Telomeric Overhang Research Articles

Human hnRNPA1 reorganizes telomere-bound replication protein A.

Human replication protein A (RPA) is a heterotrimeric ssDNA binding protein responsible for many aspects of cellular DNA metabolism. Dynamic interactions of the four RPA DNA binding domains (DBDs) with DNA control replacement of RPA by downstream proteins in various cellular metabolic pathways. RPA plays several important functions at telomeres where it binds to and melts telomeric G-quadruplexes, non-canonical DNA structures formed at the G-rich telomeric ssDNA overhangs. Here, we combine single-molecule total internal reflection fluorescence microscopy (smTIRFM) and mass photometry (MP) with biophysical and biochemical analyses to demonstrate that heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) specifically remodels RPA bound to telomeric ssDNA by dampening the RPA configurational dynamics and forming a ternary complex. Uniquely, among hnRNPA1 target RNAs, telomeric repeat-containing RNA (TERRA) is selectively capable of releasing hnRNPA1 from the RPA-telomeric DNA complex. We speculate that this telomere specific RPA-DNA-hnRNPA1 complex is an important structure in telomere protection.

Protection of the Telomeric Junction by the Shelterin Complex.

Shelterin serves critical roles in suppressing superfluous DNA damage repair pathways on telomeres. The junction between double-stranded telomeric tracts (dsTEL) and single-stranded telomeric overhang (ssTEL) is the most accessible region of the telomeric DNA. The shelterin complex contains dsTEL and ssTEL binding proteins and can protect this junction by bridging the ssTEL and dsTEL tracts. To test this possibility, we monitored shelterin binding to telomeric DNA substrates with varying ssTEL and dsTEL lengths and quantified its impact on telomere accessibility using single-molecule fluorescence microscopy methods in vitro. We identified the first dsTEL repeat nearest the junction as the preferred binding site for creating the shelterin bridge. Shelterin requires at least two ssTEL repeats, while the POT1 subunit of shelterin that binds to ssTEL requires longer ssTEL tracts for stable binding to telomeres and effective protection of the junction region. The ability of POT1 to protect the junction is significantly enhanced by the 5'-phosphate at the junction. Collectively, our results show that shelterin enhances the binding stability of POT1 to ssTEL and provides more effective protection compared with POT1 alone by bridging single- and double-stranded telomeric tracts.

The molecular mechanism for TERRA recruitment and annealing to telomeres.

Telomeric repeat containing RNA (TERRA) is a noncoding RNA that is transcribed from telomeres. Previous study showed that TERRA trans anneals by invading into the telomeric duplex to form an R-loop in mammalian cells. Here, we elucidate the molecular mechanism underlying TERRA recruitment and invasion into telomeres in the context of shelterin proteins, RAD51 and RNase H using single molecule (sm) assays. We demonstrate that TERRA trans annealing into telomeric DNA exhibits dynamic movement that is stabilized by TRF2. TERRA annealing to the telomeric duplex results in the formation of a stable triplex structure which differs from a conventional R-loop. We identified that the presence of a sub-telomeric DNA and a telomeric overhang in the form of a G-quadruplex significantly enhances TERRA annealing to telomeric duplex. We also demonstrate that RAD51-TERRA complex invades telomere duplex more efficiently than TERRA alone. Additionally, TRF2 increases TERRA affinity to telomeric duplex and protects it from RNase H digestion. In contrast, TRF1 represses TERRA annealing to telomeric duplex and fails to provide protection against RNase H digestion. Our findings provide an in-depth molecular mechanism underpinning TERRA recruitment and annealing to the telomere.

TRF2-RAP1 represses RAD51-dependent homology-directed telomere repair by promoting BLM-mediated D-loop unwinding and inhibiting BLM-DNA2-dependent 5'-end resection.

Inappropriate homology-directed repair (HDR) of telomeres results in catastrophic telomere loss and aberrant chromosome fusions, leading to genome instability. We have previously shown that the TRF2-RAP1 heterodimer protects telomeres from engaging in aberrant telomere HDR. Cells lacking the basic domain of TRF2 and functional RAP1 display HDR-mediated telomere clustering, resulting in the formation of ultrabright telomeres (UTs) and massive chromosome fusions. Using purified proteins, we uncover three distinct molecular pathways that the TRF2-RAP1 heterodimer utilizes to protect telomeres from engaging in aberrant HDR. We show mechanistically that TRF2-RAP1 inhibits RAD51-initiated telomeric D-loop formation. Both the TRF2 basic domain and RAP1-binding to TRF2 are required to block RAD51-mediated homology search. TRF2 recruits the BLM helicase to telomeres through its TRFH domain to promote BLM-mediated unwinding of telomere D-loops. In addition, TRF2-RAP1 inhibits BLM-DNA2-mediated 5' telomere end resection, preventing the generation of 3' single-stranded telomere overhangs necessary for RAD51-dependent HDR. Importantly, cells expressing BLM mutants unable to interact with TRF2 accumulate telomere D-loops and UTs. Our findings uncover distinct molecular mechanisms coordinated by TRF2-RAP1 to protect telomeres from engaging in aberrant HDR.

Slow G-Quadruplex Conformation Rearrangement and Accessibility Change Induced by Potassium in Human Telomeric Single-Stranded DNA.

The guanine-rich telomeric repeats can form G-quadruplexes (G4s) that alter the accessibility of the single-stranded telomeric overhang. In this study, we investigated the effects of Na+ and K+ on G4 folding and accessibility through cation introduction and exchange. We combined differential scanning calorimetry (DSC), circular dichroism (CD), and single molecule Förster resonance energy transfer (smFRET) to monitor the stability, conformational dynamics, and complementary strand binding accessibility of G4 formed by single-stranded telomeric DNA. Our data showed that G4 formed through heating and slow cooling in K+ solution exhibited fewer conformational dynamics than G4 formed in Na+ solution, which is consistent with the higher thermal stability of G4 in K+. Monitoring cation exchange with real time smFRET at room temperature shows that Na+ and K+ can replace each other in G4. When encountering high K+ at room or body temperature, G4 undergoes a slow conformational rearrangement process which is mostly complete by 2 h. The slow conformational rearrangement ends with a stable G4 that is unable to be unfolded by a complementary strand. This study provides new insights into the accessibility of G4 forming sequences at different time points after introduction to a high K+ environment in cells, which may affect how the nascent telomeric overhang interacts with proteins and telomerase.

Deregulated DNA ADP-ribosylation impairs telomere replication.

The recognition that DNA can be ADP ribosylated provides an unexpected regulatory level of how ADP-ribosylation contributes to genome stability, epigenetics and immunity. Yet, it remains unknown whether DNA ADP-ribosylation (DNA-ADPr) promotes genome stability and how it is regulated. Here, we show that telomeres are subject to DNA-ADPr catalyzed by PARP1 and removed by TARG1. Mechanistically, we show that DNA-ADPr is coupled to lagging telomere DNA strand synthesis, forming at single-stranded DNA present at unligated Okazaki fragments and on the 3' single-stranded telomere overhang. Persistent DNA-linked ADPr, due to TARG1 deficiency, eventually leads to telomere shortening. Furthermore, using the bacterial DNA ADP-ribosyl-transferase toxin to modify DNA at telomeres directly, we demonstrate that unhydrolyzed DNA-linked ADP-ribose compromises telomere replication and telomere integrity. Thus, by identifying telomeres as chromosomal targets of PARP1 and TARG1-regulated DNA-ADPr, whose deregulation compromises telomere replication and integrity, our study highlights and establishes the critical importance of controlling DNA-ADPr turnover for sustained genome stability.

19F Nuclear Magnetic Resonance Fingerprinting Technique for Identifying and Quantifying G-Quadruplex Topology in Human Telomeric Overhangs.

G-quadruplexes (G4s) are noncanonical nucleic acid secondary structures with diverse topological features and biological roles. Human telomeric (Htelo) overhangs consisting of TTAGGG repeats can fold into G4s that adopt different topologies under physiological conditions. These G4s are potential targets for anticancer drugs. Despite intensive research, the existence and topology of G4s at Htelo overhangs in vivo are still unclear because there is no method to distinguish and quantify the topology of Htelo overhangs with native lengths that can form more than three tandem G4s in living cells. Herein, we present a novel 19F chemical shift fingerprinting technique to identify and quantify the topology of the Htelo overhangs up to five G-quadruplexes (G4s) and 120 nucleotides long both in vitro and in living cells. Our results show that longer overhang sequences tend to form stable G4s at the 5'- and 3'-ends, while the interior G4s are dynamic and "sliding" along the sequence, with TTA or 1-3 TTAGGG repeats as a linker. Each G4 in the longer overhang is conformationally heterogeneous, but the predominant ones are hybrid-2, two- or three-tetrad antiparallel, and hybrid-1 at the 5'-terminal, interior, and 3'-terminal, respectively. Additionally, we observed a distinct behavior of different lengths of telomeric sequences in living cells, suggesting that the overhang length and protein accessibility are related to its function. This technique provides a powerful tool for quickly identifying the folding topology and relative population of long Htelo overhangs, which may provide valuable insights into telomere functionality and be beneficial for structure-based anticancer drug development targeting G4s.

POT1 mutations cause differential effects on telomere length leading to opposing disease phenotypes.

The protection of telomereprotein (POT1) is a telomere-binding protein and is an essential component of the six-memberedshelterin complex, which is associated with the telomeres. POT1 directly binds to the3' single-stranded telomeric overhang and prevents the activation of DNA damage response at telomeres thus preventing the telomere-telomere fusions and genomic instability. POT1 also plays a pivotal role in maintaining telomere length by regulating telomerase-mediated telomere elongation. Mutations in POT1 proteins resultin three different telomere phenotypes, which include long, short, or aberrant telomere length. Long telomeres predispose individuals to cancer, while short or aberrant telomere phenotypes result in pro-aging diseases referred to as telomeropathies. Here, we review the function of POT1 proteins in telomere length hemostasis and how the spectrum of mutations reported in POT1 can be segregated towarddeveloping very distinct diseasephenotypes of cancer and telomeropathies.

Abstract 1436: T-oligo's mechanism of action in melanoma cells

Abstract Telomeres have become an attractive target for anticancer therapeutics and in 2022, an estimated 99,780 new cases and 7,650 people are expected to die of melanoma. T-oligo, a hexanucleotide tandem repeat oligonucleotide that is homologous to the 3′ overhang of telomeres has been shown to have potent anticancer effects in-vitro by inhibiting the growth of cancer cells. There are three mechanisms of action proposed for T-oligo, which are shelterin dissociation model, exposed telomere mimicry model, and G-quadruplex formation. Previous studies utilizing NMR spectroscopy and immunofluorescence assays have shown that T-oligo forms G-quadruplexes in the nucleus of the melanoma cells. We hypothesize that formation of G-quadruplexes can be detected with the help of FRET analysis and T-oligo could be delivered using lipid nanoparticles. Immunofluorescence assays and an acceptor photobleaching method were used to detect FRET and confirm the formation of G-quadruplexes in the nucleus of melanoma cells. Melanoma cells were plated and grown until 70% confluent in 8-well glass chamber slides and were treated with single-labeled FITC-T-oligo (11-mer), and Cy3-T-oligo (11-mer), and double labeled T-oligo 11-mer (T-11) and 22-mer (T-22) labeled with FITC on the 5′ end and Cy3 on the 3′ end. Slides were mounted and cells were observed under a fluorescent microscope. For FRET detection, we used the acceptor photobleaching method in which the acceptor (Cy3) was completely photobleached and any corresponding increase in donor’s intensity was detected and quantified. Any significant increase in donor intensity was considered to be due to FRET. We also performed some preliminary experiments with lipid nanoparticles to study whether these lipid nanoparticles are a viable delivery system for T-oligo. We encapsulated T-oligo in lipid nanoparticles using a microfluidic mixer and studied the encapsulation efficiency, uptake, and efficacy of encapsulated T-11 and T-22 on MM-AN melanoma cells. We found a significant increase of 40% - 150% in donor intensity after acceptor photobleaching in multiple melanoma cell lines. This may be due to G-quadruplex formation causing FRET thus indicating that T-oligo may be utilizing this mechanism of action for its anticancer effects. Preliminary results indicate that lipid nanoparticles encapsulated T-oligo with an efficiency of 72%. Uptake experiments showed that encapsulated T-11 and T-22 were taken up by the cells after an incubation time of 6hrs and the encapsulated T-22 also inhibited cell proliferation by 92%. In conclusion, three melanoma cell lines showed a significant increase in donor intensity after acceptor photobleaching upon treatment with T-22 and T-11 and lipid nanoparticles might be an effective delivery system for T-oligo. Citation Format: Archit Agnihotri, Taylor Rager, Kelly Fan, Hyatt Alaraj, Lawrence Miller, Neelu Puri. T-oligo's mechanism of action in melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1436.

Impact of shelterin complex on telomere accessibility.

Telomeres terminate with a 50-300 bases long single-stranded G-rich overhang, which can be misrecognized as a DNA damage repair site. Shelterin plays critical roles in maintaining and protecting telomere ends by regulating access of various physiological agents to telomeric DNA, but the underlying mechanism is not well understood. Here, we measure how shelterin affects the accessibility of long telomeric overhangs by monitoring transient binding events of a short complementary peptide nucleic acid (PNA) probe using FRET-PAINT in vitro. We observed that the POT1 subunit of shelterin reduces the accessibility of the PNA probe by ∼2.5 fold, indicating that POT1 effectively binds to and protects otherwise exposed telomeric sequences. In comparison, a four-component shelterin stabilizes POT1 binding to the overhang by tethering POT1 to the double-stranded telomeric DNA and reduces the accessibility of telomeric overhangs by ∼5-fold. This enhanced protection suggests shelterin restructures the junction between single and double-stranded telomere, which is otherwise the most accessible part of the telomeric overhang.

The molecular mechanism for TERRA recruitment and annealing to telomere.

Telomeres are DNA-protein complexes that protect the ends of linear chromosomes. Telomeres are composed of tandem repeat sequences of duplexed (TTAGGG)n, ending with a 3’ single stranded (TTAGGG)n sequence bound to telomere specific proteins known as shelterin complex. In healthy somatic cells, progressive loss of telomeres results in cellular senescence. In 85% of cancer cells, however telomeres are elongated by telomerase, while the remaining 15% of cancers use alternative lengthening of telomeres (ALT). In ALT (+) cancers, Telomeric repeat containing RNA (TERRA), a noncoding RNA that is transcribed from telomeres is overexpressed. TERRA trans anneals by invading the telomeric duplex to form an R-loop. Recent study showed that R-loop formation is promoted by Rad51 and antagonized by RNaseH1. Despite the evidence of TERRA's role in telomere structure and function, our molecular understanding of TERRA is limited. Here, we elucidate the molecular mechanism underlying TERRA recruitment and invasion into telomeres in the context of shelterin, RAD51 and RNaseH1 using single molecule (sm) assays. Using smFRET we show that TERRA trans anneals to telomeric DNA and exhibits dynamic movement that is stabilized by TRF2. Additionally, TERRA annealing to the telomeric duplex forms a triplex structure. Sm-colocalization assays show that the presence of non-TERRA sequence, which represents the sub-telomeric DNA and the presence of G-quadruplex telomeric overhang enhances TERRA binding to telomeric duplex. We also studied the mechanism underlying TERRA recruitment and invasion by Rad51 and shelterin proteins. Our result shows that RAD51-TERRA complex invades telomere duplex more efficiently than TERRA alone. Additionally, TRF2 increases TERRA affinity to telomeric duplex and protects it from RNaseH1 digestion. In contrast, TRF1 represses TERRA binding to telomeric duplex and does not provide protection against RNaseH1. We demonstrate the molecular mechanism underpinning TERRA recruitment and annealing to telomere.

Inhibited complete folding of consecutive human telomeric G-quadruplexes.

Noncanonical DNA structures, termed G-quadruplexes, are present in human genomic DNA and are important elements in many DNA metabolic processes. Multiple sites in the human genome have G-rich DNA stretches able to support formation of several consecutive G-quadruplexes. One of those sites is the telomeric overhang region that has multiple repeats of TTAGGG and is tightly associated with both cancer and aging. We investigated the folding of consecutive G-quadruplexes in both potassium- and sodium-containing solutions using single-molecule FRET spectroscopy, circular dichroism, thermal melting and molecular dynamics simulations. Our observations show coexistence of partially and fully folded DNA, the latter consisting of consecutive G-quadruplexes. Following the folding process over hours in sodium-containing buffers revealed fast G-quadruplex folding but slow establishment of thermodynamic equilibrium. We find that full consecutive G-quadruplex formation is inhibited by the many DNA structures randomly nucleating on the DNA, some of which are off-path conformations that need to unfold to allow full folding. Our study allows describing consecutive G-quadruplex formation in both nonequilibrium and equilibrium conditions by a unified picture, where, due to the many possible DNA conformations, full folding with consecutive G-quadruplexes as beads on a string is not necessarily achieved.

POT-3 preferentially binds the terminal DNA-repeat on the telomeric G-overhang.

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.

Shelterin reduces the accessibility of telomeric overhangs.

Telomeres terminate with a 50-300 bases long single-stranded G-rich overhang, which can be misrecognized as a DNA damage repair site. Shelterin plays critical roles in maintaining and protecting telomere ends by regulating access of various physiological agents to telomeric DNA, but the underlying mechanism is not well understood. Here, we measure how shelterin affects the accessibility of long telomeric overhangs by monitoring transient binding events of a short complementary peptide nucleic acid (PNA) probe using FRET-PAINT in vitro. We observed that the POT1 subunit of shelterin reduces the accessibility of the PNA probe by ∼2.5-fold, indicating that POT1 effectively binds to and protects otherwise exposed telomeric sequences. In comparison, a four-component shelterin stabilizes POT1 binding to the overhang by tethering POT1 to the double-stranded telomeric DNA and reduces the accessibility of telomeric overhangs by ∼5-fold. This enhanced protection suggests shelterin restructures the junction between single and double-stranded telomere, which is otherwise the most accessible part of the telomeric overhang.

Emerging accessibility patterns in long telomeric overhangs

We present single-molecule experimental and computational modeling studies investigating the accessibility of human telomeric overhangs of physiologically relevant lengths. We studied 25 different overhangs that contain 4-28 repeats of GGGTTA (G-Tract) sequence and accommodate one to seven tandem G-quadruplex (GQ) structures. Using the FRET-PAINT method, we probed the distribution of accessible sites via a short imager strand, which is complementary to a G-Tract and transiently binds to available sites. We report accessibility patterns that periodically change with overhang length and interpret these patterns in terms of the underlying folding landscape and folding frustration. Overhangs that have [4n]G-Tracts, (12, 16, 20…) demonstrate the broadest accessibility patterns where the peptide nucleic acid probe accesses G-Tracts throughout the overhang. On the other hand, constructs with [4n+2]G-Tracts, (14, 18, 22…) have narrower patterns where the neighborhood of the junction between single- and double-stranded telomeres is most accessible. We interpret these results as the folding frustration being higher in [4n]G-Tract constructs compared to [4n+2]G-Tract constructs. We also developed a computational model that tests the consistency of different folding stabilities and cooperativities between neighboring GQs with the observed accessibility patterns. Our experimental and computational studies suggest the neighborhood of the junction between single- and double-stranded telomeres is least stable and most accessible, which is significant as this is a potential site where the connection between POT1/TPP1 (bound to single-stranded telomere) and other shelterin proteins (localized on double-stranded telomere) is established.

Structural insights into Pot1-ssDNA, Pot1-Tpz1 and Tpz1-Ccq1 Interactions within fission yeast shelterin complex.

The conserved shelterin complex caps chromosome ends to protect telomeres and regulate telomere replication. In fission yeast Schizosaccharomyces pombe, shelterin consists of telomeric single- and double-stranded DNA-binding modules Pot1-Tpz1 and Taz1-Rap1 connected by Poz1, and a specific component Ccq1. While individual structures of the two DNA-binding OB folds of Pot1 (Pot1OB1-GGTTAC and Pot1OB2-GGTTACGGT) are available, structural insight into recognition of telomeric repeats with spacers by the complete DNA-binding domain (Pot1DBD) remains an open question. Moreover, structural information about the Tpz1-Ccq1 interaction requires to be revealed for understanding how the specific component Ccq1 of S. pombe shelterin is recruited to telomeres to function as an interacting hub. Here, we report the crystal structures of Pot1DBD-single-stranded-DNA, Pot1372-555-Tpz1185-212 and Tpz1425-470-Ccq1123-439 complexes and propose an integrated model depicting the assembly mechanism of the shelterin complex at telomeres. The structure of Pot1DBD-DNA unveils how Pot1 recognizes S. pombe degenerate telomeric sequences. Our analyses of Tpz1-Ccq1 reveal structural basis for the essential role of the Tpz1-Ccq1 interaction in telomere recruitment of Ccq1 that is required for telomere maintenance and telomeric heterochromatin formation. Overall, our findings provide valuable structural information regarding interactions within fission yeast shelterin complex at 3' ss telomeric overhang.

Structures of the human CST-Polα-primase complex bound to telomere templates.

The mammalian DNA polymerase-α-primase (Polα-primase) complex is essential for DNA metabolism, providing the de novo RNA-DNA primer for several DNA replication pathways1-4 such as lagging-strand synthesis and telomere C-strand fill-in. The physical mechanism underlying how Polα-primase, alone or in partnership with accessory proteins, performs its complicated multistep primer synthesis function is unknown. Here we show that CST, a single-stranded DNA-binding accessory protein complex for Polα-primase, physically organizes the enzyme for efficient primer synthesis. Cryogenic electron microscopy structures of the CST-Polα-primase preinitiation complex (PIC) bound to various types of telomere overhang reveal that template-bound CST partitions the DNA and RNA catalytic centres of Polα-primase into two separate domains and effectively arranges them in RNA-DNA synthesis order. The architecture of the PIC provides a single solution for the multiple structural requirements for the synthesis of RNA-DNA primers by Polα-primase. Several insights into the template-binding specificity of CST, template requirement for assembly of the CST-Polα-primase PIC and activation are also revealed in this study.

DNA-RNA hybrid G-quadruplex tends to form near the 3′ end of telomere overhang

Telomeric repeat-containing RNA (TERRA) has been suggested to participate in telomere maintenance. TERRA consisting of UUAGGG repeats is capable of forming an intermolecular G-quadruplex (GQ) with single-stranded TTAGGG-repeat DNA in the telomere 3′ overhang. To explore the structural features and potential functions of this DNA-RNA hybrid GQ (HGQ), we used single-molecule FRET to study the folding patterns of DNA with four to seven telomeric tandem repeats annealed with a short RNA consisting of two or five telomeric repeats. Our data highlight that RNA prefers to form DNA-RNA HGQ near the 3′ end of telomeric DNA. Furthermore, the unfolding of secondary structures by a complementary C-rich sequence was observed for DNA GQ but not for DNA-RNA HGQ, which demonstrated the enhanced stability of the telomere 3′ end via hybridization with RNA. These conformational and physical properties of telomeric DNA-RNA HGQ suggest that TERRA might limit access to the 3′ end of the telomeric DNA overhang, which is known to be critical for the interaction with telomerase and other telomere-associated proteins.

Vectorial folding of telomere overhang promotes higher accessibility.

Human telomere overhang composed of tandem repeats of TTAGGG folds into G-quadruplex (G4). Unlike in an experimental setting in the test tube in which the entire length is allowed to fold at once, inside the cell, the overhang is expected to fold as it is synthesized directionally (5′ to 3′) and released segmentally by a specialized enzyme, the telomerase. To mimic such vectorial G4 folding process, we employed a superhelicase, Rep-X which can unwind DNA to release the TTAGGG repeats in 5′ to 3′ direction. We demonstrate that the folded conformation achieved by the refolding of full sequence is significantly different from that of the vectorial folding for two to eight TTAGGG repeats. Strikingly, the vectorially folded state leads to a remarkably higher accessibility to complementary C-rich strand and the telomere binding protein POT1, reflecting a less stably folded state resulting from the vectorial folding. Importantly, our study points to an inherent difference between the co-polymerizing and post-polymerized folding of telomere overhang that can impact telomere architecture and downstream processes.

Telomere dysfunction implicates POT1 in patients with idiopathic pulmonary fibrosis.

Exonic sequencing identified a family with idiopathic pulmonary fibrosis (IPF) containing a previously unreported heterozygous mutation in POT1 p.(L259S). The family displays short telomeres and genetic anticipation. We found that POT1(L259S) is defective in binding the telomeric overhang, nuclear accumulation, negative regulation of telomerase, and lagging strand maintenance. Patient cells containing the mutation display telomere loss, lagging strand defects, telomere-induced DNA damage, and premature senescence with G1 arrest. Our data suggest POT1(L259S) is a pathogenic driver of IPF and provide insights into gene therapy options.