Unlocking the potential of protein-derived peptides to target G-quadruplex DNA: from recognition to anticancer activity.

Noncanonical nucleic acid structures, particularly G-quadruplexes, have garnered significant attention as potential therapeutic targets in cancer treatment. Here, the recognition of G-quadruplex DNA by peptides derived from the Rap1 protein is explored, with the aim of developing novel peptide-based G-quadruplex ligands with enhanced selectivity and anticancer activity. Biophysical techniques were employed to assess the interaction of a peptide derived from the G-quadruplex-binding domain of the protein with various biologically relevant G-quadruplex structures. Through alanine scanning mutagenesis, key amino acids crucial for G-quadruplex recognition were identified, leading to the discovery of two peptides with improved G-quadruplex-binding properties. However, despite their in vitro efficacy, these peptides showed limited cell penetration and anticancer activity. To overcome this challenge, cell-penetrating peptide (CPP)-conjugated derivatives were designed, some of which exhibited significant cytotoxic effects on cancer cells. Interestingly, selected CPP-conjugated peptides exerted potent anticancer activity across various tumour types via a G-quadruplex-dependent mechanism. These findings underscore the potential of peptide-based G-quadruplex ligands in cancer therapy and pave the way for the development of novel therapeutic strategies targeting these DNA structures.

Human POT1 protects the telomeric ds-ss DNA junction by capping the 5' end of the chromosome.

Protection of telomeres 1 (POT1) is the 3' single-stranded overhang-binding telomeric protein that prevents an ataxia telangiectasia and Rad3-related (ATR) DNA damage response (DDR) at chromosome ends. What precludes the DDR machinery from accessing the telomeric double-stranded-single-stranded junction is unknown. We demonstrate that human POT1 binds this junction by recognizing the phosphorylated 5' end of the chromosome. High-resolution crystallographic structures reveal that the junction is capped by POT1 through a "POT-hole" surface, the mutation of which compromises junction protection in vitro and telomeric 5'-end definition and DDR suppression in human cells. Whereas both mouse POT1 paralogs bind the single-stranded overhang, POT1a, not POT1b, contains a POT-hole and binds the junction, which explains POT1a's sufficiency for end protection. Our study shifts the paradigm for DDR suppression at telomeres by highlighting the importance of protecting the double-stranded-single-stranded junction.

Complex interaction network revealed by mutation of human telomerase 'insertion in fingers' and essential N-terminal domains and the telomere protein TPP1.

In the majority of human cancer cells, cellular immortalization depends on the maintenance of telomere length by telomerase. An essential step required for telomerase function is its recruitment to telomeres, which is regulated by the interaction of the telomere protein, TPP1, with the telomerase essential N-terminal (TEN) domain of the human telomerase reverse transcriptase, hTERT. We previously reported that the hTERT 'insertion in fingers domain' (IFD) recruits telomerase to telomeres in a TPP1-dependent manner. Here, we use hTERT truncations and the IFD domain containing mutations in conserved residues or premature aging disease-associated mutations to map the interactions between the IFD and TPP1. We find that the hTERT-IFD domain can interact with TPP1. However, deletion of the IFD motif in hTERT lacking the N-terminus and the C-terminal extension does not abolish interaction with TPP1, suggesting the IFD is not essential for hTERT interaction with TPP1. Several conserved residues in the central IFD-TRAP region that we reported regulate telomerase recruitment to telomeres, and cell immortalization compromise interaction of the hTERT-IFD domain with TPP1 when mutated. Using a similar approach, we find that the IFD domain interacts with the TEN domain but is not essential for intramolecular hTERT interactions with the TEN domain. IFD-TEN interactions are not disrupted by multiple amino acid changes in the IFD or TEN, thus highlighting a complex regulation of IFD-TEN interactions as suggested by recent cryo-EM structures of human telomerase.

Shelterin is a dimeric complex with extensive structural heterogeneity.

Human shelterin is a six-subunit complex-composed of TRF1, TRF2, Rap1, TIN2, TPP1, and POT1-that binds telomeres, protects them from the DNA-damage response, and regulates the maintenance of telomeric DNA. Although high-resolution structures have been generated of the individual structured domains within shelterin, the architecture and stoichiometry of the full complex are currently unknown. Here, we report the purification of shelterin subcomplexes and reconstitution of the entire complex using full-length, recombinant subunits. By combining negative-stain electron microscopy (EM), cross-linking mass spectrometry (XLMS), AlphaFold modeling, mass photometry, and native mass spectrometry (MS), we obtain stoichiometries as well as domain-scale architectures of shelterin subcomplexes and determine that they feature extensive conformational heterogeneity. For POT1/TPP1 and POT1/TPP1/TIN2, we observe high variability in the positioning of the POT1 DNA-binding domain, the TPP1 oligonucleotide/oligosaccharide-binding (OB) fold, and the TIN2 TRFH domain with respect to the C-terminal domains of POT1. Truncation of unstructured linker regions in TIN2, TPP1, and POT1 did not reduce the conformational variability of the heterotrimer. Shelterin and TRF1-containing subcomplexes form fully dimeric stoichiometries, even in the absence of DNA substrates. Shelterin and its subcomplexes showed extensive conformational variability, regardless of the presence of DNA substrates. We conclude that shelterin adopts a multitude of conformations and argue that its unusual architectural variability is beneficial for its many functions at telomeres.

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.

Structure of Tetrahymena telomerase-bound CST with polymerase α-primase.

Telomeres are the physical ends of linear chromosomes. They are composed of short repeating sequences (such as TTGGGG in the G-strand for Tetrahymena thermophila) of double-stranded DNA with a single-strand 3' overhang of the G-strand and, in humans, the six shelterin proteins: TPP1, POT1, TRF1, TRF2, RAP1 and TIN21,2. TPP1 and POT1 associate with the 3' overhang, with POT1 binding the G-strand3 and TPP1 (in complex with TIN24) recruiting telomerase via interaction with telomerase reverse transcriptase5 (TERT). The telomere DNA ends are replicated and maintained by telomerase6, for the G-strand, and subsequently DNA polymerase α-primase7,8 (PolαPrim), for the C-strand9. PolαPrim activity is stimulated by the heterotrimeric complex CTC1-STN1-TEN110-12 (CST), but the structural basis of the recruitment of PolαPrim and CST to telomere ends remains unknown. Here we report cryo-electron microscopy (cryo-EM) structures of Tetrahymena CST in the context of the telomerase holoenzyme, in both the absence and the presence of PolαPrim, and of PolαPrim alone. Tetrahymena Ctc1 binds telomerase subunit p50, a TPP1 orthologue, on a flexible Ctc1 binding motif revealed by cryo-EM and NMR spectroscopy. The PolαPrim polymerase subunit POLA1 binds Ctc1 and Stn1, and its interface with Ctc1 forms an entry port for G-strand DNA to the POLA1 active site. We thus provide a snapshot of four key components that are required for telomeric DNA synthesis in a single active complex-telomerase-core ribonucleoprotein, p50, CST and PolαPrim-that provides insights into the recruitment of CST and PolαPrim and the handoff between G-strand and C-strand synthesis.

Shelterin Components Modulate Nucleic Acids Condensation and Phase Separation in the Context of Telomeric DNA.

Telomeres are nucleoprotein complexes that protect the ends of chromosomes and are essential for chromosome stability in Eukaryotes. In cells, individual telomeres form distinct globules of finite size that appear to be smaller than expected for bare DNA. Moreover, telomeres can cluster together, form telomere-induced-foci or co-localize with promyelocytic leukemia (PML) nuclear bodies. The physical basis for collapse of individual telomeres and coalescence of multiple ones remains unclear, as does the relationship between these two phenomena. By combining single-molecule force spectroscopy measurements, optical microscopy, turbidity assays, and simulations, we show that the telomere scaffolding protein TRF2 can condense individual DNA chains and drives coalescence of multiple DNA molecules, leading to phase separation and the formation of liquid-like droplets. Addition of the TRF2 binding protein hRap1 modulates phase boundaries and tunes the specificity of solution demixing while simultaneously altering the degree of DNA compaction. Our results suggest that the condensation of single telomeres and formation of biomolecular condensates containing multiple telomeres are two different outcomes driven by the same set of molecular interactions. Moreover, binding partners, such as other telomere components, can alter those interactions to promote single-chain DNA compaction over multiple-chain phase separation.

Insights into POT1 structural dynamics revealed by cryo-EM.

Telomeres are protein-DNA complexes that protect the ends of linear eukaryotic chromosomes. Mammalian telomeric DNA consists of 5'-(TTAGGG)n-3' double-stranded repeats, followed by up to several hundred bases of a 3' single-stranded G-rich overhang. The G-rich overhang is bound by the shelterin component POT1 which interacts with TPP1, the component involved in telomerase recruitment. A previously published crystal structure of the POT1 N-terminal half bound to the high affinity telomeric ligand 5'-TTAGGGTTAG-3' showed that the first six nucleotides, TTAGGG, are bound by the OB1 fold, while the adjacent OB2 binds the last four, TTAG. Here, we report two cryo-EM structures of full-length POT1 bound by the POT1-binding domain of TPP1. The structures differ in the relative orientation of the POT1 OB1 and OB2, suggesting that these two DNA-binding OB folds take up alternative conformations. Supporting DNA binding studies using telomeric ligands in which the OB1 and OB2 binding sites were spaced apart, show that POT1 binds with similar affinities to spaced or contiguous binding sites, suggesting plasticity in DNA binding and a role for the alternative conformations observed. A likely explanation is that the structural flexibility of POT1 enhances binding to the tandemly arranged telomeric repeats and hence increases telomere protection.

Structural basis of human telomerase recruitment by TPP1-POT1.

Telomerase maintains genome stability by extending the 3' telomeric repeats at eukaryotic chromosome ends, thereby counterbalancing progressive loss caused by incomplete genome replication. In mammals, telomerase recruitment to telomeres is mediated by TPP1, which assembles as a heterodimer with POT1. We report structures of DNA-bound telomerase in complex with TPP1 and with TPP1-POT1 at 3.2- and 3.9-angstrom resolution, respectively. Our structures define interactions between telomerase and TPP1-POT1 that are crucial for telomerase recruitment to telomeres. The presence of TPP1-POT1 stabilizes the DNA, revealing an unexpected path by which DNA exits the telomerase active site and a DNA anchor site on telomerase that is important for telomerase processivity. Our findings rationalize extensive prior genetic and biochemical findings and provide a framework for future mechanistic work on telomerase regulation.

Structures of telomerase at several steps of telomere repeat synthesis.

Telomerase is unique among the reverse transcriptases in containing a noncoding RNA (known as telomerase RNA (TER)) that includes a short template that is used for the processive synthesis of G-rich telomeric DNA repeats at the 3' ends of most eukaryotic chromosomes1. Telomerase maintains genomic integrity, and its activity or dysregulation are critical determinants of human longevity, stem cell renewal and cancer progression2,3. Previous cryo-electron microscopy structures have established the general architecture, protein components and stoichiometries of Tetrahymena and human telomerase, but our understandings of the details of DNA-protein and RNA-protein interactions and of the mechanisms and recruitment involved remain limited4-6. Here we report cryo-electron microscopy structures of active Tetrahymena telomerase with telomeric DNA at different steps of nucleotide addition. Interactions between telomerase reverse transcriptase (TERT), TER and DNA reveal the structural basis of the determination of the 5' and 3' template boundaries, handling of the template-DNA duplex and separation of the product strand during nucleotide addition. The structure and binding interface between TERT and telomerase protein p50 (a homologue of human TPP17,8) define conserved interactions that are required for telomerase activation and recruitment to telomeres. Telomerase La-related protein p65 remodels several regions of TER, bridging the 5' and 3' ends and the conserved pseudoknot to facilitate assembly of the TERT-TER catalytic core.

Structural and functional analysis of the human POT1-TPP1 telomeric complex.

POT1 and TPP1 are part of the shelterin complex and are essential for telomere length regulation and maintenance. Naturally occurring mutations of the telomeric POT1-TPP1 complex are implicated in familial glioma, melanoma and chronic lymphocytic leukaemia. Here we report the atomic structure of the interacting portion of the human telomeric POT1-TPP1 complex and suggest how several of these mutations contribute to malignant cancer. The POT1 C-terminus (POT1C) forms a bilobal structure consisting of an OB-fold and a holiday junction resolvase domain. TPP1 consists of several loops and helices involved in extensive interactions with POT1C. Biochemical data shows that several of the cancer-associated mutations, partially disrupt the POT1-TPP1 complex, which affects its ability to bind telomeric DNA efficiently. A defective POT1-TPP1 complex leads to longer and fragile telomeres, which in turn promotes genomic instability and cancer.

Dynamic peptides of human TPP1 fulfill diverse functions in telomere maintenance.

Telomeres are specialized nucleoprotein complexes that comprise the ends of linear chromosomes. Human telomeres end in a short, single-stranded DNA (ssDNA) overhang that is recognized and bound by two telomere proteins, POT1 and TPP1. Whereas POT1 binds directly to telomere ssDNA, its interaction with TPP1 is essential for localization of POT1 to the telomere. TPP1 also provides enhanced binding and sequence discrimination that regulates POT1-TPP1 interactions exclusively with telomere ssDNA. Finally, TPP1 recruits telomerase, the enzyme responsible for synthesis of telomere DNA, to the telomere. While the oligosaccharide-oligonucleotide-binding (OB)-fold domain of TPP1 has been solved by X-ray crystallography, the molecular interactions within the POT1-TPP1-ssDNA ternary complex and the conformational changes that contribute to its diverse functions remain ambiguous. We employed hydrogen/deuterium exchange combined with mass spectrometry to identify three peptides, all residing within the OB-fold of TPP1, that exhibit altered exchange rates upon complex formation or ssDNA binding. Mutation of these regions combined with functional assays revealed the diverse contributions of each moiety in protein-protein interactions, regulating telomerase activity or DNA-binding. Together, these functional data combined with biophysical analyses and homology modeling provide a molecular understanding of the diverse contributions of TPP1 in telomere maintenance.

Contributions of the TEL-patch amino acid cluster on TPP1 to telomeric DNA synthesis by human telomerase.

Telomere maintenance is a highly coordinated process, and its misregulation is linked to cancer and telomere-shortening syndromes. Recent studies have shown that the TEL-patch--a cluster of amino acids on the surface of the shelterin component TPP1--is necessary for the recruitment of telomerase to the telomere in human cells. However, there has been only basic biochemical analysis of the role of TPP1 in the telomerase recruitment process. Here we develop an in vitro assay to quantitatively measure the contribution of the TEL-patch to telomerase recruitment--binding and extension of the first telomeric repeat. We also demonstrate that the TEL-patch contributes to the translocation step of the telomerase reaction. Finally, our quantitative observations indicate that the TEL-patch stabilizes the association between telomerase and telomeric DNA substrates, providing a molecular explanation for its contributions to telomerase recruitment and action.