WRN Loss Induces Switching of Telomerase-Independent Mechanisms of Telomere Elongation

Telomere maintenance can occur in the presence of telomerase or in its absence, termed alternative lengthening of telomeres (ALT). ALT adds telomere repeats using recombination-based processes and DNA repair proteins that function in homologous recombination. Our previous work reported that the RecQ-like BLM helicase is required for ALT and that it unwinds telomeric substrates in vitro. WRN is also a RecQ-like helicase that shares many biochemical functions with BLM. WRN interacts with BLM, unwinds telomeric substrates, and co-localizes to ALT-associated PML bodies (APBs), suggesting that it may also be required for ALT processes. Using long-term siRNA knockdown of WRN in three ALT cell lines, we show that some, but not all, cell lines require WRN for telomere maintenance. VA-13 cells require WRN to prevent telomere loss and for the formation of APBs; Saos-2 cells do not. A third ALT cell line, U-2 OS, requires WRN for APB formation, however WRN loss results in p53-mediated apoptosis. In the absence of WRN and p53, U-2 OS cells undergo telomere loss for an intermediate number of population doublings (50–70), at which point they maintain telomere length even with the continued loss of WRN. WRN and the tumor suppressor BRCA1 co-localize to APBs in VA-13 and U-2 OS, but not in Saos-2 cells. WRN loss in U-2 OS is associated with a loss of BRCA1 from APBs. While the loss of WRN significantly increases telomere sister chromatid exchanges (T-SCE) in these three ALT cell lines, loss of both BRCA1 and WRN does not significantly alter T-SCE. This work demonstrates that ALT cell lines use different telomerase-independent maintenance mechanisms that variably require the WRN helicase and that some cells can switch from one mechanism to another that permits telomere elongation in the absence of WRN. Our data suggest that BRCA1 localization may define these mechanisms.     

Unwinding protein complexes in ALTernative telomere maintenance

Telomeres are composed of specialized chromatin that includes DNA repair/recombination proteins, telomere DNA‐binding proteins and a number of three dimensional nucleic acid structures including G‐quartets and D‐loops. A number of studies suggest that the BLM and WRN recQ‐like helicases play important roles in recombination‐mediated mechanisms of telomere elongation or A lternative L engthening of T elomeres (ALT), processes that maintain/elongate telomeres in the absence of telomerase. BLM and WRN localize within ALT‐associated nuclear bodies in telomerase‐negative immortalized cell lines and interact with the telomere‐specific proteins POT1, TRF1 and TRF2. Helicase activity is modulated by these interactions. BLM functions in DNA double‐strand break repair processes such as non‐homologous end joining, homologous recombination‐mediated repair, resolution of stalled replication forks and synthesis‐dependent strand annealing, although its precise functions at the telomeres are speculative. WRN also functions in DNA replication, recombination and repair, and in addition to its helicase domain, includes an exonuclease domain not found in other recQ‐like helicases. The biochemical properties of BLM and WRN are, therefore, important in biological processes other than DNA replication, recombination and repair. In this review, we discuss some previous and recent findings of human rec‐Q‐like helicases and their role in telomere elongation during ALT processes. J. Cell. Biochem. 109: 7–15, 2010. © 2009 Wiley‐Liss, Inc.     

The role of DNA damage response proteins at telomeres--an "integrative" model

Telomeres are specialized structures at chromosome ends which play the key role in chromosomal end protection. There is increasing evidence that many DNA damage response proteins are involved in telomere maintenance. For example, cells defective in DNA double strand break repair proteins including Ku, DNA-PKcs, RAD51D and the MRN (MRE11/RAD51/NBS1) complex show loss of telomere capping function. Similarly, mouse and human cells defective in ataxia telangiectasia mutated (ATM) have defective telomeres. A total of 14 mammalian DNA damage response proteins have, so far, been implicated in telomere maintenance. Recent studies indicate that three more proteins, namely BRCA1, hRad9 and PARP1 are involved in telomere maintenance. The involvement of a wide range of DNA damage response proteins at telomeres raises an important question: do telomere maintenance mechanisms constitute an integral part of DNA damage response machinery? A model termed the "integrative" model is proposed here to argue in favour of telomere maintenance being an integral part of DNA damage response. The "integrative" model is supported by the observation that a telomeric protein, TRF2, is not confined to its local telomeric environment but it migrates to the sites of DNA breakage following exposure of cells to ionizing radiation. Furthermore, even if telomeres are maintained in a non-canonical way, as in the case of Drosophila, DNA damage response proteins are still involved in telomere maintenance suggesting integration of telomere maintenance mechanisms into the DNA damage response network.     

Disruption of telomere maintenance by depletion of the MRE11/RAD50/NBS1 complex in cells that use alternative lengthening of telomeres

Immortalized human cells are able to maintain their telomeres by telomerase or by a recombination-mediated DNA replication mechanism known as alternative lengthening of telomeres (ALT). We showed previously that overexpression of Sp100 protein can suppress ALT and that this was associated with sequestration of the MRE11/RAD50/NBS1 (MRN) recombination protein complex by Sp100. In the present study, we determined whether MRN proteins are required for ALT activity. ALT cells were depleted of MRN proteins by small hairpin RNA-mediated knockdown, which was maintained for up to 100 population doublings. Knockdown of NBS1 had no effect on the level of RAD50 or MRE11, but knockdown of RAD50 also depleted cells of NBS1, and knockdown of MRE11 depleted cells of all three MRN proteins. Depletion of NBS1, with or without depletion of other members of the complex, resulted in inhibition of ALT-mediated telomere maintenance, as evidenced by decreased numbers of ALT-associated promyelocytic leukemia bodies and decreased telomere length. In some clones there was an initial period of rapid shortening followed by stabilization of telomere length, whereas in others there was continuous shortening at a rate within the reported range for normal human somatic cells lacking a telomere maintenance mechanism. In contrast, depletion of NBS1 in telomerase-positive cells did not result in telomere shortening. A recent study showed that NBS1 was required for the formation of extrachromosomal telomeric circles (Compton, S. A., Choi, J. H., Cesare, A. J., Ozgur, S., and Griffith, J. D. (2007) Cancer Res. 67, 1513-1519), also a marker for ALT. We conclude that the MRN complex, and especially NBS1, is required for the ALT mechanism.     

Mammalian 5' C-rich telomeric overhangs are a mark of recombination-dependent telomere maintenance

Recent evidence for 5'-cytosine (C)-rich overhangs at the telomeres of the nematode Caenorhabditis elegans provided the impetus to re-examine the end structure of mammalian telomeres. Two-dimensional (2D) gel electrophoresis, single telomere-length analysis (STELA), and strand-specific exonuclease assays revealed the presence of a 5'-C-rich overhang at the telomeres of human and mouse chromosomes. C-overhangs were prominent in G1/S arrested as well as terminally differentiated cells, indicating that they did not represent replication intermediates. C-rich overhangs were far more prevalent in tumor cells engaged in the alternative lengthening of telomeres (ALT) pathway of telomere maintenance, which relies on the homologous recombination (HR) machinery. Transient siRNA-based depletion of the HR-specific proteins RAD51, RAD52, and XRCC3 resulted in changes in C-overhang levels, implicating the involvement of 5'-C-overhangs in the HR-dependent pathway of telomere maintenance.     

Topoisomerase IIIalpha is required for normal proliferation and telomere stability in alternative lengthening of telomeres

Topoisomerase (Topo) IIIalpha associates with BLM helicase, which is proposed to be important in the alternative lengthening of telomeres (ALT) pathway that allows telomere recombination in the absence of telomerase. Here, we show that human Topo IIIalpha colocalizes with telomeric proteins at ALT-associated promyelocytic bodies from ALT cells. In these cells, Topo IIIalpha immunoprecipitated with telomere binding protein (TRF) 2 and BLM and was shown to be associated with telomeric DNA by chromatin immunoprecipitation, suggesting that these proteins form a complex at telomere sequences. Topo IIIalpha depletion by small interfering RNA reduced ALT cell survival, but did not affect telomerase-positive cell lines. Moreover, repression of Topo IIIalpha expression in ALT cells reduced the levels of TRF2 and BLM proteins, provoked a strong increase in the formation of anaphase bridges, induced the degradation of the G-overhang signal, and resulted in the appearance of DNA damage at telomeres. In contrast, telomere maintenance and TRF2 levels were unaffected in telomerase-positive cells. We conclude that Topo IIIalpha is an important telomere-associated factor, essential for telomere maintenance and chromosome stability in ALT cells, and speculate on its potential mechanistic function.     

The G-Quadruplex Ligand Telomestatin Impairs Binding of Topoisomerase IIIα to G-Quadruplex-Forming Oligonucleotides and Uncaps Telomeres in ALT Cells

In Alternative Lengthening of Telomeres (ALT) cell lines, specific nuclear bodies called APBs (ALT-associated PML bodies) concentrate telomeric DNA, shelterin components and recombination factors associated with telomere recombination. Topoisomerase IIIα (Topo III) is an essential telomeric-associated factor in ALT cells. We show here that the binding of Topo III to telomeric G-overhang is modulated by G-quadruplex formation. Topo III binding to G-quadruplex-forming oligonucleotides was strongly inhibited by telomestatin, a potent and specific G-quadruplex ligand. In ALT cells, telomestatin treatment resulted in the depletion of the Topo III/BLM/TRF2 complex and the disruption of APBs and led to the segregation of PML, shelterin components and Topo III. Interestingly, a DNA damage response was observed at telomeres in telomestatin-treated cells. These data indicate the importance of G-quadruplex stabilization during telomere maintenance in ALT cells. The function of TRF2/Topo III/BLM in the resolution of replication intermediates at telomeres is discussed.     

Recombination at Mammalian Telomeres: An Alternative Mechanism for Telomere Protection and Elongation

In mammalian cells, homologous recombination (HR) provides anaccurate mechanism for the repair of DNA double-strand breaks causedby replication fork breakdown or DNA damaging agents. HR also plays arole in the maintenance of eukaryotic telomeres; cells defective in therecombinational repair proteins RAD51D or RAD54 exhibit telomereshortening and end-to-end chromosome fusions. Here we discuss theway in which HR contributes to telomere protection and elongation inmammalian cells. Understanding the mechanisms by which HRpromotes telomere maintenance has important implications for genomicstability and tumorigenesis.     

Recruitment of NBS1 into PML oncogenic domains via interaction with SP100 protein

Nijmegen breakage syndrome (NBS) is an autosomal recessive disorder characterized by microcephaly, chromosomal instability, radiation sensitivity, and an increased incidence of malignancies. NBS1, the protein responsible for NBS, forms a complex with MRE11 and RAD50, and plays a vital role in DNA repair, cell cycle checkpoint, and telomere maintenance. Here, we show that a BRCA carboxyl terminus (BRCT) domain-containing region of NBS1 interacts with a nuclear dots-associated protein, SP100. The SP100 and NBS1 proteins co-localized in PODs and APBs in normal human fibroblast MRC5 and ALT line VA13 at G2 phase, respectively. Introduction of PML and SP100 into NT2 cells, which express no detectable amount of PML or SP100 proteins, resulted in localization of NBS1 in ectopically expressed PODs. These results indicate that NBS1 is recruited into PODs via interaction with SP100 protein. Thus, interaction between the NBS1 and SP100 proteins may be involved in genomic stability and telomere maintenance.     

Telomere maintenance requires the RAD51D recombination/repair protein

The five RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) are required in mammalian cells for normal levels of genetic recombination and resistance to DNA-damaging agents. We report here that RAD51D is also involved in telomere maintenance. Using immunofluorescence labeling, electron microscopy, and chromatin immunoprecipitation assays, RAD51D was shown to localize to the telomeres of both meiotic and somatic cells. Telomerase-positive Rad51d(-/-) Trp53(-/-) primary mouse embryonic fibroblasts (MEFs) exhibited telomeric DNA repeat shortening compared to Trp53(-/-) or wild-type MEFs. Moreover, elevated levels of chromosomal aberrations were detected, including telomeric end-to-end fusions, a signature of telomere dysfunction. Inhibition of RAD51D synthesis in telomerase-negative immortalized human cells by siRNA also resulted in telomere erosion and chromosome fusion. We conclude that RAD51D plays a dual cellular role in both the repair of DNA double-strand breaks and telomere protection against attrition and fusion.     

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.     

Suppression of alternative lengthening of telomeres by Sp100-mediated sequestration of the MRE11/RAD50/NBS1 complex

Approximately 10% of cancers overall use alternative lengthening of telomeres (ALT) instead of telomerase to prevent telomere shortening, and ALT is especially common in astrocytomas and various types of sarcomas. The hallmarks of ALT in telomerase-negative cancer cells include a unique pattern of telomere length heterogeneity, rapid changes in individual telomere lengths, and the presence of ALT-associated promyelocytic leukemia bodies (APBs) containing telomeric DNA and proteins involved in telomere binding, DNA replication, and recombination. The ALT mechanism appears to involve recombination-mediated DNA replication, but the molecular details are largely unknown. In telomerase-null Saccharomyces cerevisiae, an analogous survivor mechanism is dependent on the RAD50 gene. We demonstrate here that overexpression of Sp100, a constituent of promyelocytic leukemia nuclear bodies, sequestered the MRE11, RAD50, and NBS1 recombination proteins away from APBs. This resulted in repression of the ALT mechanism, as evidenced by progressive telomere shortening at 121 bp per population doubling, a rate within the range found in telomerase-negative normal cells, suppression of rapid telomere length changes, and suppression of APB formation. Spontaneously generated C-terminally truncated Sp100 that did not sequester the MRE11, RAD50, and NBS1 proteins failed to inhibit ALT. These findings identify for the first time proteins that are required for the ALT mechanism.     

The roles of WRN and BLM RecQ helicases in the Alternative Lengthening of Telomeres

Approximately 10% of all cancers, but a higher proportion of sarcomas, use the recombination-based alternative lengthening of telomeres (ALT) to maintain telomeres. Two RecQ helicase genes, BLM and WRN, play important roles in homologous recombination repair and they have been implicated in telomeric recombination activity, but their precise roles in ALT are unclear. Using analysis of sequence variation present in human telomeres, we found that a WRN– ALT+ cell line lacks the class of complex telomere mutations attributed to inter-telomeric recombination in other ALT+ cell lines. This suggests that WRN facilitates inter-telomeric recombination when there are sequence differences between the donor and recipient molecules or that sister-telomere interactions are suppressed in the presence of WRN and this promotes inter-telomeric recombination. Depleting BLM in the WRN– ALT+ cell line increased the mutation frequency at telomeres and at the MS32 minisatellite, which is a marker of ALT. The absence of complex telomere mutations persisted in BLM-depleted clones, and there was a clear increase in sequence homogenization across the telomere and MS32 repeat arrays. These data indicate that BLM suppresses unequal sister chromatid interactions that result in excessive homogenization at MS32 and at telomeres in ALT+ cells.     

Dysfunctional telomeres in human BRCA2 mutated breast tumors and cell lines

In the present study the possible involvement of telomeres in chromosomal instability of breast tumors and cell lines from BRCA2 mutation carriers was examined. Breast tumors from BRCA2 mutation carriers showed significantly higher frequency of chromosome end-to-end fusions (CEFs) than tumors from non-carriers despite normal telomere DNA content. Frequent CEFs were also found in four different BRCA2 heterozygous breast epithelial cell lines, occasionally with telomere signal at the fusion point, indicating telomere capping defects. Extrachromosomal telomeric repeat (ECTR) DNA was frequently found scattered around metaphase chromosomes and interstitial telomere sequences (ITSs) were also common. Telomere sister chromatid exchanges (T-SCEs), characteristic of cells using alternative lengthening of telomeres (ALT), were frequently detected in all heterozygous BRCA2 cell lines as well as the two ALT positive cell lines tested. Even though T-SCE frequency was similar in BRCA2 heterozygous and ALT positive cell lines they differed in single telomere signal loss and ITSs. Chromatid type alterations were more prominent in the BRCA2 heterozygous cell lines that may have propensity for telomere based chromosome healing. Telomere dysfunction-induced foci (TIFs) formation, identified by co-localization of telomeres and γ-H2AX, supported telomere associated DNA damage response in BRCA2 heterozygous cell lines. TIFs were found in interphase nuclei, at chromosome ends, ITSs and ECTR DNA. In conclusion, our results suggest that BRCA2 has an important role in telomere stabilization by repressing CEFs through telomere capping and the prevention of telomere loss by replication stabilization.     

Telomere biology: integrating chromosomal end protection with DNA damage response

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

Hyper-phosphorylated retinoblastoma protein suppresses telomere elongation

Immortalized cell lines maintain telomeres by the expression of telomerase or by a mechanism designated alternative lengthening of telomeres (ALT). Although DNA polymerase alpha (pol-alpha) is reported to be required for telomere maintenance, the critical role of pol-alpha in telomere maintenance has not been firmly determined. We examined the role of retinoblastoma protein (pRb) and pol-alpha in the regulation of telomere length, using telomere-fiber FISH. Telomere length varied dependent on the intracellular abundance of pol-alpha or pRb in HeLa cells. A proportion of hyper-phosphorylated pRb (ppRb) molecules localized to sites of telomeric DNA replication in HeLa cells. Pol-alpha might thus contribute to telomere maintenance, and might be regulated by ppRb.     

Hyper-Phosphorylated Retinoblastoma Protein Suppresses Telomere Elongation

Immortalized cell lines maintain telomeres by the expression of telomerase or by a mechanism designated alternative lengthening of telomeres (ALT). Although DNA polymerase α (pol-α) is reported to be required for telomere maintenance, the critical role of pol-α in telomere maintenance has not been firmly determined. We examined the role of retinoblastoma protein (pRb) and pol-α in the regulation of telomere length, using telomere-fiber FISH. Telomere length varied dependent on the intracellular abundance of pol-α or pRb in HeLa cells. A proportion of hyper-phosphorylated pRb (ppRb) molecules localized to sites of telomeric DNA replication in HeLa cells. Pol-α might thus contribute to telomere maintenance, and might be regulated by ppRb.     

Nucleostemin prevents telomere damage by promoting PML-IV recruitment to SUMOylated TRF1

Continuously dividing cells must be protected from telomeric and nontelomeric DNA damage in order to maintain their proliferative potential. Here, we report a novel telomere-protecting mechanism regulated by nucleostemin (NS). NS depletion increased the number of telomere damage foci in both telomerase-active (TA(+)) and alternative lengthening of telomere (ALT) cells and decreased the percentage of damaged telomeres associated with ALT-associated PML bodies (APB) and the number of APB in ALT cells. Mechanistically, NS could promote the recruitment of PML-IV to SUMOylated TRF1 in TA(+) and ALT cells. This event was stimulated by DNA damage. Supporting the importance of NS and PML-IV in telomere protection, we demonstrate that loss of NS or PML-IV increased the frequency of telomere damage and aberration, reduced telomeric length, and perturbed the TRF2(ΔBΔM)-induced telomeric recruitment of RAD51. Conversely, overexpression of either NS or PML-IV protected ALT and TA(+) cells from telomere damage. This work reveals a novel mechanism in telomere protection.     

Telomere shortening by cisplatin in yeast nucleotide excision repair mutant

Telomeres are unique DNA tandem repeats that form the ends of eukaryotic chromosomes to protect the chromosomes from degradation and illegitimate recombination. In yeast, loss of telomere may be compensated for through the acquisition of new telomere by RAD52-mediated or RAD52-independent recombinational repair. In this report, the effects of cis-dichlorodiammine-platinum (II) (cisplatin) on telomere length and the role of nucleotide excision repair in telomere maintenance were examined in the yeast Saccharomyces cerevisiae. We showed that the SSL2 (RAD25) DNA repair yeast mutant exhibited a gradual shortening of the telomere in the presence of cisplatin. Further telomere shortening was prevented upon the withdrawal of cisplatin. Complementation of the mutant with the wild-type SSL2 (RAD25) gene abolished the cisplatin-induced telomere degradation. These results suggest that telomeres are susceptible to cisplatin-induced intrastrand crosslinks and that Ssl2 (Rad25) or the nucleotide excision repair pathway may play a critical role in the repair and the maintenance of telomere integrity.     

Telomere instability in a human tumor cell line expressing NBS1 with mutations at sites phosphorylated by ATM

Nijmegen breakage syndrome (NBS) is an autosomal genetic disease demonstrating a variety of phenotypic abnormalities, including premature aging, increased cancer incidence, chromosome instability, and sensitivity to ionizing radiation. The gene involved in NBS, NBS1, is part of the MRE11/RAD50/NBS1 (MRN) complex that also includes MRE11 and RAD50, which is involved in DNA repair and cell cycle regulation in response to DNA damage. The MRN complex is also involved in telomere maintenance, as demonstrated by the shortened telomeres in NBS primary human fibroblasts and the association of NBS1 with the telomere-binding protein TRF2. To learn more about how a deficiency in telomere maintenance might contribute to chromosome instability in NBS, we have investigated the stability of telomeres in two telomerase-positive human tumor cell clones, BNmt-On and BNmt-Off, expressing an inducible NBS1(S278A/S343A) gene containing mutations at serines 278 and 343 phosphorylated by ATM. The results demonstrate an increased rate of telomere loss in both clones following expression of NBS1(S278A/S343A). The absence of detectable changes in average telomere length suggests that NBS1-associated telomere loss results from stochastic events involving complete telomere loss or loss of telomere capping function. The recombination events associated with telomere loss were found to be similar to those shown previously to result in breakage/fusion/bridge cycles, suggesting that telomere loss can contribute to chromosome instability in NBS1-deficient cells. Telomere loss showed no correlation with radiosensitivity or radioresistant DNA synthesis, demonstrating that NBS1(S278A/S343A) promotes telomere loss through a separate pathway from these other phenotypes associated with NBS.