Interaction of Human Telomerase with Its Primer Substrate

Wallweber, Gerald; Gryaznov, Sergei M.; Pongracz, Krisztina; Pruzan, Ronald

doi:10.1021/bi026914a

Cited by 55 publications

(66 citation statements)

References 28 publications

(39 reference statements)

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“…In the absence of POT1-TPP1, k off ¼ 0.012/min, in agreement with an earlier result (Wallweber et al, 2003). Unexpectedly, in the presence of a saturating quantity of POT1-TPP1, the primer dissociation rate was unchanged Figure 2 POT1-TPP1 decreases the dissociation rate of extending telomerase enzyme and increases the translocation rate.…”

Section: Pot1-tpp1 Increases the Processivity Of Telomerasesupporting

confidence: 90%

POT1–TPP1 enhances telomerase processivity by slowing primer dissociation and aiding translocation

Latrick

Cech

2010

EMBO J

165

198

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Telomerase contributes to chromosome end replication by synthesizing repeats of telomeric DNA, and the telomeric DNA-binding proteins protection of telomeres (POT1) and TPP1 synergistically increase its repeat addition processivity. To understand the mechanism of increased processivity, we measured the effect of POT1-TPP1 on individual steps in the telomerase reaction cycle. Under conditions where telomerase was actively synthesizing DNA, POT1-TPP1 bound to the primer decreased primer dissociation rate. In addition, POT1-TPP1 increased the translocation efficiency. A template-mutant telomerase that synthesizes DNA that cannot be bound by POT1-TPP1 exhibited increased processivity only when the primer contained at least one POT1-TPP1-binding site, so a single POT1-TPP1-DNA interaction is necessary and sufficient for stimulating processivity. The POT1-TPP1 effect is specific, as another single-stranded DNA-binding protein, gp32, cannot substitute. POT1-TPP1 increased processivity even when substoichiometric relative to the DNA, providing evidence for a recruitment function. These results support a model in which POT1-TPP1 enhances telomerase processivity in a manner markedly different from the sliding clamps used by DNA polymerases.

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Section: Pot1-tpp1 Increases the Processivity Of Telomerasesupporting

confidence: 90%

POT1–TPP1 enhances telomerase processivity by slowing primer dissociation and aiding translocation

Latrick

Cech

2010

EMBO J

165

198

View full text Add to dashboard Cite

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“…Evidence to support this conclusion comes from a previous study in which Harrington and Greider show that human telomerase can elongate primers lacking 3Ј telomeric DNA if G-rich or telomeric DNA is included at the 5Ј end (26). The cumulative data support a model in which telomerase can recognize telomeric DNA independently of the telomerase RNA subunit by DNA-protein recognition of G-rich sequences (26,33,43,49).…”

Section: Discussionmentioning

confidence: 72%

“…The first direct evidence for a telomerase anchor site came from photo-cross-linking studies with Euplotes aediculatus, which show an interaction between telomeric DNA substrates and (i) a TERT-sized protein and (ii) the telomerase RNA subunit (23). In vitro studies using telomerase partially purified from transformed human 293T cells demonstrate that the enzyme-primer complex is primarily stabilized by contacts between hTERT and the template-proximal region (49). Photo-cross-linking studies with S. cerevisiae suggest that the main cross-linking site on Est2p (the yeast homologue of hTERT) is within the first 190 amino acids and that TLC1 (the yeast homologue of hTR) is required to cross-link Est2p to telomeric DNA primers (36).…”

mentioning

confidence: 99%

Characterization of Physical and Functional Anchor Site Interactions in Human Telomerase

Wyatt

Lobb

Beattie

2007

Molecular and Cellular Biology

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Telomerase is a ribonucleoprotein reverse transcriptase (RT) that processively synthesizes telomeric repeats onto the ends of linear chromosomes to maintain genomic stability. It has been proposed that the N terminus of the telomerase protein subunit, telomerase RT (TERT), contains an anchor site that forms stable interactions with DNA to prevent enzyme-DNA dissociation during translocation and to promote realignment events that accompany each round of telomere synthesis. However, it is not known whether human TERT (hTERT) can directly interact with DNA in the absence of the telomerase RNA subunit. Here we use a novel primer binding assay to establish that hTERT forms stable and specific contacts with telomeric DNA in the absence of the human telomerase RNA component (hTR). We show that hTERT-mediated primer binding can be functionally uncoupled from telomerase-mediated primer extension. Our results demonstrate that the first 350 amino acids of hTERT have a critical role in regulating the strength and specificity of protein-DNA interactions, providing additional evidence that the TERT N terminus contains an anchor site. Furthermore, we establish that the RT domain of hTERT mediates important protein-DNA interactions. Collectively, these data suggest that hTERT contains distinct anchor regions that cooperate to help regulate telomerase-mediated DNA recognition and elongation.

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“…The dissociation rate of telomerase ðk off Þ has been measured in vitro; at 37°C it has a value of 0.01 min −1 (30). We estimate a value k on of 5 × 10 −3 ðnm · nMÞ −1 from the values for K m and k off reported by ref.…”

Section: Methodsmentioning

confidence: 94%

“…We estimate a value k on of 5 × 10 −3 ðnm · nMÞ −1 from the values for K m and k off reported by ref. 30. The value of c off ¼ k off .…”

Section: Methodsmentioning

confidence: 99%

Quantitative theory of telomere length regulation and cellular senescence

Rodriguez-Brenes

Peskin

2010

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

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In normal somatic cells, telomere length shortens with each cell replication. This progressive shortening is associated with cellular senescence and apoptosis. Germ cells, stem cells, and the majority of cancer cells express telomerase, an enzyme that extends telomere length and, when expressed at sufficient levels, can immortalize or extend the life span of a cell line. It is believed that telomeres switch between two states: capped and uncapped. The telomere state determines its accessibility to telomerase and also the onset of senescence. One hypothesis is that the t loop, a large lariat-like structure, represents the capped state. In this paper we model a telomere state on the basis of the biophysics of t-loop formation, allowing us to develop a single mathematical model that accounts for two processes: telomere length regulation for telomerase positive cells and cellular senescence in somatic cells. The model predicts the steady-state length distribution for telomerase positive cells, describes the time evolution of telomere length, and computes the life span of a cell line on the basis of the levels of TRF2 and telomerase expression. The model reproduces a wide range of experimental behavior and fits data from immortal cell lines (HeLa S3 and 293T) and somatic cells (human diploid fibroblasts) well. We conclude that the t loop as the capped state is a quantitatively reasonable model of telomere length regulation and cellular senescence. mathematical model | telomerase | telomeres T elomeres are noncoding repetitive sequences of DNA at the end of the chromosomes of eukaryotic cells. They play an important role in "hiding" the end of the chromosomes from the DNA damage response pathway. Dysfunctional telomeres may be interpreted as DNA breaks that may lead to nonhomologous end joining and may trigger senescence or apoptosis through the p53 and p16-RB pathways (1, 2).Because of the linear nature of eukaryotic DNA and the unidirectional synthesis of DNA polymerases, normal human somatic cells are unable to replicate their DNA completely (3). It is also believed that telomeres are actively degraded by an unknown exonuclease (4). These processes together with the action of oxidative stress (5) result in the progressive shortening of telomeres. Both in vivo and in vitro telomere lengths correlate with cellular life span.Germ cells, stem cells, and cancer cells (in approximately 85% of cancer types) express telomerase, an enzyme that extends telomere length (6). Cells that express telomerase at sufficient levels maintain a stable telomere length and have an unlimited replication capacity (7). Furthermore, it has been shown that the introduction of telomerase into normal human cells can extend the life span of a cell culture (8).The function and dynamics of telomeres depend on the action of several proteins that interact with chromosome ends. Particular attention has been given to shelterin, a complex of six telomere-specific proteins that protect and reshape telomeres (9). A key feature of shelterin is its ability...

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Interaction of Human Telomerase with Its Primer Substrate

Cited by 55 publications

References 28 publications

POT1–TPP1 enhances telomerase processivity by slowing primer dissociation and aiding translocation

POT1–TPP1 enhances telomerase processivity by slowing primer dissociation and aiding translocation

Characterization of Physical and Functional Anchor Site Interactions in Human Telomerase

Quantitative theory of telomere length regulation and cellular senescence

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