Telomere Recombination Research Articles

Abstract 2993: Homologous recombination between telomeres is present in ALT and telomerase-positive immortal cells

Abstract Telomere homeostasis is a hallmark of malignancy and occurs via two different mechanisms. The first mechanism is telomere length control via telomerase activity, the second mechanism is characterized by the lack of any detectable telomerase activity and is known as the alternative lengthening of telomeres (ALT) pathway. There is good evidence that homologous recombination between telomeric DNA is responsible for telomere elongation in these cells and therefore enables infinite growth. Telomeres resemble fragile DNA sites, and their repetitive sequence poses a challenge for the DNA replication machinery. Replication fork stallments have been shown to occur in mammalian telomeres, giving rise to DNA lesions and genetic instability. DNA replication can resume via a homologous recombination process, which could potentially cause telomere elongation. Although telomerase-positive human cells are believed to not recombine their telomeres, we were challenged by the question whether telomeric recombination is a rare event that is missed with an insensitive detection technique. In order to address this issue we designed a novel, sensitive and specific recombination reporter system that demonstrates recombination events that have become detectable by gaining antibiotic resistance. For this purpose cells were first tagged with a subtelomeric and subsequently with a telomeric reporter cassette, after which individual clones were obtained. Once telomeres recombined, both cassettes co-localize on the same telomere. Alternative splicing of intervening sequences between two portions of a resistance gene results in antibiotic resistance, hence allowing the specific and sensitive detection of a telomeric recombination event occurring upstream of the telomeric tag. With this reporter system we show that telomeric recombination is present in ALT and telomerase-positive human fibroblasts. The frequency of recombination does not differ between these two cell lines. DNA damage caused by UV irradiation, change in heterochromatin formation from HDAC inhibition or by demethylating DNA with azacitidine did not change the frequency of homologous recombination in either ALT or telomerase-positive clones. In contrast, treatment of cells with aphidicolin, which renders telomeres fragile, significantly increased the frequency of telomeric recombination in ALT, but not in telomerase-positive cells. The introduction of telomerase into these ALT cell clones abolished the effect of the aphidicolin-induced homologous recombination. Our data show that in telomerase-positive cells and ALT cells homologous recombination between telomeres occurs with nearly the same frequency, but that the increase in telomeric recombination via fragile telomeres is dependent on the absence of telomerase activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2993. doi:10.1158/1538-7445.AM2011-2993

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 down-regulation 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.

The Smc5/6 complex and the difficulties cutting the ties of twin sisters

The Smc5/6 complex and the difficulties cutting the ties of twin sisters

Effects of BRCA2 deficiency on telomere recombination in non-ALT and ALT cells

BackgroundRecent studies suggest that BRCA2 affects telomere maintenance. Interestingly, anti cancer treatments that involve BRCA2 and telomerase individually are currently being explored. In the light of the above recent studies their combinatorial targeting may be justified in the development of future treatments. In order to investigate effects of BRCA2 that can be explored for this combinatorial targeting we focused on the analysis of recombination rates at telomeres by monitoring T-SCEs (Telomere Sister Chromatid Exchanges).ResultsWe observed a significant increase in T-SCE frequencies in four BRCA2 defective human cell lines thus suggesting that BRCA2 suppresses recombination at telomeres. To test this hypothesis further we analyzed T-SCE frequencies in a set of Chinese hamster cell lines with or without functional BRCA2. Our results indicate that introduction of functional BRCA2 normalizes frequencies of T-SCEs thus supporting the notion that BRCA2 suppresses recombination at telomeres. Given that ALT (Alternative Lengthening of Telomeres) positive cells maintain telomeres by recombination we investigated the effect of BRCA2 depletion in these cells. Our results show that this depletion causes a dramatic reduction in T-SCE frequencies in ALT positive cells, but not in non-ALT cells.ConclusionBRCA2 suppresses recombination at telomeres in cells that maintain them by conventional mechanisms. Furthermore, BRCA2 depletion in ALT positive cells reduces high levels of T-SCEs normally found in these cells. Our results could be potentially important for refining telomerase-based anti-cancer therapies.

Pilocytic Astrocytomas Have Telomere-Associated Promyelocytic Leukemia Bodies without Alternatively Lengthened Telomeres

Telomere maintenance by either telomerase activity or the recombination-mediated alternative lengthening of telomeres (ALT) mechanism is a hallmark of cancer. Tumors that use ALT as their telomere maintenance mechanism are characterized by long telomeres of great heterogeneity in length and by specific nuclear structures of co-localized promyelocytic leukemia protein and telomere DNA, called ALT-associated promyelocytic leukemia bodies (APBs). Recent advances have revealed a direct role for APBs in telomere recombination in ALT-positive cells. In this study, we investigated the possibility that APBs could occur before the long 'alternatively' lengthened telomeres arise, particularly in low-grade tumors. We measured APBs, telomere length, and telomerase activity in 64 astrocytomas inclusive of grade 1-4 tumors. Almost all grade 1-3 tumors (93%) were APB-positive using published criteria. Grade 2-3 APB-positive tumors also had long telomeres and were confirmed as ALT positive. However, grade 1 tumors lacked long telomeres and were therefore classified as ALT negative, but positive for telomere-associated promyelocytic leukemia bodies (TPB). This is the first report of a TPB-positive but ALT-negative tumor, and suggests that low-grade tumors have the foundation for recombinational telomere repair, as in ALT. Further work is warranted to characterize the TPB-positive phenotype in other early malignancies, as well as to determine whether TPBs predispose to telomere maintenance by ALT.

Telomeric recombination induced by dysfunctional telomeres

Telomere maintenance is essential for cellular immortality, and most cancer cells maintain their telomeres through the enzyme telomerase. Telomeres and telomerase represent promising anticancer targets. However, 15% of cancer cells maintain their telomeres through alternative recombination-based mechanisms, and previous analyses showed that recombination-based telomere maintenance can be activated after telomerase inhibition. We determined whether telomeric recombination can also be promoted by telomere dysfunction. We report for the first time that telomeric recombination can be induced in human telomerase-positive cancer cells with dysfunctional telomeres.

Tobacco GTBP1, a Homolog of Human Heterogeneous Nuclear Ribonucleoprotein, Protects Telomeres from Aberrant Homologous Recombination

Telomeres are nucleoprotein complexes essential for the integrity of eukaryotic chromosomes. Cellular roles of single-stranded telomeric DNA binding proteins have been extensively described in yeast and animals, but our knowledge about plant single-strand telomeric factors is rudimentary. Here, we investigated Nicotiana tabacum G-strand-specific single-stranded telomere binding proteins (GTBPs), homologs of a human heterogeneous nuclear ribonucleoprotein. GTBPs bound specifically to the plant single-stranded (TTTAGGG)(4) telomeric repeat element in vitro and were associated with telomeric sequences in tobacco BY-2 suspension cells. Transgenic plants (35S:RNAi-GTBP1), in which GTBP1 was suppressed, exhibited severe developmental anomalies. In addition, the chromosomes of 35S:RNAi-GTBP1 cells displayed elongated telomeres, frequent formation of extrachromosomal telomeric circles, and numerous abnormal anaphase bridges, indicating that GTBP1 knockdown tobacco plants experienced genome instability. GTBP1 inhibited strand invasion, an initial step in interchromosomal homologous recombination. We propose that GTBP1 plays a critical role in telomere structure and function by preventing aberrant interchromosomal telomeric homologous recombination in tobacco.

Hyper telomere recombination accelerates replicative senescence and may promote premature aging

Werner syndrome and Bloom syndrome result from defects in the RecQ helicases Werner (WRN) and Bloom (BLM), respectively, and display premature aging phenotypes. Similarly, XFE progeroid syndrome results from defects in the ERCC1-XPF DNA repair endonuclease. To gain insight into the origin of cellular senescence and human aging, we analyzed the dependence of sister chromatid exchange (SCE) frequencies on location [i.e., genomic (G-SCE) vs. telomeric (T-SCE) DNA] in primary human fibroblasts deficient in WRN, BLM, or ERCC1-XPF. Consistent with our other studies, we found evidence of elevated T-SCE in telomerase-negative but not telomerase-positive backgrounds. In telomerase-negative WRN-deficient cells, T-SCE-but not G-SCE-frequencies were significantly increased compared with controls. In contrast, SCE frequencies were significantly elevated in BLM-deficient cells irrespective of genome location. In ERCC1-XPF-deficient cells, neither T- nor G-SCE frequencies differed from controls. A theoretical model was developed that allowed an in silico investigation into the cellular consequences of increased T-SCE frequency. The model predicts that in cells with increased T-SCE, the onset of replicative senescence is dramatically accelerated even though the average rate of telomere loss has not changed. Premature cellular senescence may act as a powerful tumor-suppressor mechanism in telomerase-deficient cells with mutations that cause T-SCE levels to rise. Furthermore, T-SCE-driven premature cellular senescence may be a factor contributing to accelerated aging in Werner and Bloom syndromes, but not XFE progeroid syndrome.

DNA G‐quadruplex: structure, function and human disease

The role of non-B DNA in the function and stability of genomes has become generally appreciated in recent years. It is now evident that DNA also encodes for spatial structures that are involved in gene regulation, replication and recombination. There are several types of secondary structures in non-B DNA. Among these noncanonical structures are hairpins and cruciforms, intramolecular triplexes or H-DNA, left-handed Z-DNA and guanine-rich repeats which have the capacity to adopt G-quadruplex (G4-DNA); the latter structure is also referred to in the literature as G-tetraplex. G4-DNA consists of a hydrogen-bonded self-assembly of four guanine bases, paired by Hoogsteen bonding, which forms planar arrangements, the G-quartets. Charge coordination by monovalent cations stabilizes G-quartet stacking, resulting in intramolecular or intermolecular association of four DNA strands in a parallel or antiparallel orientation. Genomes contain a high number of G-rich sequences that could form G4-DNA and this structure may serve important regulatory and structural functions; in addition, it can be the source of genomic instability which may lead to cancer, aging and human genetic diseases. In silico analysis using different computational methods, as well as laboratory experiments, indicates that many G-rich regions of chromosomes – rDNA, single-copy genes, recombination sites like those involved in immunoglobulin class-switching and repetitive sequences including satellite and telomeric DNA sequences – have the potential to form G4-DNA structures. Interestingly, RNA is also capable of forming G4 structures even more stable than those of DNA. In this sense, it is tempting to speculate that RNA G-quadruplexes could play a role in translation regulation, in a manner analogous to G4-DNA and transcription. In the human genome, the number of sites with the potential to form G-quadruplex is surprisingly high. Some of these sites are within genes and are potential threats to genome stability because they can alter the DNA architecture and interfere with normal DNA processes, such as replication and transcription. Although G4-DNA has been rigorously studied in vitro, whether this structure actually forms in vivo and what its cellular roles might be, remains unclear. However, recent advances have made a persuasive case for the existence of G4-DNA in living cells and its participation in the regulation of processes as varied as telomere maintenance, transcription, recombination and ribosome biogenesis. This minireview series deals with the structural and functional characteristics of G4-DNA, as well as its implication in human disease. In the first minireview, Huppert gives an introduction to the structure and genomic distribution of putative G4-DNA-forming sequences and creates a theoretical background for the other two articles in the series. He discusses the computational approaches used to predict them on a genomic scale, and how the information derived can be combined with experiments to understand their biological functions. In the second minireview, Brooks, Kendrick & Hurley examine the diversity of G4 structures and i-motifs in promoter elements and attempt to categorize the different types of arrangements in which they are found. The third minireview, by Wu & Brosh, provocatively summarizes the presumed in vivo role of G4-nucleic acid structures and discusses the consequences of human genetic defects affecting the different enzymes that manipulate G-quadruplex structures. They present all the circumstantial evidence suggesting that G-quadruplex nucleic acid structures, which have been demonstrated unambiguously to be formed on specific sequences in naked DNA in vitro, do exist in vivo in the chromatin-embedded genomic DNA. Finally, they discuss the connections of G-quadruplexes to human genetic diseases and cancer.

Mammalian Rap1 controls telomere function and gene expression through binding to telomeric and extratelomeric sites.

Rap1 is a component of the shelterin complex at mammalian telomeres, but its in vivo role in telomere biology has remained largely unknown to date. Here we show that Rap1 deficiency is dispensable for telomere capping but leads to increased telomere recombination and fragility. We generated cells and mice deleted for Rap1; mice with Rap1 deletion in stratified epithelia were viable but had shorter telomeres and developed skin hyperpigmentation in adulthood. By performing chromatin immunoprecipitation coupled with ultrahigh-throughput sequencing, we found that Rap1 binds to both telomeres and to extratelomeric sites through the (TTAGGG)(2) consensus motif. Extratelomeric Rap1-binding sites were enriched at subtelomeric regions, in agreement with preferential deregulation of subtelomeric genes in Rap1-deficient cells. More than 70% of extratelomeric Rap1-binding sites were in the vicinity of genes, and 31% of the genes deregulated in Rap1-null cells contained Rap1-binding sites, suggesting a role for Rap1 in transcriptional control. These findings place a telomere protein at the interface between telomere function and transcriptional regulation.

An mre11 Mutation That Promotes Telomere Recombination and an Efficient Bypass of Senescence

Preventing the formation of dysfunctional telomeres is essential for genomic stability. In most organisms, the ribo-nucleoprotein reverse transcriptase telomerase is responsible for telomere GT-strand elongation. However, in telomerase-negative cells, low-frequency recombination mechanisms can avert lethality by elongating critically short telomeres. This study focuses on the involvement of the budding yeast Mre11 in telomere recombination and homeostasis. We have identified a novel allele of MRE11, mre11-A470T, that, in telomerase-positive cells, confers a semidominant decrease in telomere size and a recessive defect in telomere healing. In addition, mutant cells lack normal telomere size homeostasis. Telomerase-negative mre11-A470T cells display a Rad51-dependent bypass of replicative senescence via induction of a highly efficient type I-related recombination pathway termed type IA. The type IA pathway involves an amplification of subtelomeric Y' elements, coupled with elongated and more heterogeneous telomere tracts relative to the short telomere size of type I survivors. The data have led us to propose the involvement of break-induced replication in telomere expansion. The differing phenotypes elicited by the mre11-A470T mutants in telomerase-positive and telomerase-negative cells have also led us to speculate that the telomere end structure may be modified differentially in mre11-A470T cells, directing the telomere into specific pathways.

Abstract 4888: The effect of chromatin structure in regulation of telomere length in telomerase negative ALT cells

Abstract Telomeres are DNA-protein complexes at the ends of linear eukaryotic chromosomes and composed of tandem repeats of a TTAGGG sequence ending in a 3′ single-stranded overhang, or the G-strand overhang. And telomeres confer chromosomal stability protecting chromosome ends. Most immortalized human cell lines maintain and extend their telomere by activating telomerase. A small number of cells maintain and extend their telomere lengths without telomerase by homologous recombination of telomeres and this mechanism is called as alternative lengthening of telomere (ALT). There are many factors involved in telomere regulation. Recently, it has been known that chromatin structure is closely related to regulation of telomere length. Maintaining telomere length is important process in tumorigenesis and it has been suggested that telomeres are important target for cancer therapy. However, in ALT cells the effect of chromatin structure on regulation of telomere length is not understood. In this study, we induced change of chromatin structure by treating Trichostatin A (TSA), Histone deacetylase inhibitor, in ALT cells. Then, we measured APBs (ALT associated PML bodies) patterns, T-SCE (Telomere Sister Chromatid Exchange) frequency and telomere lengths. APBs patterns were analyzed by FISH (Fluorescence in situ hybridization) combined IF (Immunofluorescence staining). We compared the number of APBs in ALT cells with or without TSA. In GM 847 (ALT cell), APBs positive cells were found to be more frequent in GM 847 with TSA (63/100) compared to GM 847 without TSA (30/100) (p<0.001). A similar result was found in other ALT cell line, WI38-VA13. APB positive cells were more frequent in WI38-VA13 with TSA (38/100) compare to WI38-VA13 without TSA (18/100), (p<0.001). T-SCE frequency was determined by CO-FISH (Chromosome Orientation-FISH). Increse of T-SCE frequency was found in GM 847 with TSA (262/1026, 25.5%) compared to GM 847 without TSA (215/1026, 20.9%), (p<0.001). Telomere length was determined by telomere-FISH. Decrease of telomere length was found in GM 847 with TSA (telomere intensity mean 25.11%) compared to GM 847 without TSA (telomere intensity mean 29.8%), (p<0.001). This study demonstrated a close association between chromatin structure and telomere recombination in ALT cells and this result might provide insight into the mechanism of regulation of telomere length in telomerase-negative cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4888.

Loss of Rap1 Induces Telomere Recombination in the Absence of NHEJ or a DNA Damage Signal

Shelterin is an essential telomeric protein complex that prevents DNA damage signaling and DNA repair at mammalian chromosome ends. Here we report on the role of the TRF2-interacting factor Rap1, a conserved shelterin subunit of unknown function. We removed Rap1 from mouse telomeres either through gene deletion or by replacing TRF2 with a mutant that does not bind Rap1. Rap1 was dispensable for the essential functions of TRF2--repression of ATM kinase signaling and nonhomologous end joining (NHEJ)--and mice lacking telomeric Rap1 were viable and fertile. However, Rap1 was critical for the repression of homology-directed repair (HDR), which can alter telomere length. The data reveal that HDR at telomeres can take place in the absence of DNA damage foci and underscore the functional compartmentalization within shelterin.

Zscan4 regulates telomere elongation and genomic stability in ES cells

Exceptional genomic stability is one of the hallmarks of mouse embryonic stem (ES) cells. However, the genes contributing to this stability remain obscure. We previously identified Zscan4 as a specific marker for 2-cell embryo and ES cells. Here we show that Zscan4 is involved in telomere maintenance and long-term-genomic stability in ES cells. Only 5% of ES cells express Zscan4 at a given time, but nearly all ES cells activate Zscan4 at least once within nine passages. The transient Zscan4-positive state is associated with rapid telomere extension by telomere recombination and upregulation of meiosis-specific homologous recombination genes, which encode proteins that are colocalized with ZSCAN4 on telomeres. Furthermore, Zscan4 knockdown shortens telomeres, increases karyotype abnormalities and spontaneous sister chromatid exchange, and slows down cell proliferation until reaching crisis by eight passages. Together, our data reveal a unique mode of genome maintenance in ES cells.

Sua5p is required for telomere recombination in Saccharomyces cerevisiae

Sua5p is required for telomere recombination in Saccharomyces cerevisiae

ATR suppresses telomere fragility and recombination but is dispensable for elongation of short telomeres by telomerase

Telomere shortening caused by incomplete DNA replication is balanced by telomerase-mediated telomere extension, with evidence indicating that the shortest telomeres are preferred substrates in primary cells. Critically short telomeres are detected by the cellular DNA damage response (DDR) system. In budding yeast, the important DDR kinase Tel1 (homologue of ATM [ataxia telangiectasia mutated]) is vital for telomerase recruitment to short telomeres, but mammalian ATM is dispensable for this function. We asked whether closely related ATR (ATM and Rad3 related) kinase, which is important for preventing replicative stress and chromosomal breakage at common fragile sites, might instead fulfill this role. The newly created ATR-deficient Seckel mouse strain was used to examine the function of ATR in telomerase recruitment and telomere function. Telomeres were recently found to resemble fragile sites, and we show in this study that ATR has an important role in the suppression of telomere fragility and recombination. We also find that wild-type ATR levels are important to protect short telomeres from chromosomal fusions but do not appear essential for telomerase recruitment to short telomeres in primary mouse embryonic fibroblasts from the ATR-deficient Seckel mouse model. These results reveal a previously unnoticed role for mammalian ATR in telomere protection and stability.

Roles of Heterochromatin and Telomere Proteins in Regulation of Fission Yeast Telomere Recombination and Telomerase Recruitment

When the telomerase catalytic subunit (Trt1/TERT) is deleted, a majority of fission yeast cells survives by circularizing chromosomes. Alternatively, a small minority survives by maintaining telomeric repeats through recombination among telomeres. The recombination-based telomere maintenance in trt1Delta cells is inhibited by the telomere protein Taz1. In addition, catalytically inactive full-length Trt1 (Trt1-CI) and truncated Trt1 lacking the T-motif and reverse transcriptase (RT) domain (Trt1-DeltaT/RT) can strongly inhibit recombination-based survival. Here, we investigated the effects of deleting the heterochromatin proteins Swi6 (HP1 ortholog) and Clr4 (Suv39 family of histone methyltransferases) and the telomere capping complex subunits Poz1 and Ccq1 on Taz1- and Trt1-dependent telomere recombination inhibition. The ability of Taz1 to inhibit telomere recombination did not require Swi6, Clr4, Poz1, or Ccq1. Although Swi6, Clr4, and Poz1 were dispensable for the inhibition of telomere recombination by Trt1-CI, Ccq1 was required for efficient telomere recruitment of Trt1 and Trt1-CI-dependent inhibition of telomere recombination. We also found that Swi6, Clr4, Ccq1, the checkpoint kinase Rad3 (ATR ortholog), and the telomerase regulatory subunit Est1 are all required for Trt1-DeltaT/RT to inhibit telomere recombination. However, because loss of Swi6, Clr4, Rad3, Ccq1, or Est1 did not significantly alter the recruitment efficiency of Trt1-DeltaT/RT to telomeres, these factors are likely to enhance the ability of Trt1-DeltaT/RT to inhibit recombination-based survival by contributing to the negative regulation of telomere recombination.

Distinct Activities of Exonuclease 1 and Flap Endonuclease 1 at Telomeric G4 DNA

BackgroundExonuclease 1 (EXO1) and Flap endonuclease 1 (FEN1) are members of the RAD2 family of structure-specific nucleases. Genetic analysis has identified roles for EXO1 and FEN1 in replication, recombination, DNA repair and maintenance of telomeres. Telomeres are composed of G-rich repeats that readily form G4 DNA. We recently showed that human EXO1 and FEN1 exhibit distinct activities on G4 DNA substrates representative of intermediates in immunoglobulin class switch recombination.Methodology/Principal FindingsWe have now compared activities of these enzymes on telomeric substrates bearing G4 DNA, identifying non-overlapping functions that provide mechanistic insight into the distinct telomeric phenotypes caused by their deficiencies. We show that hFEN1 but not hEXO1 cleaves substrates bearing telomeric G4 DNA 5′-flaps, consistent with the requirement for FEN1 in telomeric lagging strand replication. Both hEXO1 and hFEN1 are active on substrates bearing telomeric G4 DNA tails, resembling uncapped telomeres. Notably, hEXO1 but not hFEN1 is active on transcribed telomeric G-loops.Conclusion/SignificanceOur results suggest that EXO1 may act at transcription-induced telomeric structures to promote telomere recombination while FEN1 has a dominant role in lagging strand replication at telomeres. Both enzymes can create ssDNA at uncapped telomere ends thereby contributing to recombination.

FANCC suppresses short telomere-initiated telomere sister chromatid exchange

Telomere shortening has been linked to rare human disorders that present with bone marrow failure including Fanconi anemia (FA). FANCC is one of the most commonly mutated FA genes in FA patients and the FANCC subtype tends to have a relatively early onset of bone marrow failure and hematologic malignancies. Here, we studied the role of Fancc in telomere length regulation in mice. Deletion of Fancc (Fancc(-/-)) did not affect telomerase activity, telomere length or telomeric end-capping in a mouse strain possessing intrinsically long telomeres. However, ablation of Fancc did exacerbate telomere attrition when murine bone marrow cells experienced high cell turnover after serial transplantation. When Fancc(-/-) mice were crossed into a telomerase reverse transcriptase heterozygous or null background (Tert(+/-) or Tert(-/-)) with short telomeres, Fancc deficiency led to an increase in the incidence of telomere sister chromatid exchange. In contrast, these phenotypes were not observed in Tert mutant mice with long telomeres. Our data indicate that Fancc deficiency accelerates telomere shortening during high turnover of hematopoietic cells and promotes telomere recombination initiated by short telomeres.

Spontaneous occurrence of telomeric DNA damage response in the absence of chromosome fusions

Telomere dysfunction is typically studied under conditions in which a component of the six-subunit shelterin complex that protects chromosome ends is disrupted. The nature of spontaneous telomere dysfunction is less well understood. Here we report that immortalized human cell lines lacking wild-type p53 function spontaneously show many telomeres with a DNA damage response (DDR), commonly affecting only one sister chromatid and not associated with increased chromosome end-joining. DDR(+) telomeres represent an intermediate configuration between the fully capped and uncapped (fusogenic) states. In telomerase activity-positive (TA(+)) cells, DDR is associated with low TA and short telomeres. In cells using the alternative lengthening of telomeres mechanism (ALT(+)), DDR is partly independent of telomere length, mostly affects leading strand-replicated telomeres, and can be partly suppressed by TRF2 overexpression. In ALT(+) (but not TA(+)) cells, DDR(+) telomeres preferentially associate with large foci of extrachromosomal telomeric DNA and recombination proteins. DDR(+) telomeres therefore arise through different mechanisms in TA(+) and ALT(+) cells and have different consequences.