The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1

Telomeres are bound by dedicated proteins, which protect them from DNA damage and regulate telomere length homeostasis. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to identify TEBP-1 and TEBP-2, two paralogs expressed in the germline and embryogenesis that associate to telomeres in vitro and in vivo. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp-2 mutants display shorter telomeres and a Mortal Germline. Notably, tebp-1;tebp-2 double mutant animals have synthetic sterility, with germlines showing signs of severe mitotic and meiotic arrest. Furthermore, we show that POT-1 forms a telomeric complex with TEBP-1 and TEBP-2, which bridges TEBP-1/-2 with POT-2/MRT-1. These results provide insights into the composition and organization of a telomeric protein complex in C. elegans.

Inhibition of MRN activity by a telomere protein motif

The MRN complex (MRX in Saccharomyces cerevisiae) initiates the repair of DNA double-stranded breaks (DSBs) and activates the Tel1/ATM kinase, which orchestrates the DNA damage response (DDR). Telomeres prevent DDR activation at chromosome ends, partly by keeping MRN-ATM in check. We show that the multiple activities of the MRX complex are disabled by telomeric protein Rif2 through the action of a short motif (MIN, MRN/X-inhibitory motif) at the N-terminal end of the protein. MIN executes telomeric suppression of Tel1, DDR and and non-homologous end joining (NHEJ) via direct biding to the N-terminal region of Rad50. A combination of biochemical and genetic data suggests that Rif2 promotes a transition within the MRX complex that is not conductive for endonuclease activity, DNA-end tethering or Tel1 kinase activation. We suggests that the MIN motif operates in the RIF2 paralog ORC4 (Origin Recognition Complex 4) in K. lactis and in telomeric protein Taz1 in Schizoccharomyces pombe, which is not evolutionarily related to Orc4/Rif2. These results highlight a potential Achilles heel in Rad50, the regulatory subunit of MRN, which we suggest has been targeted by different telomeric factors in multiple fungal lineages, raising the possibility that analogous approaches might be deployed in other Eukaryotes as well.

The Telomeric Protein TRF2 Regulates Replication Origin Activity within Pericentromeric Heterochromatin

Heterochromatic regions render the replication process particularly difficult due to the high level of chromatin compaction and the presence of repeated DNA sequences. In humans, replication through pericentromeric heterochromatin requires the binding of a complex formed by the telomeric factor TRF2 and the helicase RTEL1 in order to relieve topological barriers blocking fork progression. Since TRF2 is known to bind the Origin Replication Complex (ORC), we hypothesized that this factor could also play a role at the replication origins (ORI) of these heterochromatin regions. By performing DNA combing analysis, we found that the ORI density is higher within pericentromeric satellite DNA repeats than within bulk genomic DNA and decreased upon TRF2 downregulation. Moreover, we showed that TRF2 and ORC2 interact in pericentromeric DNA, providing a mechanism by which TRF2 is involved in ORI activity. Altogether, our findings reveal an essential role for TRF2 in pericentromeric heterochromatin replication by regulating both replication initiation and elongation.

Non-canonical roles of canonical telomere binding proteins in cancers

Reactivation of telomerase is a major hallmark observed in 90% of all cancers. Yet paradoxically, enhanced telomerase activity does not correlate with telomere length and cancers often possess short telomeres; suggestive of supplementary non-canonical roles that telomerase might play in the development of cancer. Moreover, studies have shown that aberrant expression of shelterin proteins coupled with their release from shortening telomeres can further promote cancer by mechanisms independent of their telomeric role. While targeting telomerase activity appears to be an attractive therapeutic option, this approach has failed in clinical trials due to undesirable cytotoxic effects on stem cells. To circumvent this concern, an alternative strategy could be to target the molecules involved in the non-canonical functions of telomeric proteins. In this review, we will focus on emerging evidence that has demonstrated the non-canonical roles of telomeric proteins and their impact on tumorigenesis. Furthermore, we aim to address current knowledge gaps in telomeric protein functions and propose future research approaches that can be undertaken to achieve this.

Interstitial telomere sequences disrupt break-induced replication and drive formation of ectopic telomeres

Break-induced replication (BIR) is a mechanism used to heal one-ended DNA double-strand breaks, such as those formed at collapsed replication forks or eroded telomeres. Instead of utilizing a canonical replication fork, BIR is driven by a migrating D-loop and is associated with a high frequency of mutagenesis. Here we show that when BIR encounters an interstitial telomere sequence (ITS), the machinery frequently terminates, resulting in the formation of an ectopic telomere. The primary mechanism to convert the ITS to a functional telomere is by telomerase-catalyzed addition of telomeric repeats with homology-directed repair serving as a back-up mechanism. Termination of BIR and creation of an ectopic telomere is promoted by Mph1/FANCM helicase, which has the capacity to disassemble D-loops. Other sequences that have the potential to seed new telomeres but lack the unique features of a natural telomere sequence, do not terminate BIR at a significant frequency in wild-type cells. However, these sequences can form ectopic telomeres if BIR is made less processive. Our results support a model in which features of the ITS itself, such as the propensity to form secondary structures and telomeric protein binding, pose a challenge to BIR and increase the vulnerability of the D-loop to dissociation by helicases, thereby promoting ectopic telomere formation.

The telomeric Cdc13 protein from yeast Hansenula polymorpha

Telomeres are special structures at the ends of chromosomes that play an important role in the protection of the genetic material. Telomere composition is very diverse; noticeable differences can often be observed even among closely related species. Here, we identify the homolog of telomeric protein Cdc13 in the thermotolerant yeast Hansenula polymorpha. We show that it can specifically bind single-stranded telomeric DNA, as well as interact with the Stn1 protein. In addition, we have uncovered an interaction between Cdc13 and TERT (one of the core components of the telomerase complex), which suggests that Cdc13 is potentially involved in telomerase recruitment to telomeres in H. polymorpha.

Structural basis of G-quadruplex DNA recognition by the yeast telomeric protein Rap1

G-quadruplexes are four-stranded nucleic acid structures involved in multiple cellular pathways including DNA replication and telomere maintenance. Such structures are formed by G-rich DNA sequences typified by telomeric DNA repeats. Whilst there is evidence for proteins that bind and regulate G-quadruplex formation, the molecular basis for this remains poorly understood. The budding yeast telomeric protein Rap1, originally identified as a transcriptional regulator functioning by recognizing double-stranded DNA binding sites, was one of the first proteins to be discovered to also bind and promote G-quadruplex formation in vitro. Here, we present the 2.4 Å resolution crystal structure of the Rap1 DNA-binding domain in complex with a G-quadruplex. Our structure not only provides a detailed insight into the structural basis for G-quadruplex recognition by a protein, but also gives a mechanistic understanding of how the same DNA-binding domain adapts to specifically recognize different DNA structures. The key observation is the DNA-recognition helix functions in a bimodal manner: In double-stranded DNA recognition one helix face makes electrostatic interactions with the major groove of DNA, whereas in G-quadruplex recognition a different helix face is used to make primarily hydrophobic interactions with the planar face of a G-tetrad.

Protection of telomeres 1 (POT1) of Pinus tabuliformis bound the telomere ssDNA

Protection of telomeres 1 (POT1) is a telomeric protein that binds to the telomere single-stranded (ss) region. It plays an essential role in maintaining genomic stability in both plants and animals. In this study, we investigated the properties of POT1 in Pinus tabuliformis Carr. (PtPOT1) through electrophoretic mobility shift assay. PtPOT1 harbored affinity for telomeric ssDNA and could bind plant- and mammalian-type ssDNA sequences. Notably, there were two oligonucleotide/oligosaccharide binding (OB) folds, and OB1 or OB2 alone, or both together, could bind ssDNA, which is significantly different from human POT1. Based on our data, we hypothesized that the two OB folds of PtPOT1 bound the same ssDNA. This model not only provides new insight into the ssDNA binding of PtPOT1 but also sheds light on the functional divergence of POT1 proteins in gymnosperms and humans.

Combining conservation and species-specific differences to determine how human telomerase binds telomeres

Telomerase catalyzes telomeric DNA synthesis at chromosome ends to allow for continued cell division. The telomeric protein TPP1 is essential for enhancing the processivity of telomerase and recruiting the enzyme to telomeres. The telomerase interaction surface on human TPP1 has been mapped to 2 regions of the N-terminal oligosaccharide/oligonucleotide-binding (OB) domain, namely the TPP1 glutamate (E) and leucine (L)-rich (TEL) patch and the N terminus of TPP1-oligosaccharide/oligonucleotide-binding (NOB) region. To map the telomerase side of the interface, we exploited the predicted structural similarities for human andTetrahymena thermophilatelomerase as well as the species specificity of human and mouse telomerase for their cognate TPP1 partners. We show that swapping in the telomerase essential N-terminal (TEN) and insertions in fingers domain (IFD)-TRAP regions of the human telomerase catalytic protein subunit TERT into the mouse TERT backbone is sufficient to bias the species specificity toward human TPP1. Employing a structural homology-based mutagenesis screen focused on surface residues of the TEN and IFD regions, we identified TERT residues that are critical for contacting TPP1 but dispensable for other aspects of telomerase structure or function. We present a functionally validated structural model for how human telomerase engages TPP1 at telomeres, setting the stage for a high-resolution structure of this interface.

Two separation-of-function isoforms of human TPP1 and a novel intragenic noncoding RNA dictate telomerase regulation in somatic and germ cells

SummaryTelomerase replicates chromosome ends in germ and somatic stem cells to facilitate continued proliferation. Telomerase action depends on the telomeric protein TPP1, which recruits telomerase to telomeres and facilitates processive DNA synthesis. Here we identify separation-of-function long (TPP1-L) and short (TPP1-S) isoforms of TPP1 differing only in 86 amino acids at their N-terminus. While both isoforms retain the ability to recruit telomerase, only TPP1-S facilitates telomere synthesis. We identify a novel intragenic noncoding RNA in the 3’-UTR of the TPP1-encoding gene that specifically shuts down telomerase activation-incompatible TPP1-L to establish TPP1-S as the predominant isoform in somatic cells. Strikingly, TPP1-L is the major isoform in testes, where it can function to restrain telomerase in mature germ cells. Our studies uncover how differential expression of two isoforms allows TPP1 to perform separate functions in different cells, and demonstrate how isoform choice can be determined by an intragenic noncoding RNA.