Assays for Detection of Telomerase Activity

Skvortsov, Dmitry A.; Зверева, М.Э.; Shpanchenko, Olga V.; Dontsova, Olga А.

doi:10.32607/20758251-2011-3-1-48-68

Cited by 42 publications

(50 citation statements)

References 58 publications

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“…ddTRAP is accurate to one extension product, and is much more sensitive than both the direct methods and the conventional TRAP assays. It is linear in a broad dynamic range of the extension products being detected in our experiments 46 . On the beads, streptavidin molecules were at least 50 nm apart such that the telomerase holoenzymes tethered on the beads could not physically interact with each other.…”

Section: Resultsmentioning

confidence: 51%

A dynamic control of human telomerase holoenzyme

Sayed

Cheng

Yadav

et al. 2019

Preprint

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action, intracellular telomerase activating factors (iTAFs), telomerase reactivation by iTAFs, 20 single-run catalysis, fast and slow active sites, kinetics and thermodynamics 21 22 ABSTRACT: 23 Human telomerase functions in maintaining genome stability by adding telomeric repeats to the 24 termini of linear chromosomes. Past studies have revealed profound insights into telomerase 25 functions. However, low abundance of functional telomerase and difficulty in quantifying its 26 activity leave partially characterized its thermodynamic and kinetic properties. Using a newly 27 developed method to count individual extension products, we demonstrate that human 28 telomerase holoenzymes contain fast-and slow-acting catalytic sites. Surprisingly, both active 29 sites become inactive after two consecutive rounds of catalysis. The fast active sites turn off ~40-30 fold quicker than the slow ones and exhibit higher affinity to substrates. In dimeric enzymes, the 31 two sites work in tandem with the faster site functioning before the slower one. In monomeric 32 enzymes, the active sites also perform single-run catalysis. Interestingly, the inactive enzymes 33 can be reactivated by intracellular telomerase-activating factors (iTAFs) available in multiple 34 cell types. Together, the single-run catalysis and the iTAF-triggered reactivation serve as a novel 35 control circuit to ensure that the telomerase holoenzymes are dynamically controlled to match 36 their number of active sites with the number of telomeres they extend. Such exquisite kinetic 37 control of telomerase activity is expected to play important roles in cell division and ageing. 38 39 42problem. To maintain proper telomeric length, eukaryotic cells utilize telomerase to catalyze 43 addition of telomeric repeats using an intrinsic RNA template. In human cells, telomerase adds 44 hexameric repeats, (TTAGGG) n, to chromosomal termini 2 . During a cell cycle, a telomerase 45 enzyme is recruited to a transiently-uncapped telomere before it can function in a controlled 46 fashion 3 . It is believed that the telomerase preferentially acts on shorter telomeres 4-8 . Every 47 telomere should be acted on in order to maintain proper telomere length equilibrium; otherwise, 48 some telomeres would become shorter over time, leading to cellular senescence 9 . How 49 telomerase-expressing cells regulate telomere length in a global scale remains unclear. 51Telomerase plays a critical role in human diseases, in particular cancers and other age-related 52 diseases. With down-regulated telomerase activity, differentiating cells reach a critical state after 53 a certain number of cell divisions such that the cell cycle will be arrested and cells will enter 54 "senescence". How a cell senses changes in telomere length before senescence remains a 55 mystery. Approximately 85-90% of human cancers exhibit elevated telomerase activity 1, 10-18 . 56The importance of telomerase activity to cancer cells and other proliferative stem-like cells has 57 been well demonstrated. Chemical i...

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Section: Resultsmentioning

confidence: 51%

A dynamic control of human telomerase holoenzyme

Sayed

Cheng

Yadav

et al. 2019

Preprint

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“…Methods used for the detection of telomerase activity can be divided into two major groups as described by Skvortsov et al [83]: those based on direct detection of telomerase products, ( Table 1) and those based on different systems of amplification of the signals from DNA yield from telomerase ( Table 2). The methods discussed in this chapter (Figures 3 and 4) are suitable for testing telomerase activity in different types of samples such as; in protozoa, mammalian cells, mixed cellular populations, and tissues [83].…”

Section: Methods Of Measuring Telomerase Activitymentioning

confidence: 99%

Telomerase Structure and Function, Activity and Its Regulation with Emerging Methods of Measurement in Eukaryotes

Yadav¹,

Wakil²

2020

Telomerase and Non-Telomerase Mechanisms of Telomere Maintenance

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The telomerase reverse transcriptase has an essential role in telomere maintenance which is very important in aging process and cancer biology. Recent studies have revealed three-dimensional architecture of both human and ciliate telomerase at about 25 Å resolution, using single particle electron microscopy (EM). Telomerase supplements the tandem array of simple-sequence repeats at chromosome ends to compensate for the DNA erosion inherent in genome replication which makes it to be distinct among polymerases. Telomeres are found at the end of eukaryotic linear chromosomes and proteins that bind to them and help to protect DNA from being recognized as double-strand breaks thus preventing end-to-end fusions. The activity of telomerase is tightly regulated at multiple levels of cellular development, from transcriptional regulation of the telomerase components to holoenzyme biogenesis and recruitment to the telomere site for activation and processing. Commonly used methods in telomere biology are telomere restriction fragment (TRF), telomere repeat amplification protocol (TRAP) and telomere dysfunction induced foci (TIF) analysis. This chapter summarizes our current knowledge on the mechanisms of telomerase recruitment and activation using insights from studies in mammals and budding and fission yeasts. Finally, we discuss the differences in telomere homeostasis between different cell types and non-telomerase telomere maintenance mechanisms.

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“…Telomerase activity is most often checked by means of a TRAP assay, or telomeric repeat amplification protocol, in which the telomerase activity is (semi-)quantified with qPCR after an initial enzymatic incubation step. Also ELISA-based methods have been described, whether or not in combination with TRAP (elaborately reviewed in [54]). AP activity detection is often based on colorimetric assays, e.g.…”

Section: Overviewmentioning

confidence: 99%

Embryonic Stem Cells: Keeping Track of the Pluripotent Status

Vossaert¹,

Scheerlinck²,

Deforce³

2016

Pluripotent Stem Cells - From the Bench to the Clinic

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Embryonic stem cells are defined by their pluripotent status, which allows them to differentiate toward all cell types of an adult organism. This pluripotency can be characterized through many parameters, ranging from morphological traits, over certain enzymatic activities, to the expression of specific pluripotency factors, taken into account that these parameters may vary depending on the pluripotent stem cell type. As such, considerable differences are seen between human and mouse embryonic stem cell (ESC), or more generally stated, between primed and naïve pluripotent stem cells. This chapter offers an overview of the markers involved and the molecular biology techniques to monitor them during both ESC culture maintenance or differentiation experiments.

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Assays for Detection of Telomerase Activity

Cited by 42 publications

References 58 publications

A dynamic control of human telomerase holoenzyme

A dynamic control of human telomerase holoenzyme

Telomerase Structure and Function, Activity and Its Regulation with Emerging Methods of Measurement in Eukaryotes

Embryonic Stem Cells: Keeping Track of the Pluripotent Status

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