A mutation in the catalytic subunit of yeast telomerase alters primer–template alignment while promoting processivity and protein–DNA binding

Bairley, Robin C. B.; Guillaume, G.; Vega, Leticia R.; Friedman, Katherine L.

doi:10.1242/jcs.090761

Cited by 11 publications

(13 citation statements)

References 49 publications

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“…Several surface-exposed residues have been implicated in DNA binding based on the ttTEN structure from T. thermophila ( 12 ). The popular hypothesis that the TEN domain functions as an anchor site for DNA is not supported by the experimental data since the isolated TEN domain shows only weak, if any, affinity to single-stranded DNA fragments ( 75–78 ). By contrast, the TERT ring without TEN interacts with single-stranded DNA, although TEN further promotes this binding ( 77 , 79 ).…”

Section: Resultsmentioning

confidence: 99%

Structure and function of the N-terminal domain of the yeast telomerase reverse transcriptase

Petrova

Mantsyzov

Rodina

et al. 2017

Nucleic Acids Research

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The elongation of single-stranded DNA repeats at the 3′-ends of chromosomes by telomerase is a key process in maintaining genome integrity in eukaryotes. Abnormal activation of telomerase leads to uncontrolled cell division, whereas its down-regulation is attributed to ageing and several pathologies related to early cell death. Telomerase function is based on the dynamic interactions of its catalytic subunit (TERT) with nucleic acids—telomerase RNA, telomeric DNA and the DNA/RNA heteroduplex. Here, we present the crystallographic and NMR structures of the N-terminal (TEN) domain of TERT from the thermotolerant yeast Hansenula polymorpha and demonstrate the structural conservation of the core motif in evolutionarily divergent organisms. We identify the TEN residues that are involved in interactions with the telomerase RNA and in the recognition of the ‘fork’ at the distal end of the DNA product/RNA template heteroduplex. We propose that the TEN domain assists telomerase biological function and is involved in restricting the size of the heteroduplex during telomere repeat synthesis.

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Section: Resultsmentioning

confidence: 99%

Structure and function of the N-terminal domain of the yeast telomerase reverse transcriptase

Petrova

Mantsyzov

Rodina

et al. 2017

Nucleic Acids Research

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“…For example, de novo telomeres generated following DNA cleavage by HO endonuclease near a TG-rich seed sequence are frequently added at the 3= overhang of the HO endonuclease target site rather than within the telomeric seed itself (23). At the SiRTA on chromosome VII reported by Mangahas et al (40), telomere addition occurs at several closely spaced sequences located 37 to 49 bp distal to a 35-bp GT dinucleotide repeat.…”

Section: Discussionmentioning

confidence: 99%

Endogenous Hot Spots of De Novo Telomere Addition in the Yeast Genome Contain Proximal Enhancers That Bind Cdc13

Obodo

Platts

Seloff

et al. 2016

Molecular and Cellular Biology

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DNA double-strand breaks (DSBs) pose a threat to genome stability and are repaired through multiple mechanisms. Rarely, telomerase, the enzyme that maintains telomeres, acts upon a DSB in a mutagenic process termed telomere healing. The probability of telomere addition is increased at specific genomic sequences termed sites of repair-associated telomere addition (SiRTAs). By monitoring repair of an induced DSB, we show that SiRTAs on chromosomes V and IX share a bipartite structure in which a core sequence (Core) is directly targeted by telomerase, while a proximal sequence (Stim) enhances the probability of de novo telomere formation. The Stim and Core sequences are sufficient to confer a high frequency of telomere addition to an ectopic site. Cdc13, a single-stranded DNA binding protein that recruits telomerase to endogenous telomeres, is known to stimulate de novo telomere addition when artificially recruited to an induced DSB. Here we show that the ability of the Stim sequence to enhance de novo telomere addition correlates with its ability to bind Cdc13, indicating that natural sites at which telomere addition occurs at high frequency require binding by Cdc13 to a sequence 20 to 100 bp internal from the site at which telomerase acts to initiate de novo telomere addition. Chromosomes in the budding yeast Saccharomyces cerevisiae, as in all eukaryotes, terminate with specialized nucleoprotein structures called telomeres. S. cerevisiae telomeric DNA consists of ϳ250 to 350 bp of TG 1-3 /AC 1-3 repeats and a short (ϳ10-bp) terminal G-rich 3= overhang (1). Because the conventional DNA replication machinery cannot fully replicate chromosome ends, telomeres shorten with each cell division cycle. In most eukaryotes, telomere shortening is counteracted by the enzyme telomerase, a ribonucleoprotein complex that uses an intrinsic RNA subunit as the template for telomeric DNA synthesis. Associated with telomeric DNA are proteins that protect chromosome ends from nucleolytic resection and prevent chromosome end-to-end fusions by distinguishing natural chromosome ends from ends generated by DNA double-strand breaks (DSBs) (2). These protective functions make telomeres essential for the maintenance of genome integrity and cell viability.In S. cerevisiae, optimal telomere length requires a balance between positive and negative regulatory mechanisms mediated by telomere-binding proteins, including Cdc13 and Rap1 (3). Cdc13, a telomere sequence-specific single-stranded DNA (ssDNA) binding protein, recruits telomerase to telomeres during the S/G 2 phase of the cell cycle through interaction with Est1, a subunit of the telomerase holoenzyme (reviewed in reference 3). Rap1 binds to the double-stranded telomeric repeat, forming a telomere length-regulatory complex through interactions of its C-terminal domain with Rif1 and Rif2 (4,5). Regulation occurs through a counting mechanism in which telomere length is inversely proportional to the number of Rif1 and Rif2 molecules present at a telomere (6, 7).Cells experience insults to t...

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“…Liquid cultures were grown to saturation in selective media at the appropriate temperature. Genomic DNA was isolated from each strain by glass bead lysis (38) and prepared for ligation-mediated telomere PCR as described previously (40). In brief, after RNase treatment, genomic DNA was blunted with T4 DNA polymerase (New England BioLabs) and ligated to a double-stranded oligonucleotide.…”

Section: Methodsmentioning

confidence: 99%

Normal Telomere Length Maintenance in Saccharomyces cerevisiae Requires Nuclear Import of the Ever Shorter Telomeres 1 (Est1) Protein via the Importin Alpha Pathway

Hawkins

Friedman

2014

Eukaryot Cell

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The Est1 (ever shorter telomeres 1) protein is an essential component of yeast telomerase, a ribonucleoprotein complex that restores the repetitive sequences at chromosome ends (telomeres) that would otherwise be lost during DNA replication. Previous work has shown that the telomerase RNA component (TLC1) transits through the cytoplasm during telomerase biogenesis, but mechanisms of protein import have not been addressed. Here we identify three nuclear localization sequences (NLSs) in Est1p. Mutation of the most N-terminal NLS in the context of full-length Est1p reduces Est1p nuclear localization and causes telomere shortening-phenotypes that are rescued by fusion with the NLS from the simian virus 40 (SV40) large-T antigen. In contrast to that of the TLC1 RNA, Est1p nuclear import is facilitated by Srp1p, the yeast homolog of importin ␣. The reduction in telomere length observed at the semipermissive temperature in a srp1 mutant strain is rescued by increased Est1p expression, consistent with a defect in Est1p nuclear import. These studies suggest that at least two nuclear import pathways are required to achieve normal telomere length homeostasis in yeast.T elomeres, the heterochromatic, G/T-rich regions of DNA located at the ends of linear chromosomes, are dynamic structures that undergo multiple rounds of attrition and elongation over the lifetimes of many eukaryotic cells. Because telomeres provide an essential capping function that protects DNA ends and aids in the maintenance of genomic stability, most eukaryotes use the enzyme telomerase to elongate telomeres (1).Telomerase is a ribonucleoprotein complex in which the RNA subunit interacts with a specialized reverse transcriptase to synthesize telomeric DNA. In the yeast Saccharomyces cerevisiae, telomerase minimally consists of the TLC1 RNA, which contains the template for nucleotide addition, and three ever shorter telomere (EST) proteins (2-5). Est2p is the reverse transcriptase that, together with TLC1 RNA, is necessary and sufficient for enzyme activity in vitro (6, 7). Est1p and Est3p are essential regulatory components that stimulate the in vitro activity of telomerase and have been implicated in the recruitment and/or activation of telomerase at the telomere (5,6,8,9).Interactions between the subunits of telomerase and between telomerase and the telomere are complex. Est1p interacts with the single-stranded telomeric DNA binding protein, Cdc13p (10, 11). Ectopic expression of a Cdc13-Est2 fusion protein bypasses the requirement for EST1, suggesting that Est1p recruits telomerase to the telomere through the interaction with Cdc13p (12). TLC1 RNA possesses distinct binding sites for Est1p and Est2p, suggesting that the interaction between Est1p and Est2p is mediated by the telomerase RNA in vivo (13-16). However, an RNA-independent interaction between Est1p and Est2p has been observed (8). In live cells, persistent foci of TLC1 RNA are detected at telomeres during S phase-a phenotype greatly reduced in cells harboring the cdc13-2 mutation, in which t...

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A mutation in the catalytic subunit of yeast telomerase alters primer–template alignment while promoting processivity and protein–DNA binding

Cited by 11 publications

References 49 publications

Structure and function of the N-terminal domain of the yeast telomerase reverse transcriptase

Structure and function of the N-terminal domain of the yeast telomerase reverse transcriptase

Endogenous Hot Spots of De Novo Telomere Addition in the Yeast Genome Contain Proximal Enhancers That Bind Cdc13

Normal Telomere Length Maintenance in Saccharomyces cerevisiae Requires Nuclear Import of the Ever Shorter Telomeres 1 (Est1) Protein via the Importin Alpha Pathway

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