNA becomes chromatinized. This is a significant advance from an earlier and very popular model of sister chromatid cohesion predicted that Ctf7/Eco1 acetylated cohesin proteins before the encounter by the DNA replication fork, which was thought to permit fork progression and the proper cohesion state for sister chromatid tethering (for review, see ref. ²). Instead, the genetic evidence presented by the Skibbens lab supports a model whereby cohesion establishment is temporally coupled to lagging-strand processing.³ In support of the genetic proof, Rudra and Skibbens went on to show that both Ctf7/Eco1 and Chl1 are associated with the lagging-strand processing nuclease Fen1. Altogether, the experimental results implicate a post-fork establishment model that is analogous to how histone protein complexes are deposited onto newly synthesized sister chromatids and become posttranslationally modified to confer epigenetic status.The discovery from the Skibbens lab that cohesion establishment is closely orchestrated with Okazaki fragment processing prompts a new line of inquiry about the control of flap processing by acetylation and its dual purpose for proper sister chromatid cohesion and replication fidelity in eukaryotes (Fig. 1). The catalytic activity of human FEN-1^4,5 and a functionally related endonuclease known as Dna2⁴ have been shown to be modulated by p300 acetylation, which suggested a model for creating long flap intermediates to promote genomic stability and suppress mutagenesis. Given evidence that ChlR1 is implicated in the genetic disorder Warsaw Breakage syndrome and that the human homolog of yeast Chl1⁶ interacts with the RFC complex and Fen1,⁷ it will be informative to determine if acetyltransferases such as the human orthologs Esco1 and Esco2, the latter mutated in the cohesinopathy Roberts syndrome,⁸ and perhaps other acetyltransferases (e.g., p300) are master regulators of lagging-strand synthesis that not only affect replication fidelity and genomic stability, but also sister chromatid cohesion. Coordination of sister chromatid cohesion establishment with lagging strand synthesis may also involve replication fork stabilization by the Timeless-Tipin protein complex implicated in replication checkpoint.⁹ Defects in the efficient coupling of lagging-strand synthesis to sister chromatid cohesion may contribute to the chromosomal instability characteristic of age-related diseases and cancer.Open in a separate windowFigure 1. Interplay between acetylation, replication fork dynamics and cohesion establishment important for chromosomal integrity.     

Roles of NtGTBP1 in telomere stability

Telomeres are protective nucleoprotein structures at the ends of linear eukaryotic chromosomes. In contrast to double-stranded-specific telomere-binding proteins, the cellular roles of single-stranded-specific telomeric proteins are not well understood in higher plants. Three highly conserved tobacco G-strand-specific telomere-binding protein paralogs (NtGTBP1, NtGTBP2 and NtGTBP3) were identified and characterized. All three NtGTBPs were able to bind specifically to the plant single-stranded telomeric repeat elements in vitro with similar affinities. Suppression of NtGTBP1 by means of the RNAi-mediated gene knock-down method resulted in developmental defects in transgenic tobacco plants accompanied by lengthened telomeres, extra-chromosomal telomeric circles and abnormal anaphase bridges. These results suggest that the downregulation of NtGTBP1 results in genome instability. NtGTBP1 prevented in vitro strand invasion, a prerequisite process for inter-chromosomal telomeric recombination. Therefore, tobacco NtGTBP1 is one of the essential factors for telomere stability. Because abnormal telomeric elongation and recombination due to the suppression of NtGTBP1 are reminiscent of the recombinational telomere lengthening mechanism that purportedly operates in telomerase negative cancer cells, it is of interest to investigate whether telomeric recombination is associated with cell death in animal systems.Key words: genome stability, inter-chromosomal recombination, single-stranded telomere-binding proteinsExtreme ends of linear eukaryotic chromosomes maintain telomeres, which contain protective complexes of proteins and DNA repeats.^1,2 Telomeric DNA repeats consist of two parts: double-stranded and single-stranded DNA sequence elements. Telomere sequences are protected by specialized sequence-specific non-histone DNA binding proteins. In higher plants, Myb domain-containing double-stranded DNA binding proteins (TRFs) are relatively well characterized and appear to be functionally conserved with mammalian TRFs.^3–5 However, situation of single-stranded telomeric binding proteins is complicated. Pot1, a well-known shelterin complex protein, has single-stranded telomere repeat binding activity in yeasts and mammals but no DNA binding activity in Arabidopsis, despite the fact that it is necessary for the proper maintenance of telomere integrity.^6–8 These results led us to investigate other proteins that potentially bind to single-stranded telomeric ends. Because some reports have found that human heterogeneous nuclear ribonucleoproteins (HnRNP) homologs contain sequence-specific telomere repeat binding activity in higher plants,^9,10 we characterized tobacco NtGTBP1, a homolog of human HnRNPs, by performing in vitro gel retardation assays and phenotypic analyses of RNAi-mediated knockdown transgenic tobacco plants, in which NtGTBP1 was downregulated.¹¹     

OB Fold-containing Protein 1 (OBFC1), a Human Homolog of Yeast Stn1, Associates with TPP1 and Is Implicated in Telomere Length Regulation

The telosome/shelterin, a six-protein complex formed by TRF1, TRF2, RAP1, TIN2, POT1, and TPP1, functions as the core of the telomere interactome, acting as the molecular platform for the assembly of higher order complexes and coordinating cross-talks between various protein subcomplexes. Within the telosome, there are two oligonucleotide- or oligosaccharide-binding (OB) fold-containing proteins, TPP1 and POT1. They can form heterodimers that bind to the telomeric single-stranded DNA, an activity that is central for telomere end capping and telomerase recruitment. Through proteomic analyses, we found that in addition to POT1, TPP1 can associate with another OB fold-containing protein, OBFC1/AAF44. The yeast homolog of OBFC1 is Stn1, which plays a critical role in telomere regulation. We show here that OBFC1/AAF44 can localize to telomeres in human cells and bind to telomeric single-stranded DNA in vitro. Furthermore, overexpression of an OBFC1 mutant resulted in elongated telomeres in human cells, implicating OBFC1/AAF4 in telomere length regulation. Taken together, our studies suggest that OBFC1/AAF44 represents a new player in the telomere interactome for telomere maintenance.Telomeres are specialized linear chromosome end structures, which are regulated and protected by networks of protein complexes (1–4). Telomere length, structure, and integrity are critical for the cells and the organism as a whole. Telomere dysregulation can lead to DNA damage response, cell cycle checkpoint, genome instability, and predisposition to cancer (5–9). Mammalian telomeres are composed of double-stranded (TTAGGG)_n repeats followed by 3′-single-stranded overhangs (10). In addition to the telomerase that directly mediates the addition of telomere repeats to the end of chromosomes (11, 12), a multitude of telomere-specific proteins have been identified that form the telosome/shelterin complex and participate in telomere maintenance (9, 13). The telosome in turn acts as the platform onto which higher order telomere regulatory complexes may be assembled into the telomere interactome (14). The telomere interactome has been proposed to integrate the complex and labyrinthine network of protein signaling pathways involved in DNA damage response, cell cycle checkpoint, and chromosomal end maintenance and protection for telomere homeostasis and genome stability.Of the six telomeric proteins (TRF1, TRF2, RAP1, TIN2, POT1, and TPP1) that make up the telosome, TRF1 and TRF2 have been shown to bind telomeric double-stranded DNA (15, 16), whereas the OB³ fold-containing protein POT1 exhibits high affinities for telomeric ssDNA in vitro (17, 18). Although the OB fold of TPP1 does not show appreciable ssDNA binding activity, heterodimerization of TPP1 and POT1 enhances the POT1 ssDNA binding (17, 18). More importantly, POT1 depends on TPP1 for telomere recruitment, and the POT1-TPP1 heterodimer functions in telomere end protection and telomerase recruitment. Notably, the OB fold of TPP1 is critical for the recruitment of the telomerase (18). Disruption of POT1-TPP1 interaction by dominant negative inhibition, RNA interference, or gene targeting could lead to dysregulation of telomere length as well DNA damage responses at the telomeres (18–21).In budding yeast, the homolog of mammalian POT1, Cdc13, has been shown to interact with two other OB fold-containing proteins, Stn1 and Ten1, to form a Cdc13-Stn1-Ten1 (CST) complex (22, 23). The CST complex participates in both telomere length control and telomere end capping (22, 23). The presence of multiple OB fold-containing proteins from yeast to human suggests a common theme for telomere ssDNA protection (4). Indeed, it has been proposed that the CST complex is structurally analogous to the replication factor A complex and may in fact function as a telomere-specific replication factor A complex (23). Notably, homologs of the CST complex have been found in other species such as Arabidopsis (24), further supporting the notion that multiple OB fold proteins may be involved in evolutionarily conserved mechanisms for telomere end protection and length regulation. It remains to be determined whether the CST complex exists in mammals.Although the circuitry of interactions among telosome components has been well documented and studied, how core telosome subunits such as TPP1 help to coordinate the cross-talks between telomere-specific signaling pathways and other cellular networks remains unclear. To this end, we carried out large scale immunoprecipitations and mass spectrometry analysis of the TPP1 protein complexes in mammalian cells. Through these studies, we identified OB fold-containing protein 1 (OBFC1) as a new TPP1-associated protein. OBFC1 is also known as α-accessory factor AAF44 (36). Sequence alignment analysis indicates that OBFC1 is a homolog of the yeast Stn1 protein (25). Further biochemical and cellular studies demonstrate the association of OBFC1 with TPP1 in live cells. Moreover, we showed that OBFC1 bound to telomeric ssDNA and localized to telomeres in mammalian cells. Dominant expression of an OBFC1 mutant led to telomere length dysregulation, indicating that OBFC1 is a novel telomere-associated OB fold protein functioning in telomere length regulation.     

Establishing the human rolling circle reaction

Comment on: Kang YH, et al. Proc Natl Acad Sci USA 2012; 109:6042-7.In eukaryotes, the complex comprised of Mcm2–7, Cdc45 and GINS (CMG) is essential for DNA replication. Several lines of evidence indicate that the Mcm2–7 complex is the motor of the replicative helicase (reviewed in ref. ¹), which is activated by its association with Cdc45 and GINS.² Recently, we described the isolation and characterization of the human (h) CMG complex.³ In HeLa cells, this complex was formed only on chromatin and, following its isolation from cells, exhibited DNA helicase activity. Purified from Sf9 cells, hCMG possesses 3′→5′ DNA helicase activity, indicating that it moves ahead of the leading-strand DNA polymerase (pol). In contrast, the prokaryotic helicase DnaB, which unwinds DNA in the 5′→3′ direction, moves on the lagging strand. Detailed information about the progression of the prokaryotic replication fork was obtained using the rolling-circle method (ref. ⁴ and references therein). These studies permitted a detailed characterization of the joint action of the replicative pol and replicative helicase. In the rolling-circle reaction, the pol extends the 3′ end of a primer annealed to a minicircle that is then unwound simultaneously by the helicase (for a possible arrangement of proteins at the replication fork, see Fig. 1). The emerging single-stranded 5′-tail provides the template for lagging-strand synthesis. In most experiments, minicircles were engineered to contain only three nucleotides, allowing the distinction between leading- and lagging-strand nucleotide incorporation.Open in a separate windowFigure 1. Model of the human replication fork. The CMG complex unwinds DNA in the 3′→5′ direction. Polα/primase synthesizes primers to initiate leading- and lagging-strand synthesis. Polε and polδ are assigned as leading- and lagging-strand polymerases based on evidence in yeast.^5,6 Both pols require the processivity factor PCNA. RPA binds to single-stranded DNA. Additional proteins are required for DNA replication, of which only Ctf4 and Mcm10 are shown for simplicity.We initiated experiments to develop a eukaryotic replication fork in order to investigate whether the hCMG helicase activity could be coupled with the replicative pols.³ We set up rolling circle reactions using a 200-nt minicircle, the putative leading strand pol ε⁵ and hCMG and showed that DNA chains longer than 10 kb were produced (representing > 50 turns of the circle). The putative lagging strand pol δ,⁶ however, did not replace hpol ε in this reaction, though both pols extended primers on single-stranded M13 to full-length products (about 7 kb). It is tempting to speculate that an interaction between hCMG and hpol ε, but not hpol δ, contributes to their different activities. Specific interaction between GINS, a component of the CMG complex, and hpol ε has been demonstrated.⁷ However, it is presently unclear whether this contributes to the observed preferential role of pol ε and thus requires further examination.The processivity of the CMG complex alone was about 500 bp, which was stimulated to about 1 kbp by the addition of a single-strand DNA binding protein, either E. coli SSB or hRPA. The rolling circle reaction is also dependent on E. coli SSB, presumably to sequester the emerging single-stranded 5′ tail. Surprisingly, hRPA did not replace E. coli SSB in the rolling circle reaction. This was attributed to its inhibitory effects on pol ε activity in vitro. The influence of hRPA on eukaryotic fork progression is presently unclear. In the in vitro SV40 viral DNA replication system, hRPA is essential for DNA synthesis and cannot be replaced by E. coli SSB (reviewed in ref. ⁸). In this system, the SV40 large T-antigen acts as the replicative helicase, and hRPA is essential for its interaction with the hpolα/primase complex, which positions primase to initiate RNA chains. In the SV40 replication reaction, hpol δ synthesizes both leading and lagging strands. Surprisingly, while prokaryotic pols (and their processivity factors) can replace hpol δ and its auxiliary proteins in the in vitro SV40 elongation reaction, hpol ε does not play a role,⁸ suggesting that, in this system, the action of hpol ε is preferentially excluded. Importantly, no rolling circle synthesis was detected when hpol δ was used in lieu of hpol ε.³ Whether a similar mechanism leading to the exclusion of hpol δ from leading-strand synthesis is operational with the CMG helicase remains to be investigated. Using an archaeal system consisting of Pol B, RFC, PCNA, the 3′→5′ DNA helicase Mcm and the DNA primase, we have performed both leading- and lagging-strand synthesis on a rolling circle substrate.⁹ Currently, our efforts are focused on the synthesis of the lagging-strand with human proteins.In cells, the replication machinery duplicates chromatinized DNA. Thus, it is likely that chromatin remodeling factors and nucleosome chaperones play roles in the progression of the replication fork. In support of this notion, FACT was identified as a component of the yeast replisome progression complex.¹⁰ Various other proteins associate with the replication fork, such as Mcm10, Ctf4, Tim-Tipin and Claspin. The effects of these proteins on the in vitro replication reaction in eukaryotes remain to be examined.     

ATR and H2AX Cooperate in Maintaining Genome Stability under Replication Stress

Chromosomal abnormalities are frequently caused by problems encountered during DNA replication. Although the ATR-Chk1 pathway has previously been implicated in preventing the collapse of stalled replication forks into double-strand breaks (DSB), the importance of the response to fork collapse in ATR-deficient cells has not been well characterized. Herein, we demonstrate that, upon stalled replication, ATR deficiency leads to the phosphorylation of H2AX by ATM and DNA-PKcs and to the focal accumulation of Rad51, a marker of homologous recombination and fork restart. Because H2AX has been shown to play a facilitative role in homologous recombination, we hypothesized that H2AX participates in Rad51-mediated suppression of DSBs generated in the absence of ATR. Consistent with this model, increased Rad51 focal accumulation in ATR-deficient cells is largely dependent on H2AX, and dual deficiencies in ATR and H2AX lead to synergistic increases in chromatid breaks and translocations. Importantly, the ATM and DNA-PK phosphorylation site on H2AX (Ser¹³⁹) is required for genome stabilization in the absence of ATR; therefore, phosphorylation of H2AX by ATM and DNA-PKcs plays a pivotal role in suppressing DSBs during DNA synthesis in instances of ATR pathway failure. These results imply that ATR-dependent fork stabilization and H2AX/ATM/DNA-PKcs-dependent restart pathways cooperatively suppress double-strand breaks as a layered response network when replication stalls.Genome maintenance prevents mutations that lead to cancer and age-related diseases. A major challenge in preserving genome integrity occurs in the simple act of DNA replication, in which failures at numerous levels can occur. Besides the mis-incorporation of nucleotides, it is during this phase of the cell cycle that the relatively stable double-stranded nature of DNA is temporarily suspended at the replication fork, a structure that is susceptible to collapse into DSBs.² Replication fork stability is maintained by a variety of mechanisms, including activation of the ATR-dependent checkpoint pathway.The ATR pathway is activated upon the generation and recognition of extended stretches of single-stranded DNA at stalled replication forks (1-4). Genome maintenance functions for ATR and orthologs in yeast were first indicated by increased chromatid breaks in ATR^-/- cultured cells (5) and by the “cut” phenotype observed in Mec1 (Saccharomyces cerevisiae) and Rad3 (Schizosaccharomyces pombe) mutants (6-9). Importantly, subsequent studies in S. cerevisiae demonstrated that mutation of Mec1 or the downstream checkpoint kinase Rad53 led to increased chromosome breaks at regions of the genome that are inherently difficult to replicate (10), and a decreased ability to reinitiate replication fork progression following DNA damage or deoxyribonucleotide depletion (11-14).In vertebrates, similar replication fork stabilizing functions have been demonstrated for ATR and the downstream protein kinase Chk1 (15-20). Several possible mechanisms have been put forward to explain how ATR-Chk1 and orthologous pathways in yeast maintain replication fork stability, including maintenance of replicative polymerases (α, δ, and ε) at forks (17, 21), regulation of branch migrating helicases, such as Blm (22-25), and regulation of homologous recombination, either positively or negatively (26-29).Consistent with the role of the ATR-dependent checkpoint in replication fork stability, common fragile sites, located in late-replicating regions of the genome, are significantly more unstable (5-10-fold) in the absence of ATR or Chk1 (19, 20). Because these sites are favored regions of instability in oncogene-transformed cells and preneoplastic lesions (30, 31), it is possible that the increased tumor incidence observed in ATR haploinsufficient mice (5, 32) may be related to subtle increases in genomic instability. Together, these studies indicate that maintenance of replication fork stability may contribute to tumor suppression.It is important to note that prevention of fork collapse represents an early response to problems occurring during DNA replication. In the event of fork collapse into DSBs, homologous recombination (HR) has also been demonstrated to play a key role in genome stability during S phase by catalyzing recombination between sister chromatids as a means to re-establish replication forks (33). Importantly, a facilitator of homologous recombination, H2AX, has been shown to be phosphorylated under conditions that cause replication fork collapse (18, 34).Phosphorylation of H2AX occurs predominantly upon DSB formation (34-38) and has been reported to require ATM, DNA-PKcs, or ATR, depending on the context (37-42). Although H2AX is not essential for HR, studies have demonstrated that H2AX mutation leads to deficiencies in HR (43, 44), and suppresses events associated with homologous recombination, such as the focal accumulation of Rad51, BRCA1, BRCA2, ubiquitinated-FANCD2, and Ubc13-mediated chromatin ubiquitination (43, 45-51). Therefore, through its contribution to HR, it is possible that H2AX plays an important role in replication fork stability as part of a salvage pathway to reinitiate replication following collapse.If ATR prevents the collapse of stalled replication forks into DSBs, and H2AX facilitates HR-mediated restart, the combined deficiency in ATR and H2AX would be expected to dramatically enhance the accumulation of DSBs upon replication fork stalling. Herein, we utilize both partial and complete elimination of ATR and H2AX to demonstrate that these genes work cooperatively in non-redundant pathways to suppress DSBs during S phase. As discussed, these studies imply that the various components of replication fork protection and regeneration cooperate to maintain replication fork stability. Given the large number of genes involved in each of these processes, it is possible that combined deficiencies in these pathways may be relatively frequent in humans and may synergistically influence the onset of age-related diseases and cancer.     

Beginning at the end: DNA replication within the telomere

Using single molecule analysis of replicated DNA (SMARD), Drosopoulos et al. (2015; J. Cell Biol. http://dx.doi.org/10.1083/jcb.201410061) report that DNA replication initiates at measurable frequency within the telomere of mouse chromosome arm 14q. They demonstrate that resolution of G4 structures on the G-rich template strand of the telomere requires some overlapping functions of BLM and WRN helicase for leading strand synthesis.Double-strand breaks in DNA can wreak havoc in cells if not repaired. Therefore, it was proposed that the ends of chromosomes may be specialized cap structures that are not recognized as double-strand breaks, thus preventing cell cycle arrest, degradation, and recombinational fusion (Muller, 1938; McClintock, 1939). We now know that telomeres comprise the ends of chromosomes and are essential for genome stability. Telomeres are composed of tandem head-to-tail repeats of a short G-rich sequence; for example, human telomeres are 2–20 kb of (TTAGGG)_n repeats. The chromosome ends are not blunt, and the 3′ end (G-rich strand) overhangs in a single strand that can invade the interior of the telomere to displace the internal G-rich sequence and form a T-loop structure (Griffith et al., 1999; Cesare et al., 2003; Doksani et al., 2013), thus protecting the chromosome ends from being recognized by the cell as double-strand breaks, in addition to protection by proteins that bind the telomere.Eukaryotic chromosomes are duplicated via semiconservative replication with a leading (continuous synthesis for net growth at the 3′ end of the nascent leading strand) and lagging (discontinuous Okazaki fragment synthesis for net growth at the 5′ end of the nascent lagging strand) elongating strand as shown in Fig. 1. In chromosomal semiconservative replication, the short 5′ RNA primer is removed from the nascent strand and the gap is filled in by DNA that is ligated to the adjacent nascent DNA. However, at the end of the chromosome, the gap after removal of the 5′ terminal RNA primer on the lagging strand cannot be filled in, and the chromosome may become shorter with each ensuing round of replication. This has been termed the end-replication problem (Watson, 1972; Olovnikov, 1973), and telomerase helps to solve this problem (Greider and Blackburn, 1987; Soudet et al., 2014).Open in a separate windowFigure 1.DNA replication at the end of chromosomes. (A) DNA replication can initiate within the subtelomeric region with replication forks (green arrows) progressing bidirectionally away from the origin. Telomere DNA is replicated by a replication fork that passes through this region. In each panel, leading nascent strand synthesis is indicated by a blue line with a single arrowhead; lagging nascent strand synthesis is indicated by a blue line with multiple arrowheads. At the top of each panel, the red line indicates the signal seen by microscopy of replication that initiated and continued during administration of the first pulse (IdU, red), and the dotted green line indicates the signal seen for replication extension during the second pulse (CldU, green). (B) On some DNA molecules from mouse chromosome 14q, DNA replication initiates within the telomere itself. In practice, the second (green) pulse was often not observed in the telomere. (C) Partially overlapping functions of BLM and WRN helicases are used to resolve G-quadruplex (G4) DNA (blue structure) that can form on the G-rich parental strand of the telomeres. In cells deficient of BLM and/or WRN helicase, progression of the nascent leading strand in the telomere is impaired; the slowed replication forks are indicated by red arrows. The resulting replication stress is accompanied by activation of dormant replication origins in the subtelomere. The cartoon is not drawn to scale, and the infrequently used subtelomeric replication origin in C is closer to the telomere than the subtelomeric origin in A.Semiconservative replication occurs before the action of telomerase. Previously it was thought that DNA replication began at an origin in chromosomal DNA adjacent to the telomere repeats, with the replication forks moving bidirectionally away from the subtelomeric origin (Fig. 1 A), thus replicating the telomere. However, the question remained whether DNA replication might initiate with some frequency within the telomere itself (Fig. 1 B). This question has now been answered in the affirmative in this issue by Drosopoulos et al., who used single molecule analysis of replicated DNA (SMARD; Norio and Schildkraut, 2001). In this approach, replicating cells are sequentially labeled by two different nucleotide analogues that are subsequently identified by immunofluorescence. For example, in bidirectional replication, red signals from the first pulse will be flanked at each end by green signals from the second pulse. Earlier reports using SMARD had concluded that most replication initiates at subtelomeric regions in the mouse and human genome and rarely in the telomeres themselves (Sfeir et al., 2009; Drosopoulos et al., 2012). In the recent study by Drosopoulos et al. (2015), fluorescence in situ hybridization (FISH) using probes from the telomere region allowed the replication pattern to be analyzed for a 320 kb genomic segment from the end of mouse chromosome arm 14q. Due to the long time (4 h) for the first (red) pulse, usually only red tracts of signal within the telomere were seen, but since many such molecules did not have the red signal extend into the subtelomeric region, it can be comfortably concluded that replication must have initiated within the telomere (Fig. 1 B). Moreover, some molecules did have red signal in the telomere flanked by green signal, supporting this conclusion. Although in these cases there was chromosome-proximal green signal, chromosome-distal green signal was rarely seen. Thus, although there was limited evidence for bidirectional replication originating in the telomere, it is very clear that a replication origin can exist within the telomere proper with a replication fork that extends over time into the subtelomere. It remains to be investigated whether replication initiates at a relatively high frequency in the telomeres of chromosomes other than 14q.These findings raise the question of whether the origin for DNA replication coincides with the simple sequence repeat found in telomeres or instead if it coincides with some other sequence that might be interspersed within the telomere. The former is suggested by a study with Xenopus cell-free extracts that could assemble the pre-replication complex and undergo some DNA replication on exogenous DNA containing exclusively telomeric repeats (Kurth and Gautier, 2010). Similar conclusions that DNA replication can initiate in the simple DNA repeats found in centromeres where replication bubbles have been observed in Drosophila virilis by electron microscopy have been reached (Zakian, 1976), and a recent study suggests that DNA replication initiates within human alpha-satellite DNA (Erliandri et al., 2014).Replications forks move slowly through telomeric DNA (Ivessa et al., 2002; Makovets et al., 2004; Miller et al., 2006; Sfeir et al., 2009) due to the high thermal stability of GC-rich telomeric DNA as well as its propensity to form stable secondary structures, such as G-quadruplex (G4) DNA, which can pose problems for DNA replication (Lopes et al., 2011; Paeschke et al., 2011). Various helicases help solve this problem; for example, Pif1 helicase helps to unwind G4 (Paeschke et al., 2013). Bloom syndrome helicase (BLM) and the Werner syndrome helicase (WRN) have also been implicated in assisting telomere replication: BLM suppresses replication-dependent fragile telomeres (Sfeir et al., 2009), and WRN suppresses defects in telomere lagging strand synthesis (Crabbe et al., 2004). Drosopoulos et al. (2015) now report that leading strand synthesis that initiates within the telomere has a slower rate of progression into the subtelomere in BLM-deficient cells as visualized by SMARD. Moreover, there was a higher frequency of replication initiation in the 14q subtelomere of the BLM-deficient cells, originating closer to the telomere than in BLM-proficient cells. These observations suggest that dormant replication origins in the 14q subtelomere can be activated when fork progression is impeded in BLM-deficient cells (Fig. 1 C). Drosopoulos et al. (2015) also found an increase in subtelomeric replication initiation when replication fork progression from the telomere was hindered by aphidicolin, as an alternate means to activate dormant origins by replication stress. When cells were treated with the G4 stabilizer PhenDC3, 14q subtelomeric origin firing increased further in BLM-deficient cells. Collectively, the data suggest a slowdown of progression of leading strand synthesis from an origin in the 14q telomere (using the G-rich parental strand as the template) when G4 structures cannot be resolved in BLM-deficient cells. As further support for a role of BLM helicase to remove G4 structures, there was increased staining in BLM-deficient cells by the BG4 antibody (Biffi et al., 2013) against G4 in the whole genome and especially in telomeres.WRN helicase can unwind G4 in vitro (Fry and Loeb, 1999; Mohaghegh et al., 2001). When Drosopoulos et al. (2015) used SMARD to analyze replication in cells doubly deficient of both BLM and WRN, they found a marked decrease of red replication signal in 14q telomeres, suggesting some functional overlap between BLM and WRN with regard to leading strand synthesis off the G-rich strand of telomeres. Supporting this conclusion, there was more G4 staining by the BG4 antibody in cells doubly deficient of both BLM and WRN than in cells deficient of just BLM or just WRN. This is the first direct demonstration in vivo of a contribution of BLM and WRN helicases in the resolution of G4 structures, which is especially needed for progression of leading strand synthesis that initiates in telomeres and is copied from the G-rich strand.     

Human Primpol1: a novel guardian of stalled replication forks

Huang and colleagues identify a human primase-polymerase that is required for stalled replication fork restart and the maintenance of genome integrity.EMBO reports (2013) 14 12, 1104–1112 doi:10.1038/embor.2013.159The successful duplication of genomic DNA during S phase is essential for the proper transmission of genetic information to the next generation of cells. Perturbation of normal DNA replication by extrinsic stimuli or intrinsic stress can result in stalled replication forks, ultimately leading to abnormal chromatin structures and activation of the DNA damage response. On formation of stalled replication forks, many DNA repair and recombination pathway proteins are recruited to resolve the stalled fork and resume proper DNA synthesis. Initiation of replication at sites of stalled forks differs from traditional replication and, therefore, requires specialized proteins to reactivate DNA synthesis. In this issue of EMBO reports, Wan et al [1] introduce human primase-polymerase 1 (hPrimpol1)/CCDC111, a novel factor that is essential for the restart of stalled replication forks. This article is the first, to our knowledge, to ascertain the function of human Primpol enzymes, which were originally identified as members of the archaeao-eukaryotic primase (AEP) family [2].Single-stranded DNA (ssDNA) forms at stalled replication forks because of uncoupling of the DNA helicase from the polymerase, and is coated by replication protein A (RPA) for stabilization and recruitment of proteins involved in DNA repair and restart of replication. To identify novel factors playing important roles in the resolution of stalled replication forks, Wan and colleagues [1] used mass spectrometry to identify RPA-binding partners. Among the proteins identified were those already known to be located at replication forks, including SMARCAL1/HARP, BLM and TIMELESS. In addition they found a novel interactor, the 560aa protein CCDC111. This protein interacts with the carboxyl terminus of RPA1 through its own C-terminal region, and localizes with RPA foci in cells after hydroxyurea or DNA damage induced by ionizing irradiation. Owing to the presence of AEP and zinc-ribbon-like domains at the amino-terminal and C-terminal regions, respectively [2], CCDC111 was predicted to have both primase and polymerase enzymatic activities, which was confirmed with in vitro assays, leading to the name hPrimpol1 for this unique enzyme.The most outstanding discovery in this article is that hPrimpol1 is required for the restart of DNA synthesis from a stalled replication fork (Fig 1). With use of a single DNA fibre assay, knock down of hPrimpol1 had no effect on normal replication-fork progression or the firing of new origins in the presence of replication stress. After removal of replication stress, however, the restart of stalled forks was significantly impaired. Furthermore, the authors observed that hPrimpol1 depletion enhanced the toxicity of replication stress to human cells. Together, these data suggest that hPrimpol1 is a novel guardian protein that ensures the proper re-initiation of DNA replication by control of the repriming and repolymerization of newly synthesized DNA.Open in a separate windowFigure 1The role of hPrimpol1 in stalled replication fork restart. (A) Under normal conditions, the replicative helicase unwinds parental DNA, generating ssDNA that is coated by RPA and serves as a template for leading and lagging strand synthesis. Aside from interacting with RPA bound to the short stretches of ssDNA, the role of hPrimpol1 in normal progression of replication forks is unknown. (B) Following repair of a stalled replication fork, (1) hPrimpol1 rapidly resumes DNA synthesis of long stretches of RPA-coated ssDNA located at the stalled fork site. Later, the leading-strand polymerase (2) or lagging-strand primase and polymerase (3) replace hPrimpol1 to complete replication of genomic DNA. RPA, replication protein A; ssDNA, single-stranded DNA.Eukaryotic DNA replication is initiated at specific sites, called origins, through the help of various proteins, including ORC, CDC6, CDT1 and the MCM helicase complex [3]. On unwinding of the parental duplexed DNA, lagging strand ssDNA is coated by the RPA complex and used as a template for newly synthesized daughter DNA. DNA primase, a type of RNA polymerase, catalyses short RNA primers on the RPA-coated ssDNA that facilitate further DNA synthesis by DNA polymerase. While the use of a short RNA primer is occasionally necessary to restart leading-strand replication, such as in the case of a stalled DNA polymerase, it is primarily utilized in lagging-strand synthesis for the continuous production of Okazaki fragments. The lagging-strand DNA polymerase must efficiently coordinate its action with DNA primase and other replication factors, including DNA helicase and RPA [4]. Cooperation between DNA polymerase and primase is disturbed after DNA damage, ultimately resulting in the collapse of stalled replication forks. Until now, it was believed that DNA primase and DNA polymerase performed separate and catalytically unique functions in replication-fork progression in human cells, but this report provides the first example, to our knowledge, of a single enzyme performing both primase and polymerase functions to restart DNA synthesis at stalled replication forks after DNA damage (Fig 1).… this report provides the first example of a single enzyme performing both primase and polymerase function to restart DNA synthesis at stalled replication forksA stalled replication fork, if not properly resolved, can be extremely detrimental to a cell, causing permanent cell-cycle arrest and, ultimately, death. Therefore, eukaryotic cells have developed many pathways for the identification, repair and restart of stalled forks [5]. RPA recognizes ssDNA at stalled forks and activates the intra-S-phase checkpoint pathway, which involves various signalling proteins, including ATR, ATRIP and CHK1 [6]. This checkpoint pathway halts cell-cycle progression until the stalled forks are properly repaired and restarted. Compared with the recognition and repair of stalled forks, the mechanism of fork restart is relatively elusive. Studies have, however, begun to shed light on this process. For instance, RPA-directed SMARCAL1 has been discovered to be important for restart of DNA replication in bacteria and humans [7]. Together with the identification of hPrimpol1, these findings have helped to expand the knowledge of the mechanism of restarting DNA replication. Furthermore, both reports raise many questions regarding the cooperative mechanism of hPrimpol1 and SMARCAL1 with RPA at stalled forks to ensure genomic stability and proper fork restart [7].First, these findings raise the question of why cells need the specialized hPrimpol1 to restart DNA replication at stalled forks rather than using the already present DNA primase and polymerase. One possibility is that other DNA polymerases are functionally inhibited due to the response of the cell to DNA damage. Although the cells are ready to restart replication, the impaired polymerases might require additional time to recover after DNA damage, necessitating the use of hPrimpol1. In support of this idea, we found that the p12 subunit of DNA polymerase δ is degraded by CRL4^CDT2 E3 ligase after ultraviolet damage [8]. As a result, alternative polymerases, such as hPrimpol1, could compensate for temporarily non-functioning traditional polymerases. A second explanation is that the polymerase and helicase uncoupling after stalling of a fork results in long stretches of ssDNA that are coated with RPA. To restart DNA synthesis, cells must quickly reprime and polymerize large stretches of ssDNA to prevent renewed fork collapse. By its constant interaction with RPA1, hPrimpol1 is present on the ssDNA and can rapidly synthesize the new strand of DNA after the recovery of stalled forks. Third, the authors found that the association of hPrimpol1 with RPA1 is independent of its functional AEP and zinc-ribbon-like domains and occurs in the absence of DNA damage. These results might indicate a role for hPrimpol1 in normal replication fork progression, but further work is necessary to determine whether that is true.The discovery of hPrimpol1 is also important in an evolutionary contextSeveral questions remain. First, what is the fidelity of the polymerase activity? Other specialized polymerases that act at DNA damage sites sometimes have the ability to misincorporate a nucleotide across from a site of damage, for example pol-eta and -zeta [9]. It will be interesting to know whether hPrimpol1 is a high-fidelity polymerase or an error-prone polymerase. Second, is the polymerase only brought into action after fork stalling? If hPrimpol1 is an error-prone polymerase, one could envision other types of DNA damage that can be bypassed by hPrimpol1. Third, is the primase selective for ribonucleotides, or can it also incorporate deoxynucleotides? The requirement of the same domain—AEP—for primase and polymerase activities raises the possibility that NTPs or dNTPs could be used for primase or polymerase activities.The discovery of hPrimpol1 is also important in an evolutionary context. In 2003, an enzyme with catalytic activities like that of hPrimpol1 was discovered in a thermophilic archeaon and in Gram-positive bacteria [10]. This protein had several catalytic activities in vitro, including ATPase, primase and polymerase. In contrast to these Primpol enzymes, those capable of primase and polymerase functions had not been found in higher eukaryotes, which suggested that evolutionary pressures forced a split of these dual-function enzymes. Huang et al''s report suggests, however, that human cells do in fact retain enzymes similar to Primpol. In summary, the role of hPrimpol1 at stalled forks broadens our knowledge of the restart of DNA replication in human cells after fork stalling, allowing for proper duplication of genomic DNA, and provides insight into the evolution of primases in eukaryotes.     

DNA Damage-Induced Phosphorylation of TRF2 Is Required for the Fast Pathway of DNA Double-Strand Break Repair

Protein kinases of the phosphatidylinositol 3-kinase-like kinase family, originally known to act in maintaining genomic integrity via DNA repair pathways, have been shown to also function in telomere maintenance. Here we focus on the functional role of DNA damage-induced phosphorylation of the essential mammalian telomeric DNA binding protein TRF2, which coordinates the assembly of the proteinaceous cap to disguise the chromosome end from being recognized as a double-stand break (DSB). Previous results suggested a link between the transient induction of human TRF2 phosphorylation at threonine 188 (T188) by the ataxia telangiectasia mutated protein kinase (ATM) and the DNA damage response. Here, we report evidence that X-ray-induced phosphorylation of TRF2 at T188 plays a role in the fast pathway of DNA DSB repair. These results connect the highly transient induction of human TRF2 phosphorylation to the DNA damage response machinery. Thus, we find that a protein known to function in telomere maintenance, TRF2, also plays a functional role in DNA DSB repair.Telomeres act as protective caps to disguise the chromosome end from being recognized as a DNA double-strand break (DSB) and play other important roles in maintaining genomic integrity (2, 21, 26). Telomere capping dysfunction resulting in genomic instability is likely a major pathway leading to human cancers and other age-related diseases (8, 27).An increasing number of proteins known to play important roles in DNA repair have also been found to be critical for telomere maintenance (6). Specifically, phosphatidylinositol (PI) 3-kinase-like kinase family members, such as ataxia telangiectasia mutated protein kinase (ATM) and the DNA-dependent protein kinase catalytic subunit in mammals, originally known to act in maintaining genomic stability via DNA repair pathways, have been shown to be important in telomere maintenance (1, 4, 7, 9, 10, 16, 25). Previous reports indicate that ATM is required for the DNA damage-induced phosphorylation of two major telomere-associated proteins in mammals, human TRF1 and TRF2 (16, 28). The specific molecular roles played by the DNA damage-induced phosphorylation of TRF1 and TRF2 in telomere maintenance and/or DNA repair are unclear and under active investigation. We previously reported that upon DNA damage, human TRF2 was rapidly and transiently phosphorylated at threonine 188 (T188) (28). Here, we report that X-ray-induced phosphorylation of human TRF2 at T188 plays a functional role in the fast pathway of DNA DSB repair.     

A critical role for TORC1 in cellular senescence

Comment on: Kolesnichenko M, et al. Cell Cycle 2012; 11:2391-401 and Pospelova TV, et al. Cell Cycle 2012; 11:2402-407.Cellular senescence is a process initiated either when cells proliferate past their potential (replicative senescence) or by activation of an oncogenic stress (oncogene-induced senescence). Both of these events are characterized by the activation of a DNA damage response, which is initiated by eroded telomeres in the case of replicative senescence, and aberrant products of DNA replication in the case of oncogene induced senescence.¹ Senescence plays a critical tumor-suppression role in vivo, and alterations in the senescence program are a hallmark of cancer cells. Bypass of senescence is critical for tumor progression and involves the p53 and pRB tumor-suppressor pathways.² Indeed, expression of DNA tumor virus oncoproteins that target p53 and pRB can bypass senescence in cultured cells,³ and concomitant loss of pRB and p53 bypasses senescence in human diploid fibroblasts.⁴ In addition to being an obligatory step for tumor progression, bypass of senescence creates a favorable environment in which additional tumor-promoting mutations can be acquired. For example, inactivation of p53 in the context of telomere erosion promotes rampant genomic instability mediated by cycles of aberrant DNA damage/DNA repair events.⁵In a new study, Kolesnichenko et al. describe a critical role for the mTOR pathway in senescence induction.⁶ This work demonstrates that inhibition of mTOR is sufficient to delay RAS-induced senescence as well as replicative senescence. Using a combination of inhibitory molecules, shRNA-mediated knockdown and expression of inhibitory proteins, the authors demonstrate that inhibition of the TORC1 complex is sufficient to delay senescence induction. These findings are further corroborated by the independent work of Pospelova and colleagues showing that rapamycin treatment delays senescence induction in murine fibroblasts.⁷ These intriguing findings raise the question of why mTOR inhibition inhibits senescence induction. The work of Kolesnchenko and colleagues provides two clues to explain this phenotype. First, mTOR inhibition results in the activation of the pro-survival factor AKT, a factor that could explain how cells can proliferate in the face of an ongoing senescence-inducing signal. In addition, the authors find reduced levels of p53 and its target gene p21 upon mTOR inhibition. These findings are particularly significant considering the critical role for both p53 activation and p21 induction in senescence induction.In conclusion, the finding that inhibition of the TORC1 complex has a profound effect on the onset of senescence might explain why rapamycin treatment had limited success in the treatment of cancer.⁸ On the other hand, rapamycin slows aging and thus delays cancer in mice.⁹     

Telomerase-associated Protein 1, HSP90, and Topoisomerase II�� Associate Directly with the BLM Helicase in Immortalized Cells Using ALT and Modulate Its Helicase Activity Using Telomeric DNA Substrates

The BLM helicase associates with the telomere structural proteins TRF1 and TRF2 in immortalized cells using the alternative lengthening of telomere (ALT) pathways. This work focuses on identifying protein partners of BLM in cells using ALT. Mass spectrometry and immunoprecipitation techniques have identified three proteins that bind directly to BLM and TRF2 in ALT cells: telomerase-associated protein 1 (TEP1), heat shock protein 90 (HSP90), and topoisomerase IIα (TOPOIIα). BLM predominantly co-localizes with these proteins in foci actively synthesizing DNA during late S and G₂/M phases of the cell cycle when ALT is thought to occur. Immunoprecipitation studies also indicate that only HSP90 and TOPOIIα are components of a specific complex containing BLM, TRF1, and TRF2 but that this complex does not include TEP1. TEP1, TOPOIIα, and HSP90 interact directly with BLM in vitro and modulate its helicase activity on telomere-like DNA substrates but not on non-telomeric substrates. Initial studies suggest that knockdown of BLM in ALT cells reduces average telomere length but does not do so in cells using telomerase.Bloom syndrome (BS)⁴ is a genetic disease caused by mutation of both copies of the human BLM gene. It is characterized by sun sensitivity, small stature, immunodeficiency, male infertility, and an increased susceptibility to cancer of all sites and types. The high incidence of spontaneous chromosome breakage and other unique chromosomal anomalies in cells from BS patients indicate an increase in homologous recombination in somatic cells (1). Another notable feature of non-immortalized and immortalized cells from BS individuals is the presence of telomeric associations (TAs) between homologous chromosomes (2). Work from our group and others have suggested a role for BLM in recombination-mediated mechanisms of telomere elongation or ALT (alternative lengthening of telomeres), processes that maintain/elongate telomeres in the absence of telomerase (3–5). However, the exact mechanism by which BLM contributes to telomere stability is unknown.Several proteins interact with and regulate BLM helicase activity, including two telomere-specific proteins, TRF1 and TRF2 (6, 7). Although TRF2 stimulates BLM unwinding of telomeric and non-telomeric 3′-overhang substrates, TRF1 inhibits BLM unwinding of telomeric substrates. TRF2-mediated stimulation of BLM helicase activity on a telomeric substrate is observed when TRF2 is present in excess or with equimolar amount of TRF1 but not when TRF1 is present in molar excess. Both proteins associate with BLM specifically in ALT cells in vivo, suggesting their involvement in the ALT pathways. In addition to TRF1 and TRF2, the telomere single-strand DNA-binding protein POT1 strongly stimulates BLM helicase activity on long telomeric forked duplexes and D-loop structures (8). Other proteins also play an important role in telomere maintenance in telomerase-negative cells, including RAD50, NBS1, and MRE11, which co-localize with TRF1 and TRF2 in specialized ALT-associated promyelocytic leukemia (PML) nuclear bodies (APBs) (9–11). Thus, we hypothesize that BLM complex formation may be essential for the ALT mechanism, and its modification may occur dynamically during the specific nucleic acid transactions required to protect the telomere in cells using the ALT pathways.This study has identified previously unknown protein partners of BLM and TRF2 in ALT cells using double immunoprecipitation and mass spectrometry (MS). These include telomerase-associated protein 1 (TEP1), heat shock protein 90 (HSP90), and topoisomerase IIα (TOPOIIα). These proteins associate with BLM and TRF2 in cells using ALT but not in cells using telomerase and directly interact with BLM in vitro. This complex of proteins localizes to sites of new DNA synthesis in vivo in ALT cells, suggesting a role in telomere maintenance. We also identified HSP90 and TOPOIIα in another ALT-specific complex consisting of BLM, TRF1, and TRF2 but not TEP1. In vitro analyses demonstrate that HSP90 inhibits BLM helicase activity using both telomeric and non-telomeric substrates, whereas TEP1 and TOPOIIα initially slow the kinetics of BLM unwinding only using telomeric substrates. These findings suggest the presence of dynamic BLM-associated ALT complexes that include previously unidentified interacting proteins. The function of TEP1 in the BLM·TRF2 complex remains unclear, although its previously described interaction with the RNA subunit of telomerase (12) suggests an interesting hypothesis of cross-talk between mechanisms of telomere elongation.