Telomere Binding Research Articles

TERF2 (telomeric repeat binding factor 2)

Review on TERF2 (telomeric repeat binding factor 2), with data on DNA, on the protein encoded, and where the gene is implicated.

Abstract A63: Rsf-1, a chromatin remodeling protein, interacts with shelterin protein hRap1 and induces telomere shortening

Abstract Background: Rsf-1, a chromatin remodeling factor that forms an ISWI chromatin-remodeling complex with SNF2H, has been implicated in the oncogenesis of a variety of human cancers, yet its functions in promoting tumor development remains unclear. In this study, we found Rsf-1 interacted with hRap1, a telomere binding protein involved in telomere length regulation. We aimed at characterizing the interaction between Rsf-1 and hRap1 and exploring the effect of Rsf-1 overexpression on telomere length in ovarian cancer cells. Methods: Protein microarray and co-immunoprecipitation were employed to screen and validate Rsf-1 interaction proteins. Deletion mutants of both Rsf-1 and hRap1 were constructed to map the binding domains responsible for their interaction. To determine whether RSF complex was enriched in the telomeric regions, we performed chromatin immunoprecipitation (ChIP) -qPCR in two ovarian cancer cell lines (OVCAR3 with high RSF-1 gene amplification and Rsf-1 inducible SKOV3) with antibodies against Rsf-1 and SNF2H. A ChIP-sequencing dataset performed with Rsf-1 and SNF2H antibodies using OVCAR3 cells was also examined for enrichment of telomeric sequences. To investigate the effect of Rsf-1 overexpression on telomere length, we evaluated by qPCR the telomere length in two Rsf-1 inducible cell lines (SKOV3 and BRK). Results: We identified hRap1, a telomere binding protein, as an interaction partner of Rsf-1 by protein microarray and co-immunoprecipitation. Protein interaction domain mapping revealed that C-terminal domain of hRAP1 and PHD domains and arginine-rich region of RSF1 were critical for this interaction. Both ChIP-qPCR and ChIP-sequencing demonstrated that Rsf-1 and SNF2H were enriched in the telomeres, suggesting that RSF complex was recruited by hRAP1 to the telomeres. Induction of RSF1 expression resulted in shortened telomere length in two different cell lines. The results suggest that RSF complex may be involved in the telomere length regulation mediated by hRAP1. Conclusions: Rsf-1 interacted with hRap1 through the PHD domain and the arginine-rich region. RSF complex bound telomeres, probably through the interaction with hRap1. Rsf-1 overexpression led to telomere length shortening, which may contribute to genomic instability in cancer cells. Citation Format: Ren-Chin Wu, Bin Guan, Yusuke Kobayashi, Tian-Li Wang, Ie-Ming Shih. Rsf-1, a chromatin remodeling protein, interacts with shelterin protein hRap1 and induces telomere shortening. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A63.

RAP1: Protector of Telomeres, Defender against Obesity

Telomere dysfunction has previously been linked to metabolic disorders. In this issue of Cell Reports, Martínez etal. (2013) and Yeung etal. (2013) now extend this link, demonstrating that deletion of the telomere binding protein RAP1 leads to obesity and insulin resistance.

Mechanism of DNA damage responses induced by exposure to an oligonucleotide homologous to the telomere overhang in melanoma

T-oligo, an 11-base oligonucleotide homologous to the 3'-telomeric overhang, is a novel, potent therapeutic modality in melanoma and multiple other tumor types. T-oligo is proposed to function in a manner similar to experimental disruption of the telomere overhang and induces DNA damage responses including apoptosis, differentiation and senescence. However, important components involved in T-oligo induced responses are not defined, particularly the role of p53, TRF1 and TRF2 in mediating the T-oligo induced responses. In MU, PM-WK, and MM-MC melanoma cells, exposure to T-oligo upregulates p53 expression and phosphorylation, resulting in cellular differentiation and activation of a caspase-mediated apoptotic cascade. However, siRNA-mediated knockdown of p53 completely blocks T-oligo induced differentiation and significantly decreases apoptosis, suggesting that p53 is an important mediator of T-oligo induced responses. In addition, we characterized the roles of telomere binding proteins, TRF1, TRF2, and tankyrase-1, in T-oligo induced damage responses. We demonstrate that tankyrase-1 activity is required for initiation of T-oligo induced damage responses including p53 phosphorylation and reduction of cellular proliferation. These results highlight TRF1, TRF2, tankyrase-1 and p53 as important elements in T-oligo mediated responses and suggest new avenues for research into T-oligo's mechanism of action.

Structural and energetic properties of canonical and oxidized telomeric complexes studied by molecular dynamics simulations

The structural and energetic properties of native and oxidized telomeric complexes were defined by means of molecular dynamic (MD) simulations. As a starting point, the experimental conformation of B-DNA d(GpTpTpApGpGpGpTpTpApGpGpG) oligomer bound to human protein telomeric repeat binding factor 1 (TRF1) was used. The influence on the stability of the telomeric complex of the presence of 8-oxoguanine (8oxoG) in the central telomeric triad (CTT) was estimated based on trajectories collected during 130 ns MD runs. The data obtained indicate that the system analyzed is highly sensitive to the presence of oxidative damage in the CTT of the B-DNA telomeric sequence. The most important changes were observed in the immediate vicinity of the 8-oxoguanine molecule. The significantly higher mobility of arginine 425 interacting directly with the oxidized guanine molecule has a large influence on the structural, dynamic and energetic properties of neighboring amino acids. Local changes observed for individual hydrogen bonded interactions localized in the major groove of B-DNA also have significant impact on the properties of hydrophobic clusters, which are the second type of force responsible for stability of the studied bio-system. All the changes reported in detail here unambiguously indicate a significant decrease in telomer binding affinity after oxidation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00894-013-1859-z) contains supplementary material, which is available to authorized users.

Age-related telomere uncapping is associated with cellular senescence and inflammation independent of telomere shortening in human arteries

Arterial telomere dysfunction may contribute to chronic arterial inflammation by inducing cellular senescence and subsequent senescence-associated inflammation. Although telomere shortening has been associated with arterial aging in humans, age-related telomere uncapping has not been described in non-cultured human tissues and may have substantial prognostic value. In skeletal muscle feed arteries from 104 younger, middle-aged, and older adults, we assessed the potential role of age-related telomere uncapping in arterial inflammation. Telomere uncapping, measured by p-histone γ-H2A.X (ser139) localized to telomeres (chromatin immunoprecipitation; ChIP), and telomeric repeat binding factor 2 bound to telomeres (ChIP) was greater in arteries from older adults compared with those from younger adults. There was greater tumor suppressor protein p53 (P53)/cyclin-dependent kinase inhibitor 1A (P21)-induced senescence, measured by P53 bound to P21 gene promoter (ChIP), and greater expression of P21, interleukin 8, and monocyte chemotactic protein 1 mRNA (RT-PCR) in arteries from older adults compared with younger adults. Telomere uncapping was a highly influential covariate for the age-group difference in P53/P21-induced senescence. Despite progressive age-related telomere shortening in human arteries, mean telomere length was not associated with telomere uncapping or P53/P21-induced senescence. Collectively, these findings demonstrate that advancing age is associated with greater telomere uncapping in arteries, which is linked to P53/P21-induced senescence independent of telomere shortening.

Cancer chromosomes going to POT1

Alterations in the single-stranded telomere-binding protein POT1 have recently been identified in chronic lymphocytic leukemia. This discovery provides novel insights into how genomic instability induced by dysfunctional telomeres contributes to tumorigenesis.

Abstract 1746: Coordination of overexpressed NIMA-related kinase 2 and TRF1 results in mitotic abnormalities in breast cancer cells.

Abstract NIMA-related kinase 2 (NEK2), a serine-threonine protein kinase, plays a role in features of mitotic progression including timing of mitotic entry, chromatin condensation, spindle organization and cytokinesis. Kinase death NEK2 mutant expression or NEK2 depleted cells lead to failure of centrosome separation while NEK2 overexpression results in premature centrosome separation. In addition, it has been revealed that telomeric repeat binding factor 1 (TRF1) interacts directly with NEK2. TRF1 not only regulates telomere length, but is also associated with cell cycle regulation. However, the interactions and correlations between NEK2 and TRF1 are far from clear. Here, we show that mitotic aberrations through NEK2 overexpression are likely to require TRF1. Our results demonstrate that NEK2 directly binds and phosphorylates TRF1 in vitro and in vivo through multiple sites on TRF1. NEK2 overexpression in breast cancer cells, MCF 7 and MDA-MB-231, results in increased numbers of centrosomes and multinucleated cells, which leads to cytokinetic failure and aneuploidization. Additionally, TRF1 depletion by siRNA prevents the occurrence of unaligned chromosomes by NEK2 overexpression during metaphase. Concurrent Nek2 overexpression and TRF1 depleted cells demonstrated ≤ 2 centrosomes per cell, similar to mock plasmid and negative control siRNA transfected cells. Therefore, we propose that TRF1 is required for overexpressed NEK2 to trigger abnormal mitosis and chromosomal instability. Citation Format: Jaehyung Lee. Coordination of overexpressed NIMA-related kinase 2 and TRF1 results in mitotic abnormalities in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1746. doi:10.1158/1538-7445.AM2013-1746

Abstract 4049: The gain-of-function properties of mutant p53 rescues cells from telomere dysfunction-induced crisis and promotes tumorigenesis.

Abstract Telomeres are nucleoprotein complexes that cap the ends of eukaryotic chromosomes and maintain chromosomal stability. Telomeres are protected by shelterin, a complex of six core proteins (TRF1, TRF2, RAP1, TIN2, TPP1 and POT1) that prevents the chromosome ends from being mistakenly recognized as damaged DNA. Telomeres that can no longer exert end-protective functions are said to be dysfunctional. Telomere dysfunction can be caused by progressive telomere attrition (telomere shortening) or through disruption of the shelterin complex (telomere uncapping). To study whether chromosomal instability caused by telomere uncapping leads to tumorigenesis, we used the Cre-lox system to delete the telomere binding protein TRF2 in mouse embryonic fibroblasts (MEFs). In the presence of tumor suppressor protein p53, loss of TRF2 inhibited cell proliferation via induction of cellular senescence. In contrast, the absence of p53 allowed the TRF2-deficient cells to bypass senescence as these cells were negative for SA-β-galactosidase staining. However, these p53-deficient cells stopped proliferating ten days after TRF2 depletion and eventually entered telomere uncapping-induced crisis (indicated by the significant appearance of chromosomal end to end fusions, mitotic failure and induction of apoptosis). Our mechanistic studies revealed that these p53-deficient cells underwent a novel ATM-caspase 2-dependent apoptosis upon loss of TRF2. Accordingly, knocking out caspase 2 rescued these cells from apoptosis, and thus increased long-term survival of the cell colonies as well as promoted cell transformation. Mutations of p53 are predominantly missense mutations that result in accumulation of mutant p53 proteins with loss of wild-type p53 tumor-suppressive functions and gain of oncogenic activities. To study whether the gain-of-function properties of mutant p53 can rescue cells undergoing telomere crisis from cell death, we generated MEFs expressing mutant p53R172H that corresponds to the p53R175H “hot spot” mutation in human cancers. We observed that mutant p53R172H did not prevent TRF2 depletion-induced chromosomal instability as indicated by extensive chromosome fusions. However, expression of mutant p53R172H significantly rescued cells from TRF2 loss-initiated cell apoptosis and promoted cell transformation as determined by anchorage-independent soft agar assay. More importantly, mutant p53R172H promoted tumor development of TRF2-deficient MEFs in immunodeficient mice. Collectively, our findings provide compelling genetic evidence that telomere uncapping-driven chromosomal instability activates ATM-caspase 2-dependent apoptosis to suppress tumorigenesis. However, the gain-of-function properties of mutant p53 can rescue the cells undergoing telomere crisis from cell death and thus promote tumorigenesis. Citation Format: Li-Ju Chang, Fengxia Wu, Xiaolan Guo, Yibin Deng. The gain-of-function properties of mutant p53 rescues cells from telomere dysfunction-induced crisis and promotes tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4049. doi:10.1158/1538-7445.AM2013-4049

The F-box protein β-TrCP promotes ubiquitination of TRF1 and regulates the ALT-associated PML bodies formation in U2OS cells

The telomeric repeat binding protein 1 (TRF1) is a major factor of the mammalian telosome/shelterin and negatively regulates telomere length by inhibiting access of telomerase at telomere termini in telomerase-positive cells. In telomerase-negative cancer cells, TRF1 also plays a critical role in the mechanism called alternative lengthening of telomeres (ALT) and is essential for formation of the ALT-associated PML bodies (APBs). It was reported that TRF1 can be degraded by the ubiquitin–proteasome pathway, involving in two regulation factors, Fbx4 and RLIM. Here, we reported that β-TrCP1, a member of the F-box family protein with ubiquitin ligase activity, is a novel TRF1-associating protein. β-TrCP1 interacts with TRF1 in vivo and in vitro and promotes its ubiquitination. Overexpression of β-TrCP1 reduces endogenous TRF1 protein levels, while inhibition of β-TrCP1 by siRNA stabilizes TRF1. Moreover, we found that β-TrCP1 is essential for regulation of promyelocytic leukemia body recruitment of TRF1 in U2OS cells. These results reveal that β-TrCP1 is involved in the negative regulation of TRF1 and represents a new pathway for APB formation in telomerase-negative cells.

Telomerase-Dependent 3′ G-Strand Overhang Maintenance Facilitates GTBP1-Mediated Telomere Protection from Misplaced Homologous Recombination

At the 3'-end of telomeres, single-stranded G-overhang telomeric repeats form a stable T-loop. Many studies have focused on the mechanisms that generate and regulate the length of telomere 3' G-strand overhangs, but the roles of G-strand overhang length control in proper T-loop formation and end protection remain unclear. Here, we examined functional relationships between the single-stranded telomere binding protein GTBP1 and G-strand overhang lengths maintained by telomerase in tobacco (Nicotiana tabacum). In tobacco plants, telomerase reverse transcriptase subunit (TERT) repression severely worsened the GTBP1 knockdown phenotypes, which were formally characterized as an outcome of telomere destabilization. TERT downregulation shortened the telomere 3' G-overhangs and increased telomere recombinational aberrations in GTBP1-suppressed plants. Correlatively, GTBP1-mediated inhibition of single-strand invasion into the double-strand telomeric sequences was impaired due to shorter single-stranded telomeres. Moreover, TERT/GTBP1 double knockdown amplified misplaced homologous recombination of G-strand overhangs into intertelomeric regions. Thus, proper G-overhang length maintenance is required to protect telomeres against intertelomeric recombination, which is achieved by the balanced functions of GTBP1 and telomerase activity.

TERT promoter mutations occur frequently in gliomas and a subset of tumors derived from cells with low rates of self-renewal

Malignant cells, like all actively growing cells, must maintain their telomeres, but genetic mechanisms responsible for telomere maintenance in tumors have only recently been discovered. In particular, mutations of the telomere binding proteins alpha thalassemia/mental retardation syndrome X-linked (ATRX) or death-domain associated protein (DAXX) have been shown to underlie a telomere maintenance mechanism not involving telomerase (alternative lengthening of telomeres), and point mutations in the promoter of the telomerase reverse transcriptase (TERT) gene increase telomerase expression and have been shown to occur in melanomas and a small number of other tumors. To further define the tumor types in which this latter mechanism plays a role, we surveyed 1,230 tumors of 60 different types. We found that tumors could be divided into types with low (<15%) and high (≥15%) frequencies of TERT promoter mutations. The nine TERT-high tumor types almost always originated in tissues with relatively low rates of self renewal, including melanomas, liposarcomas, hepatocellular carcinomas, urothelial carcinomas, squamous cell carcinomas of the tongue, medulloblastomas, and subtypes of gliomas (including 83% of primary glioblastoma, the most common brain tumor type). TERT and ATRX mutations were mutually exclusive, suggesting that these two genetic mechanisms confer equivalent selective growth advantages. In addition to their implications for understanding the relationship between telomeres and tumorigenesis, TERT mutations provide a biomarker that may be useful for the early detection of urinary tract and liver tumors and aid in the classification and prognostication of brain tumors.

A yeast telomerase complex containing the Est1 recruitment protein is assembled early in the cell cycle.

In budding yeast, association of the Est1 regulatory protein with telomerase is thought to be limited to the late S phase, when telomere elongation occurs. By monitoring the stoichiometry of telomerase subunits, we show instead that a telomerase complex containing Est1 is assembled much earlier in the cell cycle. We also report a biochemical interaction between Est1 and the telomere binding protein Cdc13 that recapitulates the previously observed genetic relationship between EST1 and CDC13. This supports a model in which regulated binding of Cdc13 to chromosome termini dictates subsequent interaction of a recruitment-competent telomerase complex with telomeres.

Telomeres are guardians of chromosomal integrity

Telomeres are DNA-protein complexes located at the ends of linear chromosomes in most eukaryotic cells. Telomeres are vital components of chromosome integrity and cell proliferation. They protect internally located genes from degradation due to incomplete DNA replication, and they also mask natural DNA ends from being recognized as DNA breaks. While being guardians of genome integrity, telomeres can also be vulnerable to damage and inappropriate repair. Dysfunction in telomere maintenance can lead to serious illnesses in humans, including cancer. Cancers achieve immortalization through telomere elongation either through the activation of specialized enzyme telomerase or Alternative Lengthening of Telomeres (ALT) pathway. This review highlights the structure and the function of telomeres in yeast and humans, discusses telomere binding proteins and their roles in telomere maintenance, and briefly describes the implications of telomere dysfunction on human health.

P300-mediated acetylation of TRF2 is required for maintaining functional telomeres

The human telomeric protein TRF2 is required to protect chromosome ends by facilitating their organization into the protective capping structure. Post-translational modifications of TRF2 such as phosphorylation, ubiquitination, SUMOylation, methylation and poly(ADP-ribosyl)ation have been shown to play important roles in telomere function. Here we show that TRF2 specifically interacts with the histone acetyltransferase p300, and that p300 acetylates the lysine residue at position 293 of TRF2. We also report that p300-mediated acetylation stabilizes the TRF2 protein by inhibiting its ubiquitin-dependent proteolysis and is required for efficient telomere binding of TRF2. Furthermore, overexpression of the acetylation-deficient mutant, K293R, induces DNA-damage response foci at telomeres, thereby leading to induction of impaired cell growth, cellular senescence and altered cell cycle distribution. A small but significant number of metaphase chromosomes show no telomeric signals at chromatid ends, suggesting an aberrant telomere structure. These findings demonstrate that acetylation of TRF2 by p300 plays a crucial role in the maintenance of functional telomeres as well as in the regulation of the telomere-associated DNA-damage response, thus providing a new route for modulating telomere protection function.

Analysis of telomere length and function in radiosensitive mouse and human cells in response to DNA-PKcs inhibition

BackgroundTelomeres, the physical ends of chromosomes, play an important role in preserving genomic integrity. This protection is supported by telomere binding proteins collectively known as the shelterin complex. The shelterin complex protects chromosome ends by suppressing DNA damage response and acting as a regulator of telomere length maintenance by telomerase, an enzyme that elongates telomeres. Telomere dysfunction manifests in different forms including chromosomal end-to-end fusion, telomere shortening and p53-dependent apoptosis and/or senescence. An important shelterin-associated protein with critical role in telomere protection in human and mouse cells is the catalytic subunit of DNA-protein kinase (DNA-PKcs). DNA-PKcs deficiency in mouse cells results in elevated levels of spontaneous telomeric fusion, a marker of telomere dysfunction, but does not cause telomere length shortening. Similarly, inhibition of DNA-PKcs with chemical inhibitor, IC86621, prevents chromosomal end protection through mechanism reminiscent of dominant-negative reduction in DNA-PKcs activity.ResultsWe demonstrate here that the IC86621 mediated inhibition of DNA-PKcs in two mouse lymphoma cell lines results not only in elevated frequencies of chromosome end-to-end fusions, but also accelerated telomere shortening in the presence of telomerase. Furthermore, we observed increased levels of spontaneous telomeric fusions in Artemis defective human primary fibroblasts in which DNA-PKcs was inhibited, but no significant changes in telomere length.ConclusionThese results confirm that DNA-PKcs plays an active role in chromosome end protection in mouse and human cells. Furthermore, it appears that DNA-PKcs is also involved in telomere length regulation, independently of telomerase activity, in mouse lymphoma cells but not in human cells.

Oligonucleotide Models of Telomeric DNA and RNA Form a Hybrid G-quadruplex Structure as a Potential Component of Telomeres

Telomeric repeat-containing RNA, a non-coding RNA molecule, has recently been found in mammalian cells. The detailed structural features and functions of the telomeric RNA at human chromosome ends remain unclear, although this RNA molecule may be a key component of the telomere machinery. In this study, using model human telomeric DNA and RNA sequences, we demonstrated that human telomeric RNA and DNA oligonucleotides form a DNA-RNA G-quadruplex. We next employed chemistry-based oligonucleotide probes to mimic the naturally formed telomeric DNA-RNA G-quadruplexes in living cells, suggesting that the process of DNA-RNA G-quadruplex formation with oligonucleotide models of telomeric DNA and RNA could occur in cells. Furthermore, we investigated the possible roles of this DNA-RNA G-quadruplex. The formation of the DNA-RNA G-quadruplex causes a significant increase in the clonogenic capacity of cells and has an effect on inhibition of cellular senescence. Here, we have used a model system to provide evidence about the formation of G-quadruplex structures involving telomeric DNA and RNA sequences that have the potential to provide a protective capping structure for telomere ends.

Human Rap1 Interacts Directly with Telomeric DNA and Regulates TRF2 Localization at the Telomere

The TRF2-Rap1 complex suppresses non-homologous end joining and interacts with DNAPK-C to prevent end joining. We previously demonstrated that hTRF2 is a double strand telomere binding protein that forms t-loops in vitro and recognizes three- and four-way junctions independent of DNA sequence. How the DNA binding characteristics of hTRF2 to DNA is altered in the presence of hRap1 however is not known. Here we utilized EM and quantitative gel retardation to characterize the DNA binding properties of hRap1 and the TRF2-Rap1 complex. Both gel filtration chromatography and mass analysis from two-dimensional projections showed that the TRF2-Rap1 complex exists in solution and binds to DNA as a complex consisting of four monomers each of hRap1 and hTRF2. EM revealed for the first time that hRap1 binds to DNA templates in the absence of hTRF2 with a preference for double strand-single strand junctions in a sequence independent manner. When hTRF2 and hRap1 are in a complex, its affinity for ds telomeric sequences is 2-fold higher than TRF2 alone and more than 10-fold higher for telomeric 3' ends. This suggests that as hTRF2 recruits hRap1 to telomeric sequences, hRap1 alters the affinity of hTRF2 and its binding preference on telomeric DNA. Moreover, the TRF2-Rap1 complex has higher ability to re-model telomeric DNA than either component alone. This finding underlies the importance of complex formation between hRap1 and hTRF2 for telomere function and end protection.

Urokinase Receptor Mediates Doxorubicin-Induced Vascular Smooth Muscle Cell Senescence via Proteasomal Degradation of TRF2

The anthracycline doxorubicin is a widely used effective anti-cancer drug. However, its application and dosage are severely limited due to its cardiotoxicity. The exact mechanisms of doxorubicin-induced cardiotoxic side effects remain poorly understood. Even less is known about the impact of doxorubicin treatment on vascular damage. We found that low doses of doxorubicin induced a senescent response in human primary vascular smooth muscle cells (VSMC). We observed that expression of urokinase receptor (uPAR) was upregulated in response to doxorubicin. Furthermore, the level of uPAR expression played a decisive role in developing doxorubicin-induced senescence. uPAR silencing in human VSMC by means of RNA interference as well as uPAR knockout in mouse VSMC resulted in abrogation of doxorubicin-induced cellular senescence. On the contrary, uPAR overexpression promoted VSMC senescence. We further found that proteasomal degradation of telomeric repeat binding factor 2 (TRF2) mediates doxorubicin-induced VSMC senescence. Our results demonstrate that uPAR controls the ubiquitin-proteasome system in VSMC and regulates doxorubicin-induced TRF2 ubiquitination and proteasomal degradation via this mechanism. Therefore, VSMC senescence induced by low doses of doxorubicin may contribute to vascular damage upon doxorubicin treatment. uPAR-mediated TRF2 ubiquitination and proteasomal degradation are further identified as a molecular mechanism underlying this process.

Cdc13 OB2 Dimerization Required for Productive Stn1 Binding and Efficient Telomere Maintenance

Cdc13 is an essential yeast protein required for telomere length regulation and genome stability. It does so via its telomere-capping properties and by regulating telomerase access to the telomeres. The crystal structure of the Saccharomyces cerevisiae Cdc13 domain located between the recruitment and DNA binding domains reveals an oligonucleotide-oligosaccharide binding fold (OB2) with unusually long loops extending from the core of the protein. These loops are involved in extensive interactions between two Cdc13 OB2 folds leading to stable homodimerization. Interestingly, the functionally impaired cdc13-1 mutation inhibits OB2 dimerization. Biochemical assays indicate OB2 is not involved in telomeric DNA or Stn1 binding. However, disruption of the OB2 dimer in full-length Cdc13 affects Cdc13-Stn1 association, leading to telomere length deregulation, increased temperature sensitivity, and Stn1 binding defects. We therefore propose that dimerization of the OB2 domain of Cdc13 is required for proper Cdc13, Stn1, Ten1 (CST) assembly and productive telomere capping.