Telomere DNA G-quadruplex folding within actively extending human telomerase

Jansson, Linnea I.; Hentschel, Jendrik; Parks, Joshua W.; Chang, Terren; Lü, Cheng; Baral, Rishika; Bagshaw, Clive R.; Stone, Michael D.

doi:10.1073/pnas.1814777116

Cited by 100 publications

(59 citation statements)

References 77 publications

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“…The overall extension rate was slightly reduced, consistent with bulk experiments (Extended Data Fig. 3b) 20 . However, lithium eliminated the peaks centered at 15 nt in the step size distribution in both the forward and reverse directions ( Fig.…”

supporting

confidence: 87%

“…We next sought to determine the molecular mechanisms that underlie the reverse steps observed in telomerase catalysis trajectories. Reverse steps could be the consequence of two distinct processes: 1) Rebinding of the product to the anchor site, or 2) folding of product into a G-quadruplex (GQ) structure, which has been shown to affect telomerase catalysis 20 . The most common reverse step size is approximately 15 nt ( Fig.…”

mentioning

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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supporting

confidence: 87%

mentioning

confidence: 99%

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

Patrick

Slivka

Payne

et al. 2019

Preprint

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“…1C) [25][26][27][28]. Extensive research indicated that G-quadruplexes were involved in many critical biological processes, including DNA replication [29][30][31][32], telomere regulation [33][34][35][36][37], and RNA translation [38][39][40][41]. It has been proved that G-quadruplexes existed in the viral genome and can regulate the viral biological processes, which made it possible to function as potential drug targets for antiviral strategy [42][43][44].…”

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confidence: 99%

Whole genome identification of potential G-quadruplexes and analysis of the G-quadruplex binding domain for SARS-CoV-2

Zhang

Xiao

et al. 2020

Preprint

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The Coronavirus Disease 2019 pandemic caused by SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) quickly become a global public health emergency. Gquadruplex, one of the non-canonical secondary structures, has shown potential antiviral values. However, little is known about G-quadruplexes on the emerging SARS-CoV-2. Herein, we characterized the potential G-quadruplexes both in the positive and negative-sense viral stands. The identified potential G-quadruplexes exhibits similar features to the G-quadruplexes detected in the human transcriptome. Within some bat and pangolin related beta coronaviruses, the G-quartets rather than the loops are under heightened selective constraints. We also found that the SUD-like sequence is retained in the SARS-CoV-2 genome, while some other coronaviruses that can infect humans are depleted. Further analysis revealed that the SARS-CoV-2 SUD-like sequence is almost conserved among 16,466 SARS-CoV-2 samples. And the SARS-CoV-2 SUDcore-like dimer displayed similar electrostatic potential pattern to the SUD dimer. Considering the potential value of G-quadruplexes to serve as targets in antiviral strategy, we hope our fundamental research could provide new insights for the SARS-CoV-2 drug discovery. Respiratory Syndrome (MERS) in 2012 [2,5], and COVID-19. Scientists identified and sequenced the virus early in this outbreak, and named it SARS-CoV-2[6]. The symptoms of the patients infected with the novel coronavirus vary from person to person, and fever, cough, and fatigue are the most common ones[7-10]. The clinical chest CT (Computed tomography) and nucleic acid testing are the most typical methods of diagnosing 10]. It is worth noting that the recent achievements in AI (Artificial Intelligence) aid diagnosis technology[11] and CRISPR-Cas12-based detection methods [12] are expected to expand the diagnosis of COVID-19. Despite the great efforts of the researchers, so far, no specific clinical drugs or vaccines have been developed to cope with COVID-19. SARS-CoV-2 is a Betacoronavirus within the Coronaviridae family that is the culprit responsible for the COVID-19 pandemic [13,14] (Fig. 1A). Studies have confirmed that SARS-CoV-2 is a positive-sense single-stranded RNA ((+)ssRNA) virus with a total length of approximately 30k. The positive-sense RNA strand of SARS-CoV-2 can serve as a template to produce viral proteins related to replication, structure composition, and other functions or events [15,16]. One of the hotspots is how the SARS-CoV-2 entry the host cells. SARS-CoV-2 has shown a great affinity to the angiotensin-converting enzyme 2 (ACE2), which has been proved to be the binding receptor for SARS-CoV-2 [17,18]. After entering the host cells, the viral genomic RNA will be released to the cytoplasm, and the ORF1a/ORF1ab are subsequently translated into replicase polyproteins of pp1a/pp1ab, which will be cleaved into some non-structural proteins (nsps). These non-structure proteins ultimately form the replicase-transcriptase complex for replication and transcription....

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“…2a). GQ folding in nascent telomeric DNA synthesized during telomerase extension has been directly observed by Jansson et al (43).…”

Section: Vectorial Folding Of Htel28 Mimicking Telomerase Actionmentioning

confidence: 85%

Streamlining effects of extra telomeric repeat on telomere folding revealed by fluorescence-force spectroscopy

Mitra

2019

Preprint

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Tandem repeats of guanine rich sequences are ubiquitous in the eukaryotic genome. For example, in the human cells, telomeres at the chromosomal ends comprise of kilobases repeats of T2AG3. Four such repeats can form G-quadruplexes (GQs). Biophysical studies have shown that GQs formed from four consecutive repeats possess high diversity both in their structure and in their response to tension. In principle, a GQ can form from any four repeats that may not even be consecutive. In order to investigate the dynamics of GQ possessing such positional multiplicity, we studied five and six repeats human telomeric sequence using single molecule FRET as well as its combination with optical tweezers. Our results suggest preferential formation of GQs at the 3' end both in K + and Na + solutions although minority populations with a 5' GQ or long-loop GQs were also observed. Using a vectorial folding assay which mimics the directional nature of telomere extension, we found that the 3' preference holds even when folding is allowed to begin from the 5' side. Interestingly, the unassociated T2AG3 segment has a streamlining effect in that one or two mechanically distinct species was observed at a single position instead of six or more observed without an unassociated repeat. Location of GQ on a long G-rich telomeric overhang and reduction in diversity of GQ conformations and mechanical responses through adjacent sequences have important implications in processes such as telomerase inhibition, alternative lengthening of telomeres, T-loop formation, telomere end protection and replication.

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Telomere DNA G-quadruplex folding within actively extending human telomerase

Cited by 100 publications

References 77 publications

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

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

Whole genome identification of potential G-quadruplexes and analysis of the G-quadruplex binding domain for SARS-CoV-2

Streamlining effects of extra telomeric repeat on telomere folding revealed by fluorescence-force spectroscopy

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