Yeast telomerase is capable of limited repeat addition processivity

Bosoy, Dimitry; Lue, Neal F.

doi:10.1093/nar/gkg943

Cited by 31 publications

(25 citation statements)

References 63 publications

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“…Specifically, we find that the length of yeast telomeres and nucleotide addition processivity of telomerase positively correlate with intracellular dGTP levels (Figures 3 and 4). Consistent with our observations, yeast telomerase mutants that alter nucleotide addition processivity, as measured in vitro, positively correlate with the in vivo length of the telomeres (Peng et al 2001), and this processivity is enhanced by increasing dGTP concentrations (Bosoy and Lue 2004). However, these in vitro telomerase assays examined only the processivity of nucleotide addition using the 59 portion of the TLC1 template region, whereas we were able to examine in vivo both the 39 and 59 portions and have found that reverse transcription of the 39 portion is more dramatically affected by changes in dGTP levels.…”

Section: Mec1 Mediates Telomere Length Homeostasis By Regulating Dntpsupporting

confidence: 90%

“…Furthermore, by using rnr1 mutants that perturb the balance of the four dNTPs, we find dramatic effects on telomere length homeostasis, both positive and negative. In particular, intracellular dGTP levels positively correlate with telomere length and telomerase nucleotide addition processivity in vivo, consistent with in vitro data showing that dGTP levels can stimulate telomerase activity in yeast (Peng et al 2001;Bosoy and Lue 2004), ciliates Cech 1997, 1998;Hardy et al 2001), and mammals (Morin 1989;Maine et al 1999). Thus, our findings reveal an important link between telomere length homeostasis and dNTP pools and show that alterations in intracellular dGTP levels can modulate telomerase activity.…”

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confidence: 90%

“…All of these observations suggest a possible link between telomere length homeostasis and regulation of dNTP pools. In vitro studies of telomerase from S. cerevisiae (Peng et al 2001;Bosoy and Lue 2004), Euplotes aediculatus Cech 1997, 1998), Tetrahymena thermophila (Hardy et al 2001), and mammals (Morin 1989;Maine et al 1999) also suggest that dGTP levels influence telomerase activity, but the in vivo relevance of these observations is unclear.…”

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Telomere Length Homeostasis Responds to Changes in Intracellular dNTP Pools

et al. 2013

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Telomeres, the ends of linear eukaryotic chromosomes, shorten due to incomplete DNA replication and nucleolytic degradation. Cells counteract this shortening by employing a specialized reverse transcriptase called telomerase, which uses deoxyribonucleoside triphosphates (dNTPs) to extend telomeres. Intracellular dNTP levels are tightly regulated, and perturbation of these levels is known to affect DNA synthesis. We examined whether altering the levels of the dNTP pools or changing the relative ratios of the four dNTPs in Saccharomyces cerevisiae would affect the length of the telomeres. Lowering dNTP levels leads to a modest shortening of telomeres, while increasing dNTP pools has no significant effect on telomere length. Strikingly, altering the ratio of the four dNTPs dramatically affects telomere length homeostasis, both positively and negatively. Specifically, we find that intracellular deoxyguanosine triphosphate (dGTP) levels positively correlate with both telomere length and telomerase nucleotide addition processivity in vivo. Our findings are consistent with in vitro data showing dGTP-dependent stimulation of telomerase activity in multiple organisms and suggest that telomerase activity is modulated in vivo by dGTP levels.A LL eukaryotes, as well as some prokaryotes with linear chromosomes, contain repetitive sequences called telomeres at the ends of their DNA. Telomeric DNA is bound by proteins that protect chromosome ends from being recognized as genotoxic DNA double-strand breaks in need of repair (Jain and Cooper 2010). However, telomeres shorten due to incomplete DNA replication and nucleolytic degradation. Left unchecked, this telomere erosion eventually results in very short, unprotected telomeres, leading to cell-cycle arrest and replicative senescence (Lundblad and Szostak 1989;Harley et al. 1990;Yu et al. 1990).Telomere shortening is counteracted by a specialized reverse transcriptase called telomerase (Greider and Blackburn 1985), whose core consists of a protein catalytic subunit and an RNA moiety-hTERT and hTR, respectively, in humans (Feng et al. 1995;Nakamura et al. 1997), and Est2 and TLC1, respectively, in the budding yeast Saccharomyces cerevisiae (Singer and Gottschling 1994;Lingner et al. 1997). Telomerase extends telomeres by repeated reverse transcription of a short sequence to the 39 ends of telomeres, using the RNA subunit as a template (Greider and Blackburn 1989;Yu et al. 1990;Singer and Gottschling 1994). Although the sequence of the telomeric repeats differs between species, a common feature is that they are all G-rich. In vertebrates, the repeat sequence is TTAGGG (Meyne et al. 1989), while in S. cerevisiae, the telomeric repeats have a consensus sequence of (TG) 0-6 TGGGTGTG(G) 0-1 (Forstemann and Lingner 2001).Deoxyribonucleoside triphosphates (dNTPs) are the building blocks of DNA, and their production needs to be tightly regulated as imbalances in dNTP pools can be mutagenic (Reichard 1988). In S. cerevisiae, the sole mode of dNTP production is through de novo dN...

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Section: Mec1 Mediates Telomere Length Homeostasis By Regulating Dntpsupporting

confidence: 90%

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Telomere Length Homeostasis Responds to Changes in Intracellular dNTP Pools

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“…We next assessed the impact of adding Mn 2ϩ , whose mutagenic effects on other polymerases are most extensively characterized (21,28). Interestingly, in the presence of the standard combination of 0.2 M dTTP and 50 M dGTP, which enables yeast telomerase to add Ϸ15 nucleotides (29) to the telomeric primer, the inclusion of 3 mM Mn 2ϩ resulted in a slight reduction in total DNA synthesis and a mild reduction in the processivity of telomerase (Fig. 1A, lanes 1-4).…”

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Telomerase can act as a template- and RNA-independent terminal transferase

Lue

Bosoy

Moriarty

et al. 2005

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

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Telomerase is a special reverse transcriptase that extends one strand of the telomere repeat by using a template embedded in an RNA subunit. Like other polymerases, telomerase is believed to use a pair of divalent metal ions (coordinated by a triad of aspartic acid residues) for catalyzing nucleotide addition. Here we show that, in the presence of manganese, both yeast and human telomerase can switch to a template-and RNA-independent mode of DNA synthesis, acting in effect as a terminal transferase. Even as a terminal transferase, yeast telomerase retains a species-dependent preference for GT-rich, telomere-like DNA on the 5 end of the substrate. The terminal transferase activity of telomerase may account for some of the hitherto unexplained effects of telomerase overexpression on cell physiology.reverse transcriptase ͉ telomere ͉ divalent metal ion T elomerase is a ribonucleoprotein responsible for the synthesis of one strand of the telomere terminal repeat. The catalytic core of telomerase consists minimally of two components: an RNA in which the template is embedded (named TLC1 in the budding yeast Saccharomyces cerevisiae), and a reverse transcriptase (RT)-like protein that mediates catalysis (named TERT in general and Est2p in yeast) (1-4). Telomerase RNAs from different organisms are divergent in sequence but share conserved secondary structural elements (5, 6). Many mutations in nontemplate regions of telomerase RNA reduced enzyme activity, suggesting that these regions contribute important catalytic functions [although the precise nature of the contribution(s) is not clear] (7-9). Other RNA structures act to define the boundary of the template segment that supports reverse transcription (10, 11). Thus, the RNA subunit serves multiple essential roles in the course of a normal telomerase reaction. The TERT protein is well conserved in evolution and comprises a central RT domain that is flanked on both the N-and C-terminal side by telomerase-specific motifs (4, 12, 13). Mutational analysis indicates that these motifs are likely to mediate binding to telomeric DNA and telomerase RNA. Direct recognition of telomeric DNA by TERT is believed to be sequence-dependent and to allow telomerase to catalyze the addition of multiple repeats without dissociation from the DNA (13,14).In unicellular organisms, telomerase is constitutively active and required for the long-term proliferation of cells. In some multicellular organisms, including humans, telomerase is repressed in normal somatic tissues but activated in cancer cells, and is believed to promote tumor growth (15). Although the two core components of telomerase are generally assumed to act in concert to replenish telomeres, there is increasing evidence that the TERT protein can have physiologic effects that are independent of telomere length and telomerase . For instance, in mice, TERT overexpression in the absence of telomerase RNA was reported to have an inhibitory effect on tumorigenesis and wound healing (20). The mechanisms for such effects are currently no...

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“…Earlier analysis suggests that for yeast telomerase polymerization beyond a single repeat can be most easily observed by using primers that form short hybrids with telomerase RNA (33,34). Thus, to assess nucleotide and repeat addition processivity simultaneously, I used one such primer, named OXYT1.…”

Section: Mutations In the N-gq Domain Caused A Primer-specificmentioning

confidence: 99%

A Physical and Functional Constituent of Telomerase Anchor Site

Lue

2005

Journal of Biological Chemistry

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Telomerase is a ribonucleoprotein reverse transcriptase responsible for the maintenance of one strand of the telomere terminal repeats. It consists minimally of a catalytic protein component (TERT) and an RNA subunit that provides the template. Compared with prototypical reverse transcriptases, telomerase is unique in possessing a DNA binding domain (anchor site) that is distinct from the catalytic site. Yeast TERT mutants bearing deletion or point mutations in an N-terminal domain (known as N-GQ) were found to be selectively impaired in extending primers that form short hybrids with telomerase RNA. The mutants also suffered a significant loss of repeat addition processivity but displayed an enhancement in nucleotide addition processivity. Furthermore, the mutants manifested altered primer utilization properties for oligonucleotides containing non-telomeric residues in the 5-region. Crosslinking studies indicate that the N-GQ domain physically contacts the 5-region of the DNA substrate in the context of a telomerase-telomere complex. Together, these results implicate the N-GQ domain of TERT as a physical and functional constituent of the telomerase anchor site. Coupled with previous genetic analysis, our data confirm that anchor site interaction is indeed important for telomerase function in vivo.Telomerase is a ribonucleoprotein that is responsible for maintaining the terminal repeats of telomeres in most organisms (1). It acts as an unusual reverse transcriptase, using a small segment of an integral RNA component as template for the synthesis of the dG-rich strand of telomeres (2).The enzymatic core of telomerase consists minimally of two components, a reverse transcriptase (RT) 1 -like protein that catalyzes nucleotide addition (named TERT), and an RNA in which the template is embedded (for reviews see Refs. 3-6). Telomerase RNAs are evolutionarily divergent in sequence, but recent studies suggest that they share a number of key structural elements, including a template segment, a pseudoknot, and a long-range base-pairing element (for reviews, see Refs. 7 and 8). The TERT protein, on the other hand, is well conserved in evolution and possesses a central domain with significant sequence similarity to prototypical RTs (Fig. 1A) (9, 10). Flanking the RT domain are a large N-terminal extension and a short C-terminal extension (CTE). By analogy with prototypical RTs, the CTE is likely to constitute the so-called thumb domain of the polymerase (11,12). The N-terminal extension consists of a non-conserved region (named N-region), four conserved motifs (named GQ, CP, QFP, and T), and a flexible linker (located between the GQ and CP motif) (13, 14). The CP, QFP, and T motifs have been shown to mediate interaction with telomerase RNA (15-18). The N-region and GQ motif together apparently comprise a stable domain (named N-GQ) that is required for telomerase function both in vitro and in vivo (see below).An unusual property of telomerase is its ability to mediate realignment of the DNA product relative to the RNA templat...

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Yeast telomerase is capable of limited repeat addition processivity

Cited by 31 publications

References 63 publications

Telomere Length Homeostasis Responds to Changes in Intracellular dNTP Pools

Telomere Length Homeostasis Responds to Changes in Intracellular dNTP Pools

Telomerase can act as a template- and RNA-independent terminal transferase

A Physical and Functional Constituent of Telomerase Anchor Site

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