Reconstitution of a telomeric replicon organized by CST

Telomeres, the natural ends of linear chromosomes, are comprised of repeat-sequence DNA and associated proteins1. Replication of telomeres allows continued proliferation of human stem cells and immortality of cancer cells2. Replication begins with telomerase3 extending the single-stranded DNA (ssDNA) of the telomeric G-strand [(TTAGGG)n]; the synthesis of the complementary C-strand [(CCCTAA)n] is much less well characterized. The CST (CTC1-STN1-TEN1) protein complex, a DNA Polymerase α-primase accessory factor4,5, is known to be required for telomere replication in vivo6,7,8,9, and the molecular analysis presented here reveals key features of its mechanism. We find that CST uses its ssDNA-binding activity to specify the origins for telomeric C-strand synthesis by bound Polα-primase. CST-organized DNA polymerization can copy a telomeric DNA template that folds into G-quadruplex structures, but the suboptimality of this template likely contributes to telomere replication problems observed in vivo. Combining telomerase, a short telomeric ssDNA primer, and CST-Polα-primase gives complete telomeric DNA replication, resulting in the same sort of ssDNA 3’-overhang found naturally on human telomeres. We conclude that the CST complex not only terminates telomerase extension10,11 and recruits Polα-primase to telomeric ssDNA4,12,13, but it also orchestrates C-strand synthesis. Because replication of the telomere has features distinct from replication of the rest of the genome, targeting telomere-replication components including CST holds promise for cancer therapeutics.

TRF1 poly(ADP-ribosyl)ation by PARP1 allows proper telomere replication through helicase recruitment in non-ALT cells

Telomeres are nucleoprotein structures at eukaryotic chromosome termini. Their stability is preserved by a six-protein complex named shelterin. Among these, TRF1 binds telomere duplex and assists DNA replication with mechanisms only partly clarified. Poly (ADP-ribose) polymerase 1 (PARP1) is a chromatin associated enzyme which adds poly (ADP-ribose) polymers (PARs) to acceptor proteins by covalent hetero-modification. Here we found that TRF1 is covalently PARylated by PARP1 during DNA synthesis. PARP1 downregulation perturbs bromodeoxyuridine incorporation at telomeres in S-phase, triggering replication-dependent DNA damage and telomere fragility. PARylated TRF1 recruits WRN and BLM helicases in S-phase in a PARP1-dependent manner, probably through non-covalent PAR binding to solve secondary structures during telomere replication. ALT telomeres are less affected by PARP1 downregulation and are less sensitive to PARP inhibitors. This work unveils an unprecedented role for PARP1 as a "surveillant" of telomere replication, in absence of exogenous DNA insults, which orchestrates protein dynamics at proceeding replication fork.

Dynamic interaction of BRCA2 with telomeric G-quadruplexes underlies telomere replication homeostasis

BRCA2-deficient cells undergo telomere shortening upon collapse of stalled replication forks, particularly during lagging-strand telomere synthesis. The molecular mechanism underlying fork collapse remains unclear. Here we find that the BRCA2 C-terminus, which includes an OB-fold, specifically interacts with G-quadruplex (G4) structures generated during lagging-strand telomere replication. We demonstrate that BRCA2 associates with G-triplex (G3)-derived intermediates using electrophoretic mobility shift assay and single-molecule FRET. These G3 intermediates form during direct interconversion between parallel and non-parallel G4 structures. Intriguingly, MRE11 nuclease can resect G4-forming telomere sequences, a function that is inhibited by BRCA2. BRCA2 depletion consistently resulted in increased telomeric damage, which was relieved by MRE11 knockdown. These data suggest that BRCA2 interaction with telomeric G4 prevents MRE11-mediated resection. The specific interaction between BRCA2 and G4 therefore contributes to telomere stability and genome integrity.

TAZ maintains telomere length in TNBC cells by mediating Rad51C expression

Background Telomere maintenance is crucial for the unlimited proliferation of cancer cells and essential for the “stemness” of multiple cancer cells. TAZ is more extensively expressed in triple negative breast cancers (TNBC) than in other types of breast cancers, and promotes proliferation, transformation and EMT of cancer cells. It was reported that TAZ renders breast cancer cells with cancer stem cell features. However, whether TAZ regulates telomeres is still unclear. In this study, we explored the roles of TAZ in the regulation of telomere maintenance in TNBC cells. Methods siRNA and shRNA was used to generate TAZ-depleted TNBC cell lines. qPCR and Southern analysis of terminal restriction fragments techniques were used to test telomere length. Co-immunoprecipitation, Western blotting, immunofluorescence, Luciferase reporter assay and Chromatin-IP were conducted to investigate the underlying mechanism. Results By knocking down the expression of TAZ in TNBC cells, we found, for the first time, that TAZ is essential for the maintenance of telomeres in TNBC cells. Moreover, loss of TAZ causes senescence phenotype of TNBC cells. The observed extremely shortened telomeres in late passages of TAZ knocked down cells correlate with an elevated hTERT expression, reductions of shelterin proteins, and an activated DNA damage response pathway. Our data also showed that depletion of TAZ results in overexpression of TERRAs, which are a group of telomeric repeat‐containing RNAs and regulate telomere length and integrity. Furthermore, we discovered that TAZ maintains telomere length of TNBC cells likely by facilitating the expression of Rad51C, a crucial element of homologous recombination pathway that promotes telomere replication. Conclusions This study supports the notion that TAZ is an oncogenic factor in TNBC, and further reveals a novel telomere-related pathway that is employed by TAZ to regulate TNBC.

miR-376a Provokes Rectum Adenocarcinoma Via CTC1 Depletion-Induced Telomere Dysfunction

CTC1 is a component of the mammalian CST (CTC1–STN1–TEN1) complex which plays essential roles in resolving replication problems to facilitate telomeric DNA and genomic DNA replication. We previously reported that the depletion of CTC1 leads to stalled replication fork restart defects. Moreover, the mutation in CTC1 caused cancer-prone diseases including Coats plus (CP) or dyskeratosis congenita (DC). To better understand the CTC1 regulatory axis, the microRNAs (miRNAs) targeting to CTC1 were predicted by a bioinformatics tool, and the selected candidates were further confirmed by a dual-luciferase reporter assay. Here, our current results revealed that miR-376a significantly reduced CTC1 expression at the transcription level by recognizing CTC1 3′-UTR. In addition, the overexpression of miR-376a induced telomere replication defection and resulted in direct replicative telomere damage, which could be rescued by adding back CTC1. Telomere shortening was also observed upon miR-376a treatment. Furthermore, for the clinical patient samples, the high expression of miR-376a was associated with the deregulation of CTC1 and a poor outcome for the rectum adenocarcinoma patients. Together, our results uncovered a novel role of miR-376a in stimulating rectum adenocarcinoma progression via CTC1 downregulating induced telomere dysfunction.

Telomere Replication: Solving Multiple End Replication Problems

Eukaryotic genomes are highly complex and divided into linear chromosomes that require end protection from unwarranted fusions, recombination, and degradation in order to maintain genomic stability. This is accomplished through the conserved specialized nucleoprotein structure of telomeres. Due to the repetitive nature of telomeric DNA, and the unusual terminal structure, namely a protruding single stranded 3′ DNA end, completing telomeric DNA replication in a timely and efficient manner is a challenge. For example, the end replication problem causes a progressive shortening of telomeric DNA at each round of DNA replication, thus telomeres eventually lose their protective capacity. This phenomenon is counteracted by the recruitment and the activation at telomeres of the specialized reverse transcriptase telomerase. Despite the importance of telomerase in providing a mechanism for complete replication of telomeric ends, the majority of telomere replication is in fact carried out by the conventional DNA replication machinery. There is significant evidence demonstrating that progression of replication forks is hampered at chromosomal ends due to telomeric sequences prone to form secondary structures, tightly DNA-bound proteins, and the heterochromatic nature of telomeres. The telomeric loop (t-loop) formed by invasion of the 3′-end into telomeric duplex sequences may also impede the passage of replication fork. Replication fork stalling can lead to fork collapse and DNA breaks, a major cause of genomic instability triggered notably by unwanted repair events. Moreover, at chromosomal ends, unreplicated DNA distal to a stalled fork cannot be rescued by a fork coming from the opposite direction. This highlights the importance of the multiple mechanisms involved in overcoming fork progression obstacles at telomeres. Consequently, numerous factors participate in efficient telomeric DNA duplication by preventing replication fork stalling or promoting the restart of a stalled replication fork at telomeres. In this review, we will discuss difficulties associated with the passage of the replication fork through telomeres in both fission and budding yeasts as well as mammals, highlighting conserved mechanisms implicated in maintaining telomere integrity during replication, thus preserving a stable genome.

Naked mole rat TRF1 safeguards glycolytic capacity and telomere replication under low oxygen

The naked mole rat (NMR), a long-lived and cancer-resistant rodent, is highly resistant to hypoxia. Here, using robust cellular models wherein the mouse telomeric protein TRF1 is substituted by NMR TRF1 or its mutant forms, we show that TRF1 supports maximal glycolytic capacity under low oxygen, shows increased nuclear localization and association with telomeres, and protects telomeres from replicative stress. We pinpoint this evolutionary gain of metabolic function to specific amino acid changes in the homodimerization domain of this protein. We further find that NMR TRF1 accelerates telomere shortening. These findings reveal an evolutionary strategy to adapt telomere biology for metabolic control under an extreme environment.

Structurally distinct telomere-binding proteins in Ustilago maydis execute non-overlapping functions in telomere replication, recombination, and protection

Duplex telomere binding proteins exhibit considerable structural and functional diversity in fungi. Herein we interrogate the activities and functions of two Myb-containing, duplex telomere repeat-binding factors in Ustilago maydis, a basidiomycete that is evolutionarily distant from the standard fungi. These two telomere-binding proteins, UmTay1 and UmTrf2, despite having distinct domain structures, exhibit comparable affinities and sequence specificity for the canonical telomere repeats. UmTay1 specializes in promoting telomere replication and an ALT-like pathway, most likely by modulating the helicase activity of Blm. UmTrf2, in contrast, is critical for telomere protection; transcriptional repression of Umtrf2 leads to severe growth defects and profound telomere aberrations. Comparative analysis of UmTay1 homologs in different phyla reveals broad functional diversity for this protein family and provides a case study for how DNA-binding proteins can acquire and lose functions at various chromosomal locations. Our findings also point to stimulatory effect of telomere protein on ALT in Ustilago maydis that may be conserved in other systems.

Microcephalin 1/BRIT1-TRF2 interaction promotes telomere replication and repair, linking telomere dysfunction to primary microcephaly

Telomeres protect chromosome ends from inappropriately activating the DNA damage and repair responses. Primary microcephaly is a key clinical feature of several human telomere disorder syndromes, but how microcephaly is linked to dysfunctional telomeres is not known. Here, we show that the microcephalin 1/BRCT-repeats inhibitor of hTERT (MCPH1/BRIT1) protein, mutated in primary microcephaly, specifically interacts with the TRFH domain of the telomere binding protein TRF2. The crystal structure of the MCPH1–TRF2 complex reveals that this interaction is mediated by the MCPH1 330YRLSP334 motif. TRF2-dependent recruitment of MCPH1 promotes localization of DNA damage factors and homology directed repair of dysfunctional telomeres lacking POT1-TPP1. Additionally, MCPH1 is involved in the replication stress response, promoting telomere replication fork progression and restart of stalled telomere replication forks. Our work uncovers a previously unrecognized role for MCPH1 in promoting telomere replication, providing evidence that telomere replication defects may contribute to the onset of microcephaly.

Structurally distinct duplex telomere repeat-binding proteins in Ustilago maydis execute specialized, non-overlapping functions in telomere recombination and telomere protection

Duplex telomere binding proteins exhibit considerable structural and functional diversity in different phyla. Herein we address the distinct properties and functions of two Myb-containing, duplex telomere repeat-binding factors in Ustilago maydis, a basidiomycete fungus that is evolutionarily distant from the standard budding and fission yeasts. The two telomere-binding proteins in U. maydis, named UmTrf1 and UmTrf2, have different domain organizations and belong to distinct protein families with different phylogenetic distributions. Despite these differences, they exhibit comparable affinities and similar sequence specificity for the canonical, 6-base-pair telomere repeats. Deletion of trf1 triggers preferential loss of long telomere tracts, suggesting a role for the encoded protein in promoting telomere replication. Trf1 loss also partially suppresses the ALT-like phenotypes of ku70-deficient mutants, suggesting a novel role for a telomere protein in stimulating ALT-related pathways. In keeping with these ideas, we found that purified Trf1 can modulate the helicase activity of Blm, a conserved telomere replication and recombination factor. In contrast, trf2 appears to be essential and transcriptional repression of this gene leads to severe growth defects and profound telomere aberrations that encompass telomere length heterogeneity, accumulation of extrachromosomal telomere repeats such as C-circles, and high levels of single-stranded telomere DNA. These observations support a critical role for UmTrf2 in telomere protection. Together, our findings point to a unique, unprecedented division of labor between the two major duplex telomere repeat-binding factors in Ustilago maydis. Comparative analysis of UmTrf1 homologs in different phyla reveals a high degree of functional diversity for this protein family, and provides a case study for how a sequence-specific DNA binding protein can acquire and lose functions at different chromosomal locations.