Telomerase Is Required to Protect Chromosomes with Vertebrate-type T2AG3 3′ Ends in Saccharomyces cerevisiae

Département de Microbiologie et d'Infectiologie, Groupe ARN/RNA Group, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, 3001 12e Ave. Nord, Sherbrooke, Quebec J1H 5N4, Canada.
Journal of Biological Chemistry (Impact Factor: 4.57). 06/2011; 286(31):27132-8. DOI: 10.1074/jbc.M111.220186
Source: PubMed


Telomeres containing vertebrate-type DNA repeats can be stably maintained in Saccharomyces cerevisiae cells. We show here that telomerase is required for growth of yeast cells containing these vertebrate-type telomeres. When present at the chromosome termini, these heterologous repeats elicit a DNA damage response and a certain deprotection of telomeres. The data also show that these phenotypes are due only to the terminal localization of the vertebrate repeats because if they are sandwiched between native yeast repeats, no phenotype is observed. Indeed and quite surprisingly, in this latter situation, telomeres are of virtually normal lengths, despite the presence of up to 50% of heterologous repeats. Furthermore, the presence of the distal vertebrate-type repeats can cause increased problems of the replication fork. These results show that in budding yeast the integrity of the 3' overhang is required for proper termination of telomere replication as well as protection.

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    • "Although the telomeric sequence in budding yeast differs from human, the telomere lagging strand template is G-rich (TG1-3) and forms G4 DNA in vivo (31). Furthermore, replication forks stall at human telomeric repeats in yeast whether the TTAGGG repeats are engineered into a plasmid or at chromosome ends (32,33). Circular ssDNA templates were prepared from phagemids containing 10 telomeric repeats inserted in either orientation to generate lagging (G-rich) or leading (C-rich) strand templates (Figure 1A). "
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    ABSTRACT: Previous evidence indicates that telomeres resemble common fragile sites and present a challenge for DNA replication. The precise impediments to replication fork progression at telomeric TTAGGG repeats are unknown, but are proposed to include G-quadruplexes (G4) on the G-rich strand. Here we examined DNA synthesis and progression by the replicative DNA polymerase δ/proliferating cell nuclear antigen/replication factor C complex on telomeric templates that mimic the leading C-rich and lagging G-rich strands. Increased polymerase stalling occurred on the G-rich template, compared with the C-rich and nontelomeric templates. Suppression of G4 formation by substituting Li(+) for K(+) as the cation, or by using templates with 7-deaza-G residues, did not alleviate Pol δ pause sites within the G residues. Furthermore, we provide evidence that G4 folding is less stable on single-stranded circular TTAGGG templates where ends are constrained, compared with linear oligonucleotides. Artificially stabilizing G4 structures on the circular templates with the G4 ligand BRACO-19 inhibited Pol δ progression into the G-rich repeats. Similar results were obtained for yeast and human Pol δ complexes. Our data indicate that G4 formation is not required for polymerase stalling on telomeric lagging strands and suggest that an alternative mechanism, in addition to stable G4s, contributes to replication stalling at telomeres.
    Nucleic Acids Research 09/2013; 41(22). DOI:10.1093/nar/gkt813 · 9.11 Impact Factor
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    • "This increased Cdc13 binding might be due to longer single-stranded overhangs on vertebrate-like telomeres (18,20). More recently, it has been proposed that telomerase itself is required to protect telomeres ending with T2AG3 repeats probably because they need to be elongated more frequently than canonical telomeres (21). "
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    ABSTRACT: Vertebrate-like T2AG3 telomeres in tlc1-h yeast consist of short double-stranded regions and long single-stranded overhang (G-tails) and, although based on Tbf1-capping activity, they are capping deficient. Consistent with this idea, we observe Y’ amplification because of homologous recombination, even in the presence of an active telomerase. In these cells, Y’ amplification occurs by different pathways: in Tel1+ tlc1h cells, it is Rad51-dependent, whereas in the absence of Tel1, it depends on Rad50. Generation of telomeric G-tail, which is cell cycle regulated, depends on the MRX (Mre11-Rad50-Xrs2) complex in tlc1h cells or is MRX-independent in tlc1h tel1Δ mutants. Unexpectedly, we observe telomere elongation in tlc1h lacking Rad51 that seems to act as a telomerase competitor for binding to telomeric G-tails. Overall, our results show that Tel1 and Rad51 have multiple roles in the maintenance of vertebrate-like telomeres in yeast, supporting the idea that they may participate to evolutionary conserved telomere protection mechanism/s acting at uncapped telomeres.
    Nucleic Acids Research 05/2013; 41(13). DOI:10.1093/nar/gkt365 · 9.11 Impact Factor
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    • "ARIA does not contain subtelomeric sequences and is not synthesized by RNA pol II. It should be generated via promoter sites composed of telomeric repeats (Bah et al., 2011; Greenwood and Cooper, 2012). A specific role of ARIA has not been described so far but we can hypothesize that it could act as a regulator of TERRA or of the 3′ overhang by hybridizing to these RNA/DNA single strands. "
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    ABSTRACT: A major issue in telomere research is to understand how the integrity of chromosome ends is controlled. The fact that different types of nucleoprotein complexes have been described at the telomeres of different organisms raises the question of whether they have in common a structural identity that explains their role in chromosome protection. We will review here how telomeric nucleoprotein complexes are structured, comparing different organisms and trying to link these structures to telomere biology. It emerges that telomeres are formed by a complex and specific network of interactions between DNA, RNA, and proteins. The fact that these interactions and associated activities are reinforcing each other might help to guarantee the robustness of telomeric functions across the cell cycle and in the event of cellular perturbations. We will also discuss the recent notion that telomeres have evolved specific systems to overcome the DNA topological stress generated during their replication and transcription. This will lead to revisit the way we envisage the functioning of telomeric complexes since the regulation of topology is central to DNA stability, replication, recombination, and transcription as well as to chromosome higher-order organization.
    Frontiers in Oncology 03/2013; 3:48. DOI:10.3389/fonc.2013.00048
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