Generation of Telomeric G Strand Overhangs Involves Both G and C Strand Cleavage

Jacob, Naduparambil K.; Kirk, Karen E.; Price, Carolyn M.

doi:10.1016/s1097-2765(03)00131-x

Cited by 55 publications

(40 citation statements)

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“…Although the nucleases responsible for telomere processing remain unknown, studies with yeast and vertebrate cells suggest that they are likely to be general DNA repair nucleases rather than dedicated sequence-specific telomere nucleases (4,29). If this is the case, the specificity of overhang processing may come from G-overhang binding proteins providing the boundaries for nuclease digestion (19).…”

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confidence: 99%

Tetrahymena POT1a Regulates Telomere Length and Prevents Activation of a Cell CycleCheckpoint

Jacob

Lescasse

Linger

et al. 2007

Molecular and Cellular Biology

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The POT1/TEBP telomere proteins are a group of single-stranded DNA (ssDNA)-binding proteins that have long been assumed to protect the G overhang on the telomeric 3 strand. We have found that the Tetrahymena thermophila genome contains two POT1 gene homologs, POT1a and POT1b. The POT1a gene is essential, but POT1b is not. We have generated a conditional POT1a cell line and shown that POT1a depletion results in a monster cell phenotype and growth arrest. However, G-overhang structure is essentially unchanged, indicating that POT1a is not required for overhang protection. In contrast, POT1a is required for telomere length regulation. After POT1a depletion, most telomeres elongate by 400 to 500 bp, but some increase by up to 10 kb. This elongation occurs in the absence of further cell division. The growth arrest caused by POT1a depletion can be reversed by reexpression of POT1a or addition of caffeine. Thus, POT1a is required to prevent a cell cycle checkpoint that is most likely mediated by ATM or ATR (ATM and ATR are protein kinases of the PI-3 protein kinase-like family). Our findings indicate that the essential function of POT1a is to prevent a catastrophic DNA damage response. This response may be activated when nontelomeric ssDNA-binding proteins bind and protect the G overhang.In order to maintain genome integrity, the telomeric DNA from cells with linear chromosomes is packaged into a protective nucleoprotein complex (10). In the absence of this complex, the telomeres are recognized as DNA damage and subject to repair by nonhomologous end joining (33). The resulting chromosome fusions lead to genome instability (36). The protective telomeric complex is composed of a series of unique telomere proteins that bind the double-stranded region of the telomeric DNA and/or the single-strand overhang on the 3Ј G-rich strand (37). Although the exact composition of the complex varies between species, vertebrates, yeasts, plants, and ciliates all use a series of structurally related proteins to protect their telomeres. The G-overhang binding proteins all bind Gstrand DNA through a conserved OB fold motif, while the double-stranded DNA-binding proteins bind via a conserved myb motif (23,37,46).In vertebrate cells, telomeres are packaged by a core complex of six proteins which function both in telomere protection and in telomere length regulation (10). This core complex (which is sometimes called shelterin) contains two doublestranded DNA-binding proteins, TRF1 and TRF2, which anchor the complex along the length of the telomere, and the TRF1/2 interaction partners Rap1, TIN2, and TPP1. The Gstrand binding protein POT1 is also a component of the complex. Although POT1 is secured into the complex through interactions with TPP1 (17, 31, 50), POT1 molecules are also thought to bind the G-strand overhang via their OB foldcontaining DNA-binding domain (45,46). Additional telomere-associated proteins include a number of DNA damage response factors (33). However, these factors appear to bind only transiently during replication of...

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Tetrahymena POT1a Regulates Telomere Length and Prevents Activation of a Cell CycleCheckpoint

Jacob

Lescasse

Linger

et al. 2007

Molecular and Cellular Biology

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“…Teb1⌬L 45 showed a decrease in telomere interaction but retained a ChIP signal within ϳ2-fold of the wild-type signal (Fig. 6B).…”

Section: Resultsmentioning

confidence: 93%

“…7). Tetrahymena telomeres are capped by Pot1a in complex with Tpt1, Pat1, and Pat2 (25,26), bound to an overhang of 14 to 15 or 20 to 21 nucleotides with a TGGGGT-3=OH end permutation (45,46). This length of overhang is insufficient for binding both Pot1a and Teb1 (34).…”

Section: Discussionmentioning

confidence: 99%

“…F-Teb1BC and F-Teb1C were expressed in the genetic background of TERT with a C-terminal ZZ tag (33). New strains Teb1 F590A F648A-FZZ, Teb1 ⌬555-581 GSGSG-FZZ (⌬Zn), Teb1 ⌬660 -666 AGSSG-FZZ (⌬L 45 ), Teb1 ⌬687-701-FZZ (⌬CT␣H), Teb1 F293A-FZZ, Teb1 K300A-FZZ, Teb1 F423A-FZZ, Teb1 Y450A-FZZ, Teb1 F603A-FZZ, and Teb1 K660A-FZZ were made by targeting transgene integration at the ␤-tubulin 1 locus (BTU1) under the control of the metallothionein 1 gene (MTT1) promoter using selection for blasticidin resistance conferred by the bsr2 cassette (36). Strains expressing p50N25-FZZ, ZZF-p50, F-Teb1BC, F-Teb1C, and all of the newly generated Teb1-FZZ strains retain endogenous subunit expression for viability.…”

Section: Methodsmentioning

confidence: 99%

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Direct Single-Stranded DNA Binding by Teb1 Mediates the Recruitment of Tetrahymena thermophila Telomerase to Telomeres

Upton

Hong

Collins

2014

Molecular and Cellular Biology

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The eukaryotic reverse transcriptase telomerase copies its internal RNA template to synthesize telomeric DNA repeats at chromosome ends in balance with sequence loss during cell proliferation. Previous work has established several factors involved in telomerase recruitment to telomeres in yeast and mammalian cells; however, it remains unclear what determines the association of telomerase with telomeres in other organisms. Here we investigate the cell cycle dependence of telomere binding by each of the seven Tetrahymena thermophila telomerase holoenzyme proteins TERT, p65, Teb1, p50, p75, p45, and p19. We observed coordinate cell cycle-regulated recruitment and release of all of the subunits, including the telomeric-repeat DNA-binding subunit Teb1. Using domain truncation and mutagenesis approaches, we investigated which subunits govern the interaction of telomerase holoenzyme with telomeres. Our results show that Teb1 is critical for telomere interaction of other holoenzyme subunits and demonstrate that high-affinity Teb1 DNA-binding activity is necessary and sufficient for cell cycle-regulated telomere association. Overall, these and additional findings indicate that in the ciliate Tetrahymena, telomerase recruitment to telomeres requires direct binding to single-stranded DNA, unlike the indirect DNA recognition through telomere-bound proteins essential in yeast and mammalian cells.

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“…Loss of terminal sequence could occur if second strand synthesis did not start at the extreme end of the reverse transcribed DNA or if the synthesis were primed by sequence that was removed and not replaced after replication. In Tetrahymena (19), yeast (20), and humans (21,22), the ends of telomere DNA are known to be shaped by precise postreplicative processing. Much of this processing is accomplished by as yet unidentified proteins, but one protein identified in Saccharomyces cerevisiae (20) and humans (23) is Mre11.…”

Section: Resultsmentioning

confidence: 99%

Evolution of species-specific promoter-associated mechanisms for protecting chromosome ends by Drosophila Het - A telomeric transposons

Traverse¹,

George²,

DeBaryshe³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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The non-LTR retrotransposons forming Drosophila telomeres constitute a robust mechanism for telomere maintenance, one which has persisted since before separation of the extant Drosophila species. These elements in D. melanogaster differ from nontelomeric retrotransposons in ways that give insight into general telomere biology. Here, we analyze telomere-specific retrotransposons from D. virilis, separated from D. melanogaster by 40 to 60 million years, to evaluate the evolutionary divergence of their telomeric traits. The telomeric retrotransposon HeT-A from D. melanogaster has an unusual promoter near its 3′ terminus that drives not the element in which it resides, but the adjacent downstream element in a head-to-tail array. An obvious benefit of this promoter is that it adds nonessential sequence to the 5′ end of each transcript, which is reverse transcribed and added to the chromosome. Because the 5′ end of each newly transposed element forms the end of the chromosome until another element transposes onto it, this nonessential sequence can buffer erosion of sequence essential for HeT-A. Surprisingly, we have now found that HeT-A in D. virilis has a promoter typical of non-LTR retrotransposons. This promoter adds no buffering sequence; nevertheless, the complete 5′ end of the element persists in telomere arrays, necessitating a more precise processing of the extreme end of the telomere in D. virilis.telomeres | retrotransposons | end replication problem | promoter evolution H eT-A, TART, and TAHRE are non-LTR retrotransposons that maintain the telomeres in Drosophila melanogaster. They transpose only to chromosome ends, where they form long arrays of head-to-tail repeats that make up the telomeres. Despite their differences, this retrotransposon mechanism for extending chromosome ends is functionally equivalent to the telomerase mechanism. In each case, an RNA template is reverse transcribed to add DNA repeats to the chromosome. Telomerase repeats are short sequences copied from part of the enzyme's RNA component. In Drosophila, each repeat is a copy of one of the three telomerespecific retrotransposons. These retrotransposons have unusual features, some in common, some not, but all presumably related to their role at the telomere (reviewed in refs. 1, 2). This study of species differences in the HeT-A promoters lends insight into the mechanisms by which HeT-A elements protect chromosome ends.A notable feature of D. melanogaster HeT-A is its promoter, which has unexpected similarities to promoters of retroviruses and LTR retrotransposons (3). HeT-A promoter sequences are found at the 3′ end of each element, in the 3′untranslated region (UTR). They direct transcription of the neighbor immediately downstream, rather than the element in which they reside (see Fig. 1). To an outside observer, if the downstream element is another HeT-A, the combined sequence (consisting of the 3′ sequence of the upstream element plus the entire downstream element) appears to be, and is formally equivalent to, an LTR retrotransposon ...

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Generation of Telomeric G Strand Overhangs Involves Both G and C Strand Cleavage

Cited by 55 publications

References 44 publications

Tetrahymena POT1a Regulates Telomere Length and Prevents Activation of a Cell CycleCheckpoint

Tetrahymena POT1a Regulates Telomere Length and Prevents Activation of a Cell CycleCheckpoint

Direct Single-Stranded DNA Binding by Teb1 Mediates the Recruitment of Tetrahymena thermophila Telomerase to Telomeres

Evolution of species-specific promoter-associated mechanisms for protecting chromosome ends by Drosophila Het - A telomeric transposons

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