Reconstitution of a telomeric replicon organized by CST

2021 ◽  
Author(s):  
Thomas Cech ◽  
Arthur Zaug
Keyword(s):  
Cancer Therapeutics ◽  
Binding Activity ◽  
Repeat Sequence ◽  
Human Stem Cells ◽  
Telomeric Dna ◽  
Ssdna Binding ◽  
Telomere Replication ◽  
Linear Chromosomes ◽  
Dna Template

Abstract 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.

scholarly journals G-Quadruplexes at Telomeres: Friend or Foe?

Molecules ◽  
2020 ◽  
Vol 25 (16) ◽  
pp. 3686 ◽  
Author(s):  
Tracy M. Bryan
Keyword(s):  
Tandem Repeats ◽  
Genome Instability ◽  
Protein Complexes ◽  
Telomere Maintenance ◽  
Telomeric Dna ◽  
Positive Role ◽  
Telomere Biology ◽  
Linear Chromosomes

Telomeres are DNA-protein complexes that cap and protect the ends of linear chromosomes. In almost all species, telomeric DNA has a G/C strand bias, and the short tandem repeats of the G-rich strand have the capacity to form into secondary structures in vitro, such as four-stranded G-quadruplexes. This has long prompted speculation that G-quadruplexes play a positive role in telomere biology, resulting in selection for G-rich tandem telomere repeats during evolution. There is some evidence that G-quadruplexes at telomeres may play a protective capping role, at least in yeast, and that they may positively affect telomere maintenance by either the enzyme telomerase or by recombination-based mechanisms. On the other hand, G-quadruplex formation in telomeric DNA, as elsewhere in the genome, can form an impediment to DNA replication and a source of genome instability. This review summarizes recent evidence for the in vivo existence of G-quadruplexes at telomeres, with a focus on human telomeres, and highlights some of the many unanswered questions regarding the location, form, and functions of these structures.

scholarly journals Live-cell single-molecule tracking highlights requirements for stable Smc5/6 chromatin association in vivo

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Thomas J Etheridge ◽  
Desiree Villahermosa ◽  
Eduard Campillo-Funollet ◽  
Alex David Herbert ◽  
Anja Irmisch ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Single Molecule ◽  
Chromosomal Rearrangements ◽  
Binding Activity ◽  
Single Molecule Tracking ◽  
Ssdna Binding ◽  
Dna Binding Activity ◽  
Gross Chromosomal Rearrangements ◽  
Genes Encoding

The essential Smc5/6 complex is required in response to replication stress and is best known for ensuring the fidelity of homologous recombination. Using single-molecule tracking in live fission yeast to investigate Smc5/6 chromatin association, we show that Smc5/6 is chromatin associated in unchallenged cells and this depends on the non-SMC protein Nse6. We define a minimum of two Nse6-dependent sub-pathways, one of which requires the BRCT-domain protein Brc1. Using defined mutants in genes encoding the core Smc5/6 complex subunits, we show that the Nse3 double-stranded DNA binding activity and the arginine fingers of the two Smc5/6 ATPase binding sites are critical for chromatin association. Interestingly, disrupting the single-stranded DNA (ssDNA) binding activity at the hinge region does not prevent chromatin association but leads to elevated levels of gross chromosomal rearrangements during replication restart. This is consistent with a downstream function for ssDNA binding in regulating homologous recombination.

scholarly journals Identification of telomerase RNAs in species of the Yarrowia clade provides insights into the co-evolution of telomerase, telomeric repeats and telomere-binding proteins

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Filip Červenák ◽  
Katarína Juríková ◽  
Hugo Devillers ◽  
Binyamin Kaffe ◽  
Areej Khatib ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Repeat Unit ◽  
Repeat Sequence ◽  
Telomeric Dna ◽  
Telomeric Repeats ◽  
Structure Conservation ◽  
Sequence Variations

Abstract Telomeric repeats in fungi of the subphylum Saccharomycotina exhibit great inter- and intra-species variability in length and sequence. Such variations challenged telomeric DNA-binding proteins that co-evolved to maintain their functions at telomeres. Here, we compare the extent of co-variations in telomeric repeats, encoded in the telomerase RNAs (TERs), and the repeat-binding proteins from 13 species belonging to the Yarrowia clade. We identified putative TER loci, analyzed their sequence and secondary structure conservation, and predicted functional elements. Moreover, in vivo complementation assays with mutant TERs showed the functional importance of four novel TER substructures. The TER-derived telomeric repeat unit of all species, except for one, is 10 bp long and can be represented as 5′-TTNNNNAGGG-3′, with repeat sequence variations occuring primarily outside the vertebrate telomeric motif 5′-TTAGGG-3′. All species possess a homologue of the Yarrowia lipolytica Tay1 protein, YlTay1p. In vitro, YlTay1p displays comparable DNA-binding affinity to all repeat variants, suggesting a conserved role among these species. Taken together, these results add significant insights into the co-evolution of TERs, telomeric repeats and telomere-binding proteins in yeasts.

A Quantitative Assay for Telomere Protection in Saccharomyces cerevisiae

Genetics ◽  
2002 ◽  
Vol 161 (3) ◽  
pp. 995-1013 ◽  
Author(s):  
Michelle L DuBois ◽  
Zara W Haimberger ◽  
Martin W McIntosh ◽  
Daniel E Gottschling
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Homologous Recombination ◽  
Transcriptional Silencing ◽  
Repeat Length ◽  
Quantitative Assay ◽  
Telomeric Dna ◽  
Telomere Repeat ◽  
Telomere Protection ◽  
Linear Chromosomes

Abstract Telomeres are the protective ends of linear chromosomes. Telomeric components have been identified and described by their abilities to bind telomeric DNA, affect telomere repeat length, participate in telomeric DNA replication, or modulate transcriptional silencing of telomere-adjacent genes; however, their roles in chromosome end protection are not as well defined. We have developed a genetic, quantitative assay in Saccharomyces cerevisiae to measure whether various telomeric components protect chromosome ends from homologous recombination. This “chromosomal cap” assay has revealed that the telomeric end-binding proteins, Cdc13p and Ku, both protect the chromosome end from homologous recombination, as does the ATM-related kinase, Tel1p. We propose that Cdc13p and Ku structurally inhibit recombination at telomeres and that Tel1p regulates the chromosomal cap, acting through Cdc13p. Analysis with recombination mutants indicated that telomeric homologous recombination events proceeded by different mechanisms, depending on which capping component was compromised. Furthermore, we found that neither telomere repeat length nor telomeric silencing correlated with chromosomal capping efficiency. This capping assay provides a sensitive in vivo approach for identifying the components of chromosome ends and the mechanisms by which they are protected.

scholarly journals The U3 small nucleolar ribonucleoprotein component Imp4p is a telomeric DNA-binding protein

2007 ◽  
Vol 408 (3) ◽  
pp. 387-393 ◽  
Author(s):  
Yi-Ching Hsieh ◽  
Pei-Jung Tu ◽  
Ying-Yuan Lee ◽  
Chun-Chen Kuo ◽  
Yi-Chien Lin ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Binding Activity ◽  
Telomere Maintenance ◽  
Telomeric Dna ◽  
Mobility Shift ◽  
Telomere Function

Imp4p is a component of U3 snoRNP (small nucleolar ribonucleoprotein) involved in the maturation of 18S rRNA. We have shown that Imp4p interacts with Cdc13p, a single-stranded telomere-binding protein involved in telomere maintenance. To understand the role of Imp4p in telomeres, we purified recombinant Imp4p protein and tested its binding activity towards telomeric DNA using electrophoretic mobility-shift assays. Our results showed that Imp4p bound specifically to single-stranded telomeric DNA in vitro. The interaction of Imp4p to telomeres in vivo was also demonstrated by chromatin immunoprecipitation experiments. Significantly, the binding of Imp4p to telomeres was not limited to yeast proteins, since the hImp4 (human Imp4) also bound to vertebrate single-stranded telomeric DNA. Thus we conclude that Imp4p is a novel telomeric DNA-binding protein that, in addition to its role in rRNA processing, might participate in telomere function.

scholarly journals Novel DNA Motif Binding Activity Observed In Vivo With an Estrogen Receptor α Mutant Mouse

2014 ◽  
Vol 28 (6) ◽  
pp. 899-911 ◽  
Author(s):  
Sylvia C. Hewitt ◽  
Leping Li ◽  
Sara A. Grimm ◽  
Wipawee Winuthayanon ◽  
Katherine J. Hamilton ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Gene ◽  
Mutant Mouse ◽  
Estrogen Receptor Α ◽  
Binding Activity ◽  
Estrogen Response Element ◽  
Dna Binding Domain ◽  
Estrogen Response ◽  
Uterine Growth

Abstract Estrogen receptor α (ERα) interacts with DNA directly or indirectly via other transcription factors, referred to as “tethering.” Evidence for tethering is based on in vitro studies and a widely used “KIKO” mouse model containing mutations that prevent direct estrogen response element DNA- binding. KIKO mice are infertile, due in part to the inability of estradiol (E2) to induce uterine epithelial proliferation. To elucidate the molecular events that prevent KIKO uterine growth, regulation of the pro-proliferative E2 target gene Klf4 and of Klf15, a progesterone (P4) target gene that opposes the pro-proliferative activity of KLF4, was evaluated. Klf4 induction was impaired in KIKO uteri; however, Klf15 was induced by E2 rather than by P4. Whole uterine chromatin immunoprecipitation-sequencing revealed enrichment of KIKO ERα binding to hormone response elements (HREs) motifs. KIKO binding to HRE motifs was verified using reporter gene and DNA-binding assays. Because the KIKO ERα has HRE DNA-binding activity, we evaluated the “EAAE” ERα, which has more severe DNA-binding domain mutations, and demonstrated a lack of estrogen response element or HRE reporter gene induction or DNA-binding. The EAAE mouse has an ERα null–like phenotype, with impaired uterine growth and transcriptional activity. Our findings demonstrate that the KIKO mouse model, which has been used by numerous investigators, cannot be used to establish biological functions for ERα tethering, because KIKO ERα effectively stimulates transcription using HRE motifs. The EAAE-ERα DNA-binding domain mutant mouse demonstrates that ERα DNA-binding is crucial for biological and transcriptional processes in reproductive tissues and that ERα tethering may not contribute to estrogen responsiveness in vivo.

Regeneration of pulp-dentin complex using human stem cells of the apical papilla: in vivo interaction with two bioactive materials

2021 ◽  
Author(s):  
Diana B. Sequeira ◽  
Ana Rafaela Oliveira ◽  
Catarina M. Seabra ◽  
Paulo J. Palma ◽  
Carlos Ramos ◽  
...  
Keyword(s):  
Stem Cells ◽  
Human Stem Cells ◽  
Bioactive Materials ◽  
Apical Papilla

scholarly journals C-Terminal Deletions Can Suppress Temperature-Sensitive Mutations and Change Dominance in the Phage Mu Repressor

Genetics ◽  
1996 ◽  
Vol 142 (3) ◽  
pp. 661-672 ◽  
Author(s):  
Jodi L Vogel ◽  
Vincent Geuskens ◽  
Lucie Desmet ◽  
N Patrick Higgins ◽  
Ariane Toussaint
Keyword(s):  
Binding Activity ◽  
Temperature Sensitive ◽  
Dna Binding Activity ◽  
Heat Stable ◽  
C Terminus ◽  
Acid Domain ◽  
Mutant Forms ◽  
Amino Acid Domain

Abstract Mutations in an N-terminal 70-amino acid domain of bacteriophage Mu's repressor cause temperature-sensitive DNA-binding activity. Surprisingly, amber mutations can conditionally correct the heat-sensitive defect in three mutant forms of the repressor gene, cts25 (D43-G), cts62 (R47-Q and cts71 (M28-I), and in the appropriate bacterial host produce a heat-stable Sts phenotype (for survival of temperature shifts). Sts repressor mutants are heat sensitive when in supE or supF hosts and heat resistant when in Sup° hosts. Mutants with an Sts phenotype have amber mutations at one of three codons, Q179, Q187, or Q190. The Sts phenotype relates to the repressor size: in Sup° hosts sts repressors are shorter by seven, 10, or 18 amino acids compared to repressors in supE or supF hosts. The truncated form of the sts62-1 repressor, which lacks 18 residues (Q179–V196), binds Mu operator DNA more stably at 42° in vitro compared to its full-length counterpart (cts62 repressor). In addition to influencing temperature sensitivity, the C-terminus appears to control the susceptibility to in vivo Clp proteolysis by influencing the multimeric structure of repressor.

scholarly journals LncRNA SPOCD1-AS from ovarian cancer extracellular vesicles remodels mesothelial cells to promote peritoneal metastasis via interacting with G3BP1

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Conghui Wang ◽  
Jiaying Wang ◽  
Xiameng Shen ◽  
Mingyue Li ◽  
Yongfang Yue ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Extracellular Vesicles ◽  
Peritoneal Metastasis ◽  
Cancer Metastasis ◽  
Cancer Therapeutics ◽  
Mesothelial Cells ◽  
Ovarian Cancer Cells ◽  
Adhesion Assay ◽  
Mesenchymal Transition

Abstract Background Metastasis is the key cause of death in ovarian cancer patients. To figure out the biological nature of cancer metastasis is essential for developing effective targeted therapy. Here we investigate how long non-coding RNA (lncRNA) SPOCD1-AS from ovarian cancer extracellular vesicles (EVs) remodel mesothelial cells through a mesothelial-to-mesenchymal transition (MMT) manner and facilitate peritoneal metastasis. Methods EVs purified from ovarian cancer cells and ascites of patients were applied to mesothelial cells. The MMT process of mesothelial cells was assessed by morphology observation, western blot analysis, migration assay and adhesion assay. Altered lncRNAs of EV-treated mesothelial cells were screened by RNA sequencing and identified by qRT-PCR. SPOCD1-AS was overexpressed or silenced by overexpression lentivirus or shRNA, respectively. RNA pull-down and RNA immunoprecipitation assays were conducted to reveal the mechanism by which SPOCD1-AS remodeled mesothelial cells. Interfering peptides were synthesized and applied. Ovarian cancer orthotopic implantation mouse model was established in vivo. Results We found that ovarian cancer-secreted EVs could be taken into recipient mesothelial cells, induce the MMT phenotype and enhance cancer cell adhesion to mesothelial cells. Furthermore, SPOCD1-AS embedded in ovarian cancer-secreted EVs was transmitted to mesothelial cells to induce the MMT process and facilitate peritoneal colonization in vitro and in vivo. SPOCD1-AS induced the MMT process of mesothelial cells via interacting with G3BP1 protein. Additionally, G3BP1 interfering peptide based on the F380/F382 residues was able to block SPOCD1-AS/G3BP1 interaction, inhibit the MMT phenotype of mesothelial cells, and diminish peritoneal metastasis in vivo. Conclusions Our findings elucidate the mechanism associated with EVs and their cargos in ovarian cancer peritoneal metastasis and may provide a potential approach for metastatic ovarian cancer therapeutics.

Dynamics of Telomeric DNA Turnover in Yeast

Genetics ◽  
2002 ◽  
Vol 160 (1) ◽  
pp. 63-73
Author(s):  
Michael J McEachern ◽  
Dana Hager Underwood ◽  
Elizabeth H Blackburn
Keyword(s):  
Kluyveromyces Lactis ◽  
Mutant Gene ◽  
Dna Repeats ◽  
Wild Type ◽  
Telomeric Dna ◽  
Cell Divisions ◽  
Dna Turnover ◽  
Length Regulation ◽  
Turn Over

Abstract Telomerase adds telomeric DNA repeats to telomeric termini using a sequence within its RNA subunit as a template. We characterized two mutations in the Kluyveromyces lactis telomerase RNA gene (TER1) template. Each initially produced normally regulated telomeres. One mutation, ter1-AA, had a cryptic defect in length regulation that was apparent only if the mutant gene was transformed into a TER1 deletion strain to permit extensive replacement of basal wild-type repeats with mutant repeats. This mutant differs from previously studied delayed elongation mutants in a number of properties. The second mutation, TER1-Bcl, which generates a BclI restriction site in newly synthesized telomeric repeats, was indistinguishable from wild type in all phenotypes assayed: cell growth, telomere length, and in vivo telomerase fidelity. TER1-Bcl cells demonstrated that the outer halves of the telomeric repeat tracts turn over within a few hundred cell divisions, while the innermost few repeats typically resisted turnover for at least 3000 cell divisions. Similarly deep but incomplete turnover was also observed in two other TER1 template mutants with highly elongated telomeres. These results indicate that most DNA turnover in functionally normal telomeres is due to gradual replicative sequence loss and additions by telomerase but that there are other processes that also contribute to turnover.

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