Effect of Telomere Proximity on Telomere Position Effect,Chromosome Healing,and Sensitivity to DNA Double-Strand Breaks in a Human Tumor Cell Line

The ends of chromosomes, called telomeres, are composed of a DNA repeat sequence and associated proteins, which prevent DNA degradation and chromosome fusion. We have previously used plasmid sequences integrated adjacent to a telomere to demonstrate that mammalian telomeres suppress gene expression, called telomere position effect (TPE). We have also shown that subtelomeric regions are highly sensitive to double-strand breaks, leading to chromosome instability, and that this instability can be prevented by the addition of a new telomere to the break, a process called chromosome healing. We have now targeted the same plasmid sequences to a site 100 kb from a telomere in a human carcinoma cell line to address the effect of telomere proximity on telomere position effect, chromosome healing, and sensitivity to double-strand breaks. The results demonstrate a substantial decrease in TPE 100 kb from the telomere, demonstrating that TPE is very limited in range. Chromosome healing was also diminished 100 kb from the telomere, consistent with our model that chromosome healing serves as a repair process for restoring lost telomeres. Conversely, the region 100 kb from the telomere was highly sensitive to double-strand breaks, demonstrating that the sensitive region is a relatively large target for ionizing radiation-induced chromosome instability.Telomeres are composed of a six-base pair repeat sequence and associated proteins that together form a cap to protect the ends of chromosomes and prevent chromosome fusion (6). Telomeres are actively maintained by the enzyme telomerase in human germ line cells but shorten with age in most somatic cells due to the low level of expression of telomerase (12). When a telomere shortens to the point that it is recognized as a double-strand break (DSB), it serves as a signal for replicative cell senescence (13). Human cells that lose the ability to senesce continue to show telomere shortening and eventually enter crisis, which involves increased chromosome fusion, aneuploidy, and cell death (11, 15). An important step that is required for continued division of cancer cells is therefore that they possess the ability to maintain telomeres, not only to avoid senescence but also to avoid chromosome fusion brought on by crisis (11, 25).In addition to their role in protecting the ends of chromosomes, telomeres can also inhibit the expression of nearby genes, called telomere position effect (TPE). TPE has been proposed to have a role in the cellular response to changes in telomere length (26); however, the function of TPE remains unknown. TPE has been extensively studied in Saccharomyces cerevisiae using transgenes integrated near telomeres on truncated chromosomes (1, 2, 22, 47). These studies demonstrated that TPE involves changes in chromatin conformation and is dependent upon both the distance from the telomere and telomere length (55). Subsequent studies of endogenous yeast genes, however, revealed that the influence of TPE on gene expression varies depending on the presence of insulator sequences (18, 45). TPE also occurs in mammalian cells and has been implicated in the loss of expression of genes relocated near telomeres in a variety of human syndromes (9, 16, 28, 58, 59). As in yeast, transgenes located near telomeres have been used to study TPE in the C33-A (32) and HeLa (4) human cervical carcinoma cell lines. We have also studied TPE using transgenes located adjacent to telomeres in mouse embryonic stem (ES) cells, mouse embryo fibroblasts, and transgenic mice (43). However, none of the studies of TPE in mammalian cells has addressed the distance over which TPE extends from the telomere, and so the number of genes whose expression is likely to be affected is not known.The presence of a telomere can also influence the sensitivity of subtelomeric regions to DSBs. We previously demonstrated the sensitivity of subtelomeric regions to DSBs using selectable transgenes and a recognition site for the I-SceI endonuclease that are integrated immediately adjacent to a telomere. Unlike I-SceI-induced DSBs at most locations, which primarily result in small deletions (27, 34, 46, 50), I-SceI-induced DSBs near telomeres commonly result in large deletions, gross chromosome rearrangements (GCRs), and chromosome instability in both mouse ES cells (37) and human tumor cells (65). Therefore, depending on the size of the sensitive region, the combined targets of the subtelomeric regions on all telomeres could contribute significantly to the genomic instability caused by ionizing radiation or other agents that produce DSBs (35). This sensitivity to DSBs may result from a deficiency in DSB repair since regions near telomeres in yeast are deficient in nonhomologous end joining, resulting in an increase in GCRs (48). One possible reason for a deficiency in DSB repair near telomeres is the role of the telomere in preventing chromosome fusion. Telomeric repeat sequences in yeast have been shown to suppress the activation of cell cycle checkpoints in response to DSBs (39). Similarly, the human TRF2 protein, which is required to prevent chromosome fusion, has been demonstrated to inhibit ATM (31), whose activation is instrumental in the repair of DSBs in heterochromatin (20).One mechanism for avoiding the consequences of DSBs near telomeres is through the addition of a new telomere to the site of a DSB, termed chromosome healing (44). Studies in yeast have shown that chromosome healing occurs through the de novo addition of telomeric repeat sequences by telomerase (14, 33, 38). Chromosome healing in S. cerevisiae is inhibited by the 5′-3′ helicase, Pif1 (52), with Pif1-deficient cells showing up to a 1,000-fold increase in chromosome healing (33, 38). The ability of Pif1 to inhibit chromosome healing has been proposed to serve as a mechanism to prevent chromosome healing from interfering with DSB repair (63). Mammalian cells that express telomerase are also capable of performing chromosome healing. We have shown that chromosome healing can also occur following spontaneous telomere loss (17, 49) or DSBs near telomeres in a human cancer cell line (65) or mouse ES cells (19, 54). We have also shown that chromosome healing can prevent the chromosome instability resulting from DSBs near telomeres (19). Because the de novo addition of telomeric repeat sequences has not been observed in mammalian cells at I-SceI-induced DSBs at interstitial sites (27, 34, 46, 50), we have proposed that chromosome healing is inhibited at most locations but serves as an important mechanism for dealing with DSBs near telomeres that would otherwise result in chromosome instability. However, an alternative possibility that has not been ruled out is that chromosome healing also occurs at interstitial sites but that the large terminal deletions that it causes at these sites results in cell death.In the present study, we address several key questions regarding the importance of telomere proximity on TPE, chromosome healing, and sensitivity to DSBs by investigating how telomere proximity affects these processes. The first of these questions involves establishing the distance over which TPE extends from the telomere to gain insights into the numbers of genes that would be affected by changes in TPE. Second, we will investigate whether chromosome healing can occur at a site that is distant from a telomere but in which terminal deletions are known not to be lethal. This will determine for the first time whether chromosome healing is limited to regions near telomeres. Finally, we will investigate the size of the region near a telomere that is sensitive to DSBs, which will address the potential importance of the subtelomeric region as a target for ionizing radiation-induced genomic instability (35). The distance over which a telomere can exert its effects was investigated by comparing TPE, chromosome healing, and the sensitivity to DSBs at a site 100 kb from a telomere with a site immediately adjacent to the same telomere. As a control for the efficiency of generating DSBs at these sites, we have also analyzed the frequency of small deletions, the most common type of I-SceI-induced DNA rearrangement at interstitial sites in mammalian cells (27, 60). Small deletions serve as an excellent internal control for comparing the frequency of other types of rearrangements since we have previously observed a similar frequency of small deletions at telomeric and interstitial sites (65). The results provide important information on the distance over which a telomere can influence TPE, chromosome healing, and the sensitivity to DSBs.     

Evidence for an Additional Base-Pairing Element between the Telomeric Repeat and the Telomerase RNA Template in Kluyveromyces lactis and Other Yeasts

In all telomerases, the template region of the RNA subunit contains a region of telomere homology that is longer than the unit telomeric repeat. This allows a newly synthesized telomeric repeat to translocate back to the 3′ end of the template prior to a second round of telomeric repeat synthesis. In the yeast Kluyveromyces lactis, the telomerase RNA (Ter1) template has 30 nucleotides of perfect homology to the 25-bp telomeric repeat. Here we provide strong evidence that three additional nucleotides at positions −2 through −4 present on the 3′ side of the template form base-pairing interactions with telomeric DNA. Mutation of these bases can lead to opposite effects on telomere length depending on the sequence permutation of the template in a manner consistent with whether the mutation increases or decreases the base-pairing potential with the telomere. Additionally, mutations in the −2 and −3 positions that restore base-pairing potential can suppress corresponding sequence changes in the telomeric repeat. Finally, multiple other yeast species were found to also have telomerase RNAs that encode relatively long 7- to 10-nucleotide domains predicted to base pair, often with imperfect pairing, with telomeric DNA. We further demonstrate that K. lactis telomeric fragments produce banded patterns with a 25-bp periodicity. This indicates that K. lactis telomeres have preferred termination points within the 25-bp telomeric repeat.Telomeres are DNA and protein complexes present at the ends of eukaryotic chromosomes that function to protect chromosome ends from terminal sequence losses and fusions (3, 36). Telomeric DNA is typically composed of tandem 5- to 26-bp repeats that are sufficient for telomere function and that serve as binding sites for telomeric proteins (32). The ribonucleoprotein enzyme telomerase adds telomeric repeats to chromosome ends and prevents the gradual shortening that would otherwise occur. Telomerase synthesizes new telomeric repeats onto chromosome ends by using part of its RNA subunit as a template (13, 14, 31). Cells without telomerase encounter growth and viability problems once telomeres begin to become too short to properly function. In most human cells, telomerase activity is greatly reduced or absent and the ensuing telomere shortening functions to inhibit the formation of cancer by limiting the number of divisions that cells can undergo (4, 7, 16, 30).Recognition of a telomeric end by telomerase in vivo is complex and requires a number of different interactions between components of telomerase and components of the telomere (32). Specialized proteins that bind the 3′ single-stranded overhangs of telomeres, including the yeast Cdc13 protein, can interact directly with telomerase (9, 28). A critical aspect of telomerase''s interaction with the telomeres comes through base pairing between the telomeric overhang and the template region of the telomerase RNA. In all known telomerases, the template region of the RNA subunit contains a region of telomere homology that is longer than the unit telomeric repeat. This presence of short sequence identities at the 3′ and 5′ borders of the template allow a newly synthesized telomeric repeat to translocate back to the 3′ end of the template prior to a second round of telomeric repeat synthesis (38).Kluyveromyces lactis is an ascomycetous yeast species that is a valuable model organism for studying telomeres and telomerase. Unlike the better-studied yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, K. lactis has telomeres composed of repeats of uniform size (25 bp) and sequence (24). This indicates that the translocation step during a round of DNA synthesis by the telomerase enzyme normally occurs between precise positions at the two ends of the telomerase RNA template region. Point mutations at any of multiple positions within either of the two 5-nucleotide (nt)-long direct repeats that border the telomerase RNA template result in telomeric repeats of abnormal size (35). These appear to result from disruption of the normal base-pairing interactions between template and telomeric DNA during the translocation step.Here we present genetic data that argue strongly that three additional nucleotides 3′ of the template and outside the region of continuous homology with the telomeric repeat are involved in the base pairing between telomeric DNA and the telomerase RNA template in K. lactis. Sequence data suggest that similar extended base-pairing regions are widespread in other yeast species.     

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.     

Kinase-Independent Functions of TEL1 in Telomere Maintenance

TEL1 is important in Saccharomyces cerevisiae telomere maintenance, and its kinase activity is required. Tel1p associates with telomeres in vivo, is enriched at short telomeres, and enhances the binding of telomerase components to short telomeres. However, it is unclear how the kinase activity and telomere association contribute to Tel1p''s overall function in telomere length maintenance. To investigate this question, we generated a set of single point mutants and a double point mutant (tel1^KD) of Tel1p that were kinase deficient and two Xrs2p mutants that failed to bind Tel1p. Using these separation-of-function alleles in a de novo telomere elongation assay, we found, surprisingly, that the tel1^KD allele and xrs2 C-terminal mutants were both partially functional. Combining the tel1^KD and xrs2 C-terminal mutants had an additive effect and resembled the TEL1 null (tel1Δ) phenotype. These data indicate that Tel1p has two separate functions in telomere maintenance and that the Xrs2p-dependent recruitment of Tel1p to telomeres plays an important role even in the absence of its kinase activity.The telomere is a highly ordered complex of proteins and DNA found at the ends of linear chromosomes that functions to protect the ends and prevents them from being recognized as double-strand DNA breaks (51). Telomeres shorten gradually due to incomplete replication (1, 20), and this shortening is counteracted by telomerase, which elongates telomeres (18, 19).Saccharomyces cerevisiae telomeres are composed of 300 ± 50 bp of the sequence TG_1-3/C_1-3A. The yeast telomerase complex consists of Est2p (catalytic subunit), the RNA component TLC1, and two accessory proteins, Est1p and Est3p (50). Cells deficient for any of these telomerase components undergo progressive telomere shortening and a simultaneous decrease in growth rate, described as senescence (24, 27). Typically, a small fraction of cells, termed survivors, escape senescence and maintain telomere length by utilizing RAD52-dependent recombination (24, 26).In addition to the telomerase complex, a number of yeast proteins are important in maintaining telomere length and integrity. These include Tel1p and Mec1p, the yeast homologues of mammalian ATM and ATR, respectively (39). While deletion of TEL1 results in short but stable telomeres, MEC1 deletion has little effect on average telomere length. However, cells lacking TEL1 that have a mutant mec1-21 allele undergo senescence, similar to telomerase null cells (36), suggesting that MEC1 plays a minor but essential role in telomere length maintenance in tel1Δ cells. It has been shown that the protein kinase activities of Tel1p and Mec1p are essential in telomere maintenance, since tel1^KD cells have short telomeres and tel1Δ mec1^KD cells undergo senescence (29).In current models, Tel1p acts to maintain telomere length by regulating the access of telomerase to short telomeres. TEL1 is required for the association of Est1p and Est2p with telomeres in the late S/G₂ phase of the cell cycle (16), the time when telomeres are elongated (9, 31). Additionally, in both yeast and mammalian cells, telomerase preferentially elongates the shortest telomeres (22, 30, 47). Therefore, TEL1 seems to be required mainly for the association of telomerase to short telomeres in yeast. Indeed, Tel1p preferentially binds to short telomeres (4, 21, 38) and is essential for the increased association of Est1p and Est2p to short telomeres during late S/G₂ (38). However, the kinase activity of Tel1p is not required for the telomere association (21). In addition to its role in telomerase recruitment, TEL1 may also regulate telomere length by enhancing the processivity of telomerase at short telomeres (7).The Mre11p, Rad50p, and Xrs2p (MRX) complex also plays important roles in telomere maintenance. Cells lacking any one of these components (mrxΔ) have short and stable telomeres. Since combining mrxΔ with tel1Δ has no synergistic effect on telomere shortening and mrxΔ mec1Δ cells undergo senescence, it was proposed that the MRX complex and Tel1p function in the same telomere maintenance pathway (37). In agreement with this model, the C-terminal region of Xrs2p is essential in recruiting Tel1p both to double-strand breaks (32) and to short telomeres (38). Interestingly, the mammalian functional homologue of Xrs2p, NBS1, interacts with ATM via its extreme C terminus (13), suggesting that the recruitment of Tel1p to telomeres and the recruitment of ATM to DNA damage sites are conserved.It remains a question what exact roles the kinase activity of Tel1p and its telomere binding play in telomere maintenance. Tel1p''s telomere maintenance function seems to be dependent on its kinase activity, since tel1^KD cells have short telomeres (29). It has been proposed that Tel1p may regulate the recruitment of Est1p, and thus the rest of the telomerase complex (12, 23, 54), to telomeres by phosphorylating Cdc13p (3, 48). Other experiments suggest the association of Tel1p to the telomere plays a major role. The preferential binding of Tel1p to short telomeres is lost in xrs2-664 cells (38), which lack the C-terminal 190 amino acids of Xrs2p and have short telomeres, similar to xrs2Δ (41). It has been suggested that the association of Tel1p to telomeres is required for its substrate phosphorylation and, therefore, telomere length maintenance (3, 39).To further analyze the functions of Tel1p in telomere maintenance, we generated a novel kinase-dead allele of TEL1 and new alleles of XRS2 that do not interact with Tel1p. Through these separation-of-function mutants, we show that both sets of alleles are partially active in a de novo telomere elongation assay. However, combining both the tel1^KD and either of the Tel1p interaction-deficient xrs2 alleles resulted in a phenotype resembling the tel1Δ phenotype, suggesting that Tel1p has kinase-dependent and kinase-independent, but telomere binding-dependent, functions in telomere maintenance.     

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.     

Rad50 Is Dispensable for the Maintenance and Viability of Postmitotic Tissues

The majority of spontaneous chromosome breakage occurs during the process of DNA replication. Homologous recombination is the primary mechanism of repair of such damage, which probably accounts for the fact that it is essential for genome integrity and viability in mammalian cells. The Mre11 complex plays diverse roles in the maintenance of genomic integrity, influencing homologous recombination, checkpoint activation, and telomere maintenance. The complex is essential for cellular viability, but given its myriad influences on genomic integrity, the mechanistic basis for the nonviability of Mre11 complex-deficient cells has not been defined. In this study we generated mice carrying a conditional allele of Rad50 and examined the effects of Rad50 deficiency in proliferative and nonproliferative settings. Depletion of Rad50 in cultured cells caused extensive DNA damage and death within 3 to 5 days of Rad50 deletion. This was not associated with gross telomere dysfunction, suggesting that the telomeric functions of the Mre11 complex are not required for viability. Rad50 was also dispensable for the viability of quiescent liver and postmitotic Purkinje cells of the cerebellum. These findings support the idea that the essential functions of the Mre11 complex are associated with DNA replication and further suggest that homologous recombination is not essential in nondividing cells.The Mre11 complex regulates both DNA damage checkpoint function and repair. Its checkpoint functions appear to be primarily related to its role as a DNA double-strand break (DSB) sensor which binds DNA damage and activates ATM (ataxia-telangiectasia [AT] mutated). The ATM kinase transduces the damage signal via phosphorylating mediators of the damage response (30, 42), which promotes cell cycle arrest, DNA repair, and apoptosis. Mre11 complex functions are compromised in the human chromosome instability syndromes Nijmegen breakage syndrome and AT-like disorder, which are caused by hypomorphic mutations in Nbs1 and Mre11. Cells derived from patients and from mouse models of these diseases exhibit spontaneous DNA damage, ionizing radiation (IR) sensitivity, and checkpoint defects (25, 27, 48, 52, 57).The complex''s primary role in DNA repair is in recombinational DSB repair, and this role likely underlies its essential nature. In Saccharomyces cerevisiae, the complex governs homologous recombination (HR) and nonhomologous end joining (NHEJ) (19), whereas in vertebrate systems it primarily functions in HR (51, 61, 62). In fact, studies of Nbs1-deficient cells suggest that the Mre11 complex may inhibit NHEJ in mammals (62). Data from several species also implicate the Mre11 nuclease in the metabolism of topoisomerase adducts (40, 43, 49). This highly conserved function could also explain why the Mre11 complex is essential.The Mre11 complex''s function at telomeres may also be required for viability. Telomeres protect the ends of linear chromosomes from being recognized as DSBs and thereby activating the DNA damage response (DDR) (9). In S. cerevisiae the Mre11 complex influences telomere length maintenance (5, 28), whereas in mammals the complex interacts with the telomere binding protein Trf2 and localizes to telomeres (63). Loss of Trf2 results in telomere uncapping, causing activation of the DDR, telomere fusions, and senescence (7). Given the association of Mre11 with Trf2, it is conceivable that acute Mre11 complex deficiency in the mouse would phenocopy Trf2 loss and similarly lead to cell death as a result of telomere uncapping.Conclusions regarding the essential nature of HR in general (33, 47, 53) and the Mre11 complex specifically (10, 17, 45, 59, 62) have been derived from the analysis of proliferating cells in vitro or in vivo. The coincidence of DNA replication and the formation of spontaneous DSBs prompted us to test whether the Mre11 complex and, by extension, HR would be essential in quiescent or postmitotic tissues in which the frequency of spontaneous DSBs is significantly reduced. To examine this issue, we generated mice containing a conditional Rad50 allele in which the Rad50 gene could be inactivated in quiescent and postmitotic cells.Our results indicate that Rad50 is not required for homeostasis or viability of quiescent hepatocytes of the adult liver; nor does it appear to be required for maintenance of postmitotic Purkinje cells of the cerebellum. In contrast, Rad50 was required for viability of proliferating tissue culture and bone marrow cells. Rad50-deficient hepatocytes that were induced to divide via hepatectomy were able to achieve limited division and survived despite the presence of DNA damage that persisted long after the bulk of regeneration was complete. Rad50-deficient cells did not exhibit overtly dysfunctional telomeres, suggesting that their loss of viability was not due to acute telomere failure. These data indicate that the Mre11 complex and, by extension, HR may be dispensable in postmitotic cells and are consistent with the interpretation that the replication-associated functions of the Mre11 complex account for its essential nature.     

Telomere Organization in the Ligninolytic Basidiomycete Pleurotus ostreatus

Telomeres are structural and functional chromosome regions that are essential for the cell cycle to proceed normally. They are, however, difficult to map genetically and to identify in genome-wide sequence programs because of their structure and repetitive nature. We studied the telomeric and subtelomeric organization in the basidiomycete Pleurotus ostreatus using a combination of molecular and bioinformatics tools that permitted us to determine 19 out of the 22 telomeres expected in this fungus. The telomeric repeating unit in P. ostreatus is TTAGGG, and the numbers of repetitions of this unit range between 25 and 150. The mapping of the telomere restriction fragments to linkage groups 6 and 7 revealed polymorphisms compatible with those observed by pulsed field gel electrophoresis separation of the corresponding chromosomes. The subtelomeric regions in Pleurotus contain genes similar to those described in other eukaryotic systems. The presence of a cluster of laccase genes in chromosome 6 and a bipartite structure containing a Het-related protein and an alcohol dehydrogenase are especially relevant; this bipartite structure is characteristic of the Pezizomycotina fungi Neurospora crassa and Aspergillus terreus. As far as we know, this is the first report describing the presence of such structures in basidiomycetes and the location of a laccase gene cluster in the subtelomeric region, where, among others, species-specific genes allowing the organism to adapt rapidly to the environment usually map.Pleurotus ostreatus (Jacq.: Fr) Kumm. (Dikarya, Basidiomycota, Agaricomycotina, Agaricales) (52) is an active lignin degrader that lives as a saprophyte on dead or decaying wood. P. ostreatus (oyster mushroom) has been industrially cultivated for food production because of its flavor and its nutritional (49) and health-stimulating (8) properties. In addition, it produces various secondary metabolites of medical interest (33). P. ostreatus ligninolytic activity and enzymes have been used in the bioconversion of agricultural wastes (1); in the biodegradation of organopollutants, xenobiotics, and industrial contaminants (12); and in paper pulp bleaching (65), among other applications (10).The whole genome sequence of P. ostreatus is currently being assembled at the Joint Genome Institute (California). P. ostreatus is the first edible and the second lignin-degrading basidiomycete to be sequenced. The sequences of other basidiomycetes, such as Phanerochaete chrysosporium (48), Cryptococcus neoformans (44), Ustilago maydis (38), and Laccaria bicolor (47) have been published, and others (Postia placenta, Heterobasidion annosum, Agaricus bisporus, Serpula lacrymans, etc.) are in progress.Telomeres are the protective DNA-protein complexes found at chromosome termini (6, 13, 76). In most eukaryotes, telomeric DNA consists of tandem arrays of 5- to 8-bp direct repeats where specific telomere-capping proteins bind to ensure chromosomal-end integrity. Telomeres are essential for genome stability, and their shortening (attrition) can lead to chromosome instability, replicative senescence, and apoptosis (43), while their loss causes activation of DNA damage responses (45, 66), cell cycle arrest (28), and chromosome fusions, such as nonreciprocal translocations (7, 32). Moreover, high recombination rates are frequent near telomeres (50).Telomeres and subtelomeric regions are usually gene reservoirs that permit organisms to quickly adapt to new ecological niches (60). Two types of genes participate in this adaptive process: species-specific (18) and contingency genes (5). Species-specific genes are shorter than the core genes of the genomes in which they are present, contain fewer exons, exhibit a subtelomeric bias, and arise by duplication, diversification, and differential gene loss. The avirulence genes of some phytopathogenic fungi are contingency genes that appear near telomeres (15). Furthermore, it has recently been found in Fusarium species that pathogenicity-related genes cooccur with telomeric regions. In this case, chromosomal rearrangements (fusions) have maintained these structures. The Fusarium graminearum genome revealed a link between localized polymorphism and pathogen specialization (11). Among the genes frequently found in subtelomeric regions in Magnaporthe oryzae and Aspergillus sp., the presence of transposons, telomere-linked RecQ helicases, clusters of secondary-metabolite genes, cytochrome oxidases, hydrolases, molecular transporters, and genes encoding secreted proteins, among others, has been reported (18, 56).RecQ helicases are highly conserved in evolution and are required for genome stability. Genes coding for these enzymes have been described in prokaryotes and eukaryotes (4, 9, 39, 71). There are a minimum of five RecQ helicase-like genes in humans, and three of them (BLM, WRN, and RECQL4) are mutated in the Bloom, Werner, and Rothmund-Thomson recessive autosomal syndromes, which exhibit genomic instability leading ultimately to cancer (9). Fungal RecQ helicase-like genes have been previously found associated with chromosome ends (23, 35, 56, 61).In genome-sequencing projects, telomeres and subtelomeric regions are rarely present or assembled because of problems derived from their repetitive nature; therefore, it is necessary to perform direct cloning of the subtelomeric regions. The rice pathogen M. oryzae (56) is one of the few fungi with telomeric and subtelomeric regions characterized. Telomere-associated markers provide an accurate assessment of linkage group (LG) completeness and a better estimate of genetic size and help in establishing the synteny of LGs, especially in those organisms for which genetic-linkage maps are not available (34). Moreover, these markers inform us about the genome organization and the occurrence of species-specific and contingency genes (5, 18), as well as about the chromosome rearrangements that could have occurred in the evolution of the genome.In this work, we mapped and studied the telomeric and subtelomeric regions of most of the P. ostreatus chromosomes, and we describe the main genes present in them. The study was carried out with a combination of genetic, molecular, and bioinformatics tools. The results obtained show the high complexity of these regions and confirm the presence of RecQ helicase-like, heterokaryotic incompatibility (het), and short-chain dehydrogenase genes that have also been found in other fungi. In addition, a laccase gene cluster is described for the first time in the subtelomeric region of chromosome 6. This study is the first step toward analyzing the effects that the subtelomeric positions of some fungal-species-specific genes (such as the laccases can be in white rot lignocellulolytic fungi) could have in the adaptation to new growing substrates and in the generation of large families of apparently redundant elements.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).