Telomeres are nucleoprotein structures at the ends of linear chromosomes and present an essential feature for genome integrity. Vertebrate telomeres usually consist of hexameric TTAGGG repeats, however, in cells that use the alternative lengthening of telomeres (ALT) mechanism, variant repeat sequences are interspersed throughout telomeres. Previously, it was shown that NR2C/F transcription factors bind to TCAGGG variant repeats and contribute to telomere maintenance in ALT cells. While specific binders to other variant repeat sequences have been lacking to date, we here identify ZBTB10 as the first TTGGGG-binding protein and demonstrate direct binding via the two zinc fingers with affinity in the nanomolar range. Concomitantly, ZBTB10 co-localizes with a subset of telomeres in ALT-positive U2OS cells and interacts with TRF2/RAP1 via the N-terminal region of TRF2. Our data establishes ZBTB10 as a novel variant repeat binding protein at ALT telomeres.

Eukaryotic chromosomes typically end in 3' telomeric overhangs. The safeguarding of telomeric single-stranded DNA overhangs is carried out by factors related to the protection of telomeres 1 (POT1) protein in humans. Of the three POT1-like proteins in Caenorhabditis elegans, POT-3 was the only member thought to not play a role at telomeres. Here, we provide evidence that POT-3 is a bona fide telomere-binding protein. Using a new loss-of-function mutant, we show that the absence of POT-3 causes telomere lengthening and increased levels of telomeric C-circles. We find that POT-3 directly binds the telomeric G-strand in vitro and map its minimal DNA binding site to the six-nucleotide motif, GCTTAG. We further show that the closely related POT-2 protein binds the same motif, but that POT-3 shows higher sequence selectivity. Crucially, in contrast to POT-2, POT-3 prefers binding sites immediately adjacent to the 3' end of DNA. These differences are significant as genetic analyses reveal that pot-2 and pot-3 do not function redundantly with each other in vivo. Our work highlights the rapid evolution and specialisation of telomere binding proteins and places POT-3 in a unique position to influence activities that control telomere length.

The germline-specific role of telomeres consists of chromosome end elongation and proper chromosome segregation during early developmental stages. Despite the crucial role of telomeres in germ cells, little is known about telomere biology in the germline. We analyzed telomere homeostasis in the Drosophila female germline and early embryos. A novel germline-specific function of deadenylase complex Ccr4-Not in the telomeric transcript surveillance mechanism is reported. Depletion of Ccr4-Not complex components causes strong derepression of the telomeric retroelement HeT-A in the germ cells, accompanied by elongation of the HeT-A poly(A) tail. Dysfunction of transcription factors Woc and Trf2, as well as RNA-binding protein Ars2, also results in the accumulation of excessively polyadenylated HeT-A transcripts in ovaries. Germline knockdowns of Ccr4-Not components, Woc, Trf2 and Ars2, lead to abnormal mitosis in early embryos, characterized by chromosome missegregation, centrosome dysfunction and spindle multipolarity. Moreover, the observed phenotype is accompanied by the accumulation of HeT-A transcripts around the centrosomes in early embryos, suggesting the putative relationship between overexpression of telomeric transcripts and mitotic defects. Our data demonstrate that Ccr4-Not, Woc, Trf2 and Ars2, components of different regulatory pathways, are required for telomere protection in the germline in order to guarantee normal development.

The CST complex is a key player in telomere replication and stability, which in yeast comprises Cdc13, Stn1 and Ten1. While Stn1 and Ten1 are very well conserved across species, Cdc13 does not resemble its mammalian counterpart CTC1 either in sequence or domain organization, and Cdc13 but not CTC1 displays functions independently of the rest of CST. Whereas the structures of human CTC1 and CST have been determined, the molecular organization of Cdc13 remains poorly understood. Here, we dissect the molecular architecture of Candida glabrata Cdc13 and show how it regulates binding to telomeric sequences. Cdc13 forms dimers through the interaction between OB-fold 2 (OB2) domains. Dimerization stimulates binding of OB3 to telomeric sequences, resulting in the unfolding of ssDNA secondary structure. Once bound to DNA, Cdc13 prevents the refolding of ssDNA by mechanisms involving all domains. OB1 also oligomerizes, inducing higher-order complexes of Cdc13 in vitro. OB1 truncation disrupts these complexes, affects ssDNA unfolding and reduces telomere length in C. glabrata. Together, our results reveal the molecular organization of C. glabrata Cdc13 and how this regulates the binding and the structure of DNA, and suggest that yeast species evolved distinct architectures of Cdc13 that share some common principles.

KH (hnRNP K homology) domains, consisting of approximately 70 amino acid residues, are present in a variety of nucleic-acid-binding proteins. Among these are poly(C)-binding proteins (PCBPs), which are important regulators of mRNA stability and posttranscriptional regulation in general. All PCBPs contain three different KH domains and recognize poly(C)-sequences with high affinity and specificity. To reveal the molecular basis of poly(C)-sequence recognition, we have determined the crystal structure, at 1.6 A resolution, of PCBP2 KH3 domain in complex with a 7-nt DNA sequence (5'-AACCCTA-3') corresponding to one repeat of the C-rich strand of human telomeric DNA. The domain assumes a type-I KH fold in a betaalphaalphabetabetaalpha configuration. The protein-DNA interface could be studied in unprecedented detail and is made up of a series of direct and water-mediated hydrogen bonds between the protein and the DNA, revealing an especially dense network involving several structural water molecules for the last 2 nt in the core recognition sequence. Unlike published KH domain structures, the protein crystallizes without protein-protein contacts, yielding new insights into the dimerization properties of different KH domains. A nucleotide platform, an interesting feature found in some RNA molecules, was identified, evidently for the first time in DNA.

More than two decades of genetic research have identified and assigned main biological functions of shelterin proteins that safeguard telomeres. However, a molecular mechanism of how each protein subunit contributes to the protecting function of the whole shelterin complex remains elusive. Human Repressor activator protein 1 (Rap1) forms a multifunctional complex with Telomeric Repeat binding Factor 2 (TRF2). Rap1-TRF2 complex is a critical part of shelterin as it suppresses homology-directed repair in Ku 70/80 heterodimer absence. To understand how Rap1 affects key functions of TRF2, we investigated full-length Rap1 binding to TRF2 and Rap1-TRF2 complex interactions with double-stranded DNA by quantitative biochemical approaches. We observed that Rap1 reduces the overall DNA duplex binding affinity of TRF2 but increases the selectivity of TRF2 to telomeric DNA. Additionally, we observed that Rap1 induces a partial release of TRF2 from DNA duplex. The improved TRF2 selectivity to telomeric DNA is caused by less pronounced electrostatic attractions between TRF2 and DNA in Rap1 presence. Thus, Rap1 prompts more accurate and selective TRF2 recognition of telomeric DNA and TRF2 localization on single/double-strand DNA junctions. These quantitative functional studies contribute to the understanding of the selective recognition of telomeric DNA by the whole shelterin complex.

The ends of linear chromosomes are capped by protein-DNA complexes termed telomeres. Telomere repeat binding factors 1 and 2 (TRF1 and TRF2) bind specifically to duplex telomeric DNA and are critical components of functional telomeres. Consequences of telomere dysfunction include genomic instability, cellular apoptosis or senescence and organismal aging. Mild oxidative stress induces increased erosion and loss of telomeric DNA in human fibroblasts. We performed binding assays to determine whether oxidative DNA damage in telomeric DNA alters the binding activity of TRF1 and TRF2 proteins. Here, we report that a single 8-oxo-guanine lesion in a defined telomeric substrate reduced the percentage of bound TRF1 and TRF2 proteins by at least 50%, compared with undamaged telomeric DNA. More dramatic effects on TRF1 and TRF2 binding were observed with multiple 8-oxo-guanine lesions in the tandem telomeric repeats. Binding was likewise disrupted when certain intermediates of base excision repair were present within the telomeric tract, namely abasic sites or single nucleotide gaps. These studies indicate that oxidative DNA damage may exert deleterious effects on telomeres by disrupting the association of telomere-maintenance proteins TRF1 and TRF2.

The junction between the double-stranded and single-stranded telomeric DNA (ds-ss junction) is fundamental in the maintenance of the telomeric chromatin, as it directs the assembly of the telomere binding proteins. In budding yeast, multiple Rap1 proteins bind the telomeric dsDNA, while ssDNA repeats are bound by the Cdc13 protein. Here, we aimed to determine, for the first time, the telomeric 5' end nucleotide in a budding yeast. To this end, we developed a permutation-specific PCR-based method directed towards the regular 8-mer telomeric repeats in Naumovozyma castellii. We find that, in logarithmically growing cells, the 320 ± 30 bp long telomeres mainly terminate in either of two specific 5' end permutations of the repeat, both corresponding to a terminal adenine nucleotide. Strikingly, two permutations are completely absent at the 5' end, indicating that not all ds-ss junction structures would allow the establishment of the protective telomere chromatin cap structure. Using in vitro DNA end protection assays, we determined that binding of Rap1 and Cdc13 around the most abundant ds-ss junction ensures the protection of both 5' ends and 3' overhangs from exonucleolytic degradation. Our results provide mechanistic insights into telomere protection, and reveal that Rap1 and Cdc13 have complementary roles.

Attempts to inactivate an essential gene in the protozoan parasite Leishmania have often led to the generation of extra copies of the wild-type alleles of the gene. In experiments with Leishmania tarentolae set up to disrupt the gene encoding the J-binding protein 1 (JBP1), a protein binding to the unusual base beta-D-glucosyl-hydroxymethyluracil (J) of Leishmania, we obtained JBP1 mutants containing linear DNA elements (amplicons) of approximately 100 kb. These amplicons consist of a long inverted repeat with telomeric repeats at both ends and contain either the two different targeting cassettes used to inactivate JBP1, or one cassette and one JBP1 gene. Each long repeat within the linear amplicons corresponds to sequences covering the JBP1 locus, starting at the telomeres upstream of JBP1 and ending in a approximately 220 bp sequence repeated in an inverted (palindromic) orientation downstream of the JBP1 locus. We propose that these amplicons have arisen by a template switch inside a DNA replication fork involving the inverted DNA repeats and helped by the gene targeting.

Telomerase is a ribonucleoprotein enzyme that maintains chromosome ends through de novo addition of telomeric DNA. The ability of telomerase to interact with its DNA substrate at sites outside its catalytic centre ('anchor sites') is important for its unique ability to undergo repeat addition processivity. We have developed a direct and quantitative equilibrium primer-binding assay to measure DNA-binding affinities of regions of the catalytic protein subunit of recombinant Tetrahymena telomerase (TERT). There are specific telomeric DNA-binding sites in at least four regions of TERT (the TEN, RBD, RT and C-terminal domains). Together, these sites contribute to specific and high-affinity DNA binding, with a K(d) of approximately 8 nM. Both the K(m) and K(d) increased in a stepwise manner as the primer length was reduced; thus recombinant Tetrahymena telomerase, like the endogenous enzyme, contains multiple anchor sites. The N-terminal TEN domain, which has previously been implicated in DNA binding, shows only low affinity binding. However, there appears to be cooperativity between the TEN and RNA-binding domains. Our data suggest that different DNA-binding sites are used by the enzyme during different stages of the addition cycle.

Shelterin/telosome is a multi-protein complex at mammalian telomeres, anchored to the double-stranded region by the telomeric-repeat binding factors-1 and -2. In vitro modification of these proteins by poly(ADP-ribosyl)ation through poly(ADP-ribose) polymerases-5 (tankyrases) and -1/-2, respectively, impairs binding. Thereafter, at least telomeric-repeat binding factor-1 is degraded by the proteasome. We show that pharmacological inhibition of poly(ADP-ribose) polymerase activity in cells from two different species leads to rapid decrease in median telomere length and stabilization at a lower setting. Specific knockdown of poly(ADP-ribose) polymerase-1 by RNA interference had the same effect. The length of the single-stranded telomeric overhang as well as telomerase activity were not affected. Release of inhibition led to a fast re-gain in telomere length to control levels in cells expressing active telomerase. We conclude that poly(ADP-ribose) polymerase-1 activity and probably its interplay with telomeric-repeat binding factor-2 is an important determinant in telomere regulation. Our findings reinforce the link between poly(ADP-ribosyl)ation and aging/longevity and also impact on the use of poly(ADP-ribose) polymerase inhibitors in tumor therapy.

Protection of telomeres (POT1) binds chromosome ends, recognizing single-strand telomeric DNA via two oligonucleotide/oligosaccharide binding folds (OB-folds). The Arabidopsis thaliana POT1a and POT1b paralogs are atypical: they do not exhibit telomeric DNA binding, and they have opposing roles in regulating telomerase activity. AtPOT1a stimulates repeat addition processivity of the canonical telomerase enzyme, while AtPOT1b interacts with a regulatory lncRNA that represses telomerase activity. Here, we show that OB1 of POT1a, but not POT1b, has an intrinsic affinity for telomeric DNA. DNA binding was dependent upon a highly conserved Phe residue (F65) that in human POT1 directly contacts telomeric DNA. F65A mutation of POT1aOB1 abolished DNA binding and diminished telomerase repeat addition processivity. Conversely, AtPOT1b and other POT1b homologs from Brassicaceae and its sister family, Cleomaceae, naturally bear a non-aromatic amino acid at this position. By swapping Val (V63) with Phe, AtPOT1bOB1 gained the capacity to bind telomeric DNA and to stimulate telomerase repeat addition processivity. We conclude that, in the context of DNA binding, variation at a single amino acid position promotes divergence of the AtPOT1b paralog from the ancestral POT1 protein.

In response to metabolic or environmental stress, cells activate powerful defense mechanisms to prevent the formation and accumulation of toxic protein aggregates. The main orchestrator of this cellular response is HSF1 (heat shock factor 1), a transcription factor involved in the up-regulation of protein-coding genes with protective roles. It has become very clear that HSF1 has a broader function than initially expected. Indeed, our previous work demonstrated that, upon stress, HSF1 activates the transcription of a non-coding RNA, named Satellite III, at pericentromeric heterochromatin. Here, we observe that the function of HSF1 extends to telomeres and identify subtelomeric DNA as a new genomic target of HSF1. We show that the binding of HSF1 to subtelomeric regions plays an essential role in the upregulation of non-coding TElomeric Repeat containing RNA (TERRA) transcription upon heat shock. Importantly, our data show that telomere integrity is impacted by heat shock and that telomeric DNA damages are markedly enhanced in HSF1 deficient cells. Altogether, our findings reveal a new direct and essential function of HSF1 in the transcriptional activation of TERRA and in telomere protection upon stress.

The majority of Cockayne syndrome (CS) patients carry a mutation in Cockayne Syndrome group B (CSB), a large nuclear protein implicated in DNA repair, transcription and chromatin remodeling. However, whether CSB may play a role in telomere metabolism has not yet been characterized. Here, we report that CSB physically interacts with TRF2, a duplex telomeric DNA binding protein essential for telomere protection. We find that CSB localizes at a small subset of human telomeres and that it is required for preventing the formation of telomere dysfunction-induced foci (TIF) in CS cells. We find that CS cells or CSB knockdown cells accumulate telomere doublets, the suppression of which requires CSB. We find that overexpression of CSB in CS cells promotes telomerase-dependent telomere lengthening, a phenotype that is associated with a decrease in the amount of telomere-bound TRF1, a negative mediator of telomere length maintenance. Furthermore, we show that CS cells or CSB knockdown cells exhibit misregulation of TERRA, a large non-coding telomere repeat-containing RNA important for telomere maintenance. Taken together, these results suggest that CSB is required for maintaining the homeostatic level of TERRA, telomere length and integrity. These results further imply that CS patients carrying CSB mutations may be defective in telomere maintenance.

Telomeric repeat binding factor 2 (TRF2) folds human telomeres into loops to prevent unwanted DNA repair and chromosome end-joining. The N-terminal basic domain of TRF2 (B-domain) protects the telomeric displacement loop (D-loop) from cleavage by endonucleases. Repressor activator protein 1 (Rap1) binds TRF2 and improves telomeric DNA recognition. We found that the B-domain of TRF2 stabilized the D-loop and thus reduced unwinding by BLM and RPA, whereas the formation of the Rap1-TRF2 complex restored DNA unwinding. To understand how the B-domain of TRF2 affects DNA binding and D-loop processing, we analyzed DNA binding of full-length TRF2 and a truncated TRF2 construct lacking the B-domain. We quantified how the B-domain improves TRF2's interaction with DNA via enhanced long-range electrostatic interactions. We developed a structural envelope model of the B-domain bound on DNA. The model revealed that the B-domain is flexible in solution but becomes rigid upon binding to telomeric DNA. We proposed a mechanism for how the B-domain stabilizes the D-loop.

A variety of human tumors employ alternative and recombination-mediated lengthening for telomere maintenance (ALT). Human RecQ helicases, such as BLM and WRN, can efficiently unwind alternate/secondary structures during telomere replication and/or recombination. Here, we report a novel role for RECQL1, the most abundant human RecQ helicase but functionally least studied, in telomere maintenance. RECQL1 associates with telomeres in ALT cells and actively resolves telomeric D-loops and Holliday junction substrates. RECQL1 physically and functionally interacts with telomere repeat-binding factor 2 that in turn regulates its helicase activity on telomeric substrates. The telomeric single-stranded binding protein, protection of telomeres 1 efficiently stimulates RECQL1 on telomeric substrates containing thymine glycol, a replicative blocking lesion. Loss of RECQL1 results in dysfunctional telomeres, telomere loss and telomere shortening, elevation of telomere sister-chromatid exchanges and increased aphidicolin-induced telomere fragility, indicating a role for RECQL1 in telomere maintenance. Further, our results indicate that RECQL1 may participate in the same pathway as WRN, probably in telomere replication.

The heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a multi-tasking protein that acts in the cytoplasm and nucleus. We have explored the possibility that this protein is associated with telomeres and participates in their maintenance. Rat brain hnRNP A2 was shown to have two nucleic acid binding sites. In the presence of heparin one site binds single-stranded oligodeoxyribonucleotides irrespective of sequence but not the corresponding oligoribonucleotides. Both the hnRNP A2-binding cis-acting element for the cytoplasmic RNA trafficking element, A2RE, and the ssDNA telomere repeat match a consensus sequence for binding to a second sequence-specific site identified by mutational analysis. hnRNP A2 protected the telomeric repeat sequence, but not the complementary sequence, against DNase digestion: the glycine-rich domain was found to be necessary, but not sufficient, for protection. The N-terminal RRM (RNA recognition motif) and tandem RRMs of hnRNP A2 also bind the single-stranded, template-containing segment of telomerase RNA. hnRNP A2 colocalizes with telomeric chromatin in the subset of PML bodies that are a hallmark of ALT cells, reinforcing the evidence for hnRNPs having a role in telomere maintenance. Our results support a model in which hnRNP A2 acts as a molecular adapter between single-stranded telomeric repeats, or telomerase RNA, and another segment of ssDNA.

Telomeres, the physical ends of eukaryotic chromosomes, are transcribed into telomeric repeat-containing RNA (TERRA), a large non-coding RNA, which forms an integral part of telomeric heterochromatin. In vitro, naked TERRA molecules are efficient inhibitors of human telomerase, base-pairing via their 5'-UUAGGG-3' repeats with the template sequence of telomerase RNA, in addition to contacting the telomerase reverse transcriptase protein subunit. In vivo, however, TERRA-mediated inhibition of telomerase can be prevented by unknown mechanisms. Also, heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) has been implicated in telomere length control. In vivo, TERRA is partially associated with hnRNPA1, and hnRNPA1 is also detected at telomeres. We demonstrate that on binding of TERRA, hnRNPA1 can alleviate the TERRA-mediated inhibition of telomerase. However, when in excess over TERRA, hnRNPA1 becomes itself an inhibitor of telomere extension, on binding of the telomeric DNA substrate. Yet, hnRNPA1 has no notable direct effects on the telomerase catalysis. Our in vitro results suggest that TERRA-mediated telomerase inhibition may be prevented by hnRNPA1 in vivo. Telomere extension by telomerase may require balanced levels of TERRA and hnRNPA1 at telomeres. Thus, TERRA and hnRNPA1 can function as a bimolecular regulator to turn telomerase and the telomere on and off.

BLM and WRN are members of the RecQ family of DNA helicases, and in humans their loss is associated with syndromes characterized by genome instability and cancer predisposition. As the only RecQ DNA helicase in the yeast Saccharomyces cerevisiae, Sgs1 is known to safeguard genome integrity through its role in DNA recombination. Interestingly, WRN, BLM and Sgs1 are all known to be modified by the small ubiquitin-related modifier (SUMO), although the significance of this posttranslational modification remains elusive. Here, we demonstrate that Sgs1 is specifically sumoylated under the stress of DNA double strand breaks. The major SUMO attachment site in Sgs1 is lysine 621, which lies between the Top3 binding domain and the DNA helicase domain. Surprisingly, sumoylation of K621 was found to be uniquely required for Sgs1's role in telomere-telomere recombination. In contrast, sumoylation was dispensable for Sgs1's roles in DNA damage tolerance, supppression of direct repeat and rDNA recombination, and promotion of top3Delta slow growth. Our results demonstrate that although modification by SUMO is a conserved feature of RecQ family DNA helicases, the major sites of modification are located on different domains of the protein in different organisms. We suggest that sumoylation of different domains of RecQ DNA helicases from different organisms contributes to conserved roles in regulating telomeric recombination.

Histone H3.3 is an H3 variant which differs from the canonical H3.1/2 at four residues, including a serine residue at position 31 which is evolutionarily conserved. The H3.3 S31 residue is phosphorylated (H3.3 S31Ph) at heterochromatin regions including telomeres and pericentric repeats. However, the role of H3.3 S31Ph in these regions remains unknown. In this study, we find that H3.3 S31Ph regulates heterochromatin accessibility at telomeres during replication through regulation of H3K9/K36 histone demethylase KDM4B. In mouse embryonic stem (ES) cells, substitution of S31 with an alanine residue (H3.3 A31 -phosphorylation null mutant) results in increased KDM4B activity that removes H3K9me3 from telomeres. In contrast, substitution with a glutamic acid (H3.3 E31, mimics S31 phosphorylation) inhibits KDM4B, leading to increased H3K9me3 and DNA damage at telomeres. H3.3 E31 expression also increases damage at other heterochromatin regions including the pericentric heterochromatin and Y chromosome-specific satellite DNA repeats. We propose that H3.3 S31Ph regulation of KDM4B is required to control heterochromatin accessibility of repetitive DNA and preserve chromatin integrity.