Dynamics of human telomerase recruitment depend on template-telomere base pairing

Schmidt, Jens C.; Zaug, Arthur J.; Kufer, Regina; Cech, Thomas R.

doi:10.1091/mbc.e17-11-0637

Cited by 30 publications

(28 citation statements)

References 35 publications

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“…The different dynamic properties could be explained by a faster rate of acquisition during imaging of HaLo-hTERT compared to MS2-hTR. Although, we cannot rule out that compromised telomerase activity due to hTERT tagging (Chiba et al, 2017; Schmidt et al, 2018) could have altered the dynamic behavior of the enzyme at telomeres.…”

Section: Discussionmentioning

confidence: 98%

“…In contrast HaLo-hTERT interactions are either probing (transient) or stably associated (localized) at telomeres. Furthermore, while the kinetic of hTERT binding to telomeres was not affected by GRN163L treatments (Schmidt et al, 2018), the drug significantly impacted the dwell time and off-rate of MS2-hTR (Figure 5). The different dynamic properties could be explained by a faster rate of acquisition during imaging of HaLo-hTERT compared to MS2-hTR.…”

Section: Discussionmentioning

confidence: 98%

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Single-molecule imaging of telomerase RNA reveals a Recruitment – Retention model for telomere elongation

Laprade

Querido

Smith³

et al. 2020

Preprint

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Extension of telomeres is a critical step in the immortalization of cancer cells. This complex reaction requires proper spatio-temporal coordination of telomerase and telomeres, and remains poorly understood at the cellular level. To understand how cancer cells execute this process, we combined CRISPR genome editing and MS2 RNA-tagging to image single-molecules of telomerase RNA (hTR). Real-time dynamics and photoactivation experiments of hTR in Cajal bodies (CBs) reveal that hTERT controls the exit of hTR from CBs. Single-molecule tracking of hTR at telomeres shows that TPP1-mediated recruitment results in short telomere-telomerase scanning interactions, then base-pairing between hTR and telomere ssDNA promotes long interactions required for stable telomerase retention. Interestingly, POT1 OB-fold mutations that result in abnormally long telomeres in cancers act by enhancing this retention step. In summary, single-molecule imaging unveils the life-cycle of telomerase RNA and provides a framework to understand how cancer-associated mutations mechanistically drive defects in telomere homeostasis.

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Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 98%

Single-molecule imaging of telomerase RNA reveals a Recruitment – Retention model for telomere elongation

Laprade

Querido

Smith³

et al. 2020

Preprint

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“…Together, our direct observation of telomerase cataly-sis using high-resolution optical tweezers reveals fundamental principles of the molecular mechanisms by which human telomerase processively elongates DNA (Fig. 4c), a process critical for telomere length maintenance 8,9 . Telomerase contains a secondary substrate binding site to prevent premature dissociation from the chromosome end.…”

mentioning

confidence: 71%

Section: Mjcomsto@msuedumentioning

confidence: 89%

“…We observed folding of the released product DNA into G-quadruplex structures. Our results provide detailed mechanistic insights into processive telomerase catalysis, a process critical for telomere length maintenance and therefore cancer cell survival 8,9 .To investigate substrate extension by a single telomerase ribonucleoprotein (RNP), we developed a single molecule telomerase activity assay using dual-trap high-resolution optical tweezers (Fig. 1a).…”

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confidence: 99%

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Mechanism of processive telomerase catalysis revealed by high-resolution optical tweezers

Patrick

Slivka

Payne

et al. 2019

Preprint

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Telomerase | Telomere | Optical Tweezers | Single-Molecule Fluorescence | G-quadruplex | Cancer | AgingCorrespondence:schmi706@msu.edu (Twitter: @jenscs83) and mjcomsto@msu.eduTelomere maintenance by telomerase is essential for continuous proliferation of human cells and is vital for the survival of stem cells and 90% of cancer cells 1 . Telomerase is a reverse transcriptase composed of telomerase reverse transcriptase (TERT) 2 , telomerase RNA (TR) 3 , and several accessory proteins 4 . To compensate for telomeric DNA lost during DNA replication, telomerase processively adds GGTTAG repeats to the singlestranded overhangs at chromosome ends by copying the template region within its RNA subunit 5,6 . Between repeat additions, the RNA template must be recycled, which requires disrupting the base-pairing between TR and the substrate DNA 6 . How telomerase remains associated with the substrate DNA during this critical translocation step remains unknown. Here, we demonstrate that stable substrate DNA binding at an anchor site within telomerase facilitates the processive synthesis of telomeric repeats. Using a newly developed single molecule telomerase activity assay utilizing high-resolution optical tweezers, we directly measured stepwise, processive telomerase activity. We found that telomerase tightly associates with its DNA substrate, synthesizing multiple telomeric repeats before releasing them in a single large step. The rate at which product is released from the anchor site closely corresponds to the overall rate of product dissociation from elongating telomerase 7 , suggesting that it is a key parameter controlling telomerase processivity. We observed folding of the released product DNA into G-quadruplex structures. Our results provide detailed mechanistic insights into processive telomerase catalysis, a process critical for telomere length maintenance and therefore cancer cell survival 8,9 .To investigate substrate extension by a single telomerase ribonucleoprotein (RNP), we developed a single molecule telomerase activity assay using dual-trap high-resolution optical tweezers (Fig. 1a). Telomerase and its substrate were attached to separate polystyrene beads (Fig. 1b). The connection between the two beads was formed by the association of telomerase with its substrate DNA ( Fig. 1a,b). When applying a low constant force (4.0-4.5 pN) to the tether, substrate elongation by telomerase was measured as an increase in dis-tance between the two beads ( Fig. 1a). To attach telomerase to the bead, we utilized a 3xFLAG-HaloTag on TERT, modified with biotin ( Fig. 1b,c). This tag did not affect telomerase assembly, catalytic activity, processivity, or stimulation by POT1/TPP1 ( Fig. 1c,d, Extended Data Fig. 1a-c), indicating that it is fully functional.Using this experimental approach, we set out to analyze how telomerase processively synthesizes telomeric repeats. If substrate DNA is only bound to telomerase by basepairing to TR we would expect to observe a sequence of single nucleotide (nt) addition events as a result o...

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Occurrence, functionality and abundance of the TERT promoter mutations

Rachakonda

Hoheisel

Kumar

2021

Intl Journal of Cancer

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Telomere shortening at chromosomal ends due to the constraints of the DNA replication process acts as a tumor suppressor by restricting the replicative potential in primary cells. Cancers evade that limitation primarily through the reactivation of telomerase via different mechanisms. Mutations within the promoter of the telomerase reverse transcriptase (TERT) gene represent a definite mechanism for the ribonucleic enzyme regeneration predominantly in cancers that arise from tissues with low rates of self-renewal. The promoter mutations cause a moderate increase in TERT transcription and consequent telomerase upregulation to the levels sufficient to delay replicative senescence but not prevent bulk telomere shortening and genomic instability. Since the discovery, a staggering number of studies have resolved the discrete aspects, effects and clinical relevance of the TERT promoter mutations. The promoter mutations link transcription of TERT with oncogenic pathways, associate with markers of poor outcome and define patients with reduced survivals in several cancers. In this review, we discuss the occurrence and impact of the promoter mutations and highlight the mechanism of TERT activation. We further deliberate on the foundational question of the abundance of the TERT promoter mutations and a general dearth of functional mutations within noncoding sequences, as evident from pan-cancer analysis of the whole-genomes. We posit that the favorable genomic constellation within the TERT promoter may be less than a common occurrence in other noncoding functional elements. Besides, the evolutionary constraints limit the functional fraction within the human genome, hence the lack of abundant mutations outside the coding sequences.

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Dynamics of human telomerase recruitment depend on template-telomere base pairing

Cited by 30 publications

References 35 publications

Single-molecule imaging of telomerase RNA reveals a Recruitment – Retention model for telomere elongation

Single-molecule imaging of telomerase RNA reveals a Recruitment – Retention model for telomere elongation

Mechanism of processive telomerase catalysis revealed by high-resolution optical tweezers

Occurrence, functionality and abundance of the TERT promoter mutations

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