scholarly journals Streamlining effects of extra telomeric repeat on telomeric DNA folding revealed by fluorescence-force spectroscopy

2019 ◽  
Vol 47 (21) ◽  
pp. 11044-11056 ◽  
Author(s):  
Jaba Mitra ◽  
Taekjip Ha
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Distinct Species ◽  
Telomeric Sequence ◽  
Loop Formation ◽  
Telomeric Dna ◽  
Telomerase Inhibition ◽  
Preferential Formation ◽  
Single Molecule Fret ◽  
Conformational Diversity

Abstract A human telomere ends in a single-stranded 3′ tail, composed of repeats of T2AG3. G-quadruplexes (GQs) formed from four consecutive repeats have been shown to possess high-structural and mechanical diversity. In principle, a GQ can form from any four repeats that are not necessarily consecutive. To understand the dynamics of GQs with positional multiplicity, we studied five and six repeats human telomeric sequence using a combination of single molecule FRET and optical tweezers. Our results suggest preferential formation of GQs at the 3′ end both in K+ and Na+ solutions, with minor populations of 5′-GQ or long-loop GQs. A vectorial folding assay which mimics the directional nature of telomere extension showed that the 3′ preference holds even when folding is allowed to begin from the 5′ side. In 100 mM K+, 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. We did not observe such streamlining effect in 100 mM Na+. Location of GQ and reduction in conformational diversity in the presence of extra repeats have implications in telomerase inhibition, T-loop formation and telomere end protection.

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

2019 ◽  
Author(s):  
Jaba Mitra ◽  
Taekjip Ha
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Tandem Repeats ◽  
Distinct Species ◽  
Telomeric Sequence ◽  
Loop Formation ◽  
Preferential Formation ◽  
Mechanical Responses ◽  
Single Molecule Fret ◽  
Alternative Lengthening

AbstractTandem 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.

scholarly journals Extreme mechanical diversity of human telomeric DNA revealed by fluorescence-force spectroscopy

2019 ◽  
Vol 116 (17) ◽  
pp. 8350-8359 ◽  
Author(s):  
Jaba Mitra ◽  
Monika A. Makurath ◽  
Thuy T. M. Ngo ◽  
Alice Troitskaia ◽  
Yann R. Chemla ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Force Spectroscopy ◽  
Telomeric Dna ◽  
Telomeric Sequences ◽  
Substitution Mutant

G-quadruplexes (GQs) can adopt diverse structures and are functionally implicated in transcription, replication, translation, and maintenance of telomere. Their conformational diversity under physiological levels of mechanical stress, however, is poorly understood. We used single-molecule fluorescence-force spectroscopy that combines fluorescence resonance energy transfer with optical tweezers to measure human telomeric sequences under tension. Abrupt GQ unfolding with K+in solution occurred at as many as four discrete levels of force. Added to an ultrastable state and a gradually unfolding state, there were six mechanically distinct structures. Extreme mechanical diversity was also observed with Na+, although GQs were mechanically weaker. Our ability to detect small conformational changes at low forces enabled the determination of refolding forces of about 2 pN. Refolding was rapid and stochastically redistributed molecules to mechanically distinct states. A single guanine-to-thymine substitution mutant required much higher ion concentrations to display GQ-like unfolding and refolded via intermediates, contrary to the wild type. Contradicting an earlier proposal, truncation to three hexanucleotide repeats resulted in a single-stranded DNA-like mechanical behavior under all conditions, indicating that at least four repeats are required to form mechanically stable structures.

scholarly journals A comprehensive evaluation of a typical plant telomeric G-quadruplex (G4) DNA reveals the dynamics of G4 formation, rearrangement, and unfolding

2020 ◽  
Vol 295 (16) ◽  
pp. 5461-5469 ◽  
Author(s):  
Wen-Qiang Wu ◽  
Ming-Li Zhang ◽  
Chun-Peng Song
Keyword(s):  
Single Molecule ◽  
Comprehensive Evaluation ◽  
Cd Spectroscopy ◽  
Telomeric Sequence ◽  
Single Molecule Fret ◽  
G4 Dna ◽  
G Quadruplex ◽  
Simple Repeats ◽  
Plant Telomeres ◽  
Shed Light

Telomeres are specific nucleoprotein structures that are located at the ends of linear eukaryotic chromosomes and play crucial roles in genomic stability. Telomere DNA consists of simple repeats of a short G-rich sequence: TTAGGG in mammals and TTTAGGG in most plants. In recent years, the mammalian telomeric G-rich repeats have been shown to form G-quadruplex (G4) structures, which are crucial for modulating telomere functions. Surprisingly, even though plant telomeres are essential for plant growth, development, and environmental adaptions, only few reports exist on plant telomeric G4 DNA (pTG4). Here, using bulk and single-molecule assays, including CD spectroscopy, and single-molecule FRET approaches, we comprehensively characterized the structure and dynamics of a typical plant telomeric sequence, d[GGG(TTTAGGG)3]. We found that this sequence can fold into mixed G4s in potassium, including parallel and antiparallel structures. We also directly detected intermediate dynamic transitions, including G-hairpin, parallel G-triplex, and antiparallel G-triplex structures. Moreover, we observed that pTG4 is unfolded by the AtRecQ2 helicase but not by AtRecQ3. The results of our work shed light on our understanding about the existence, topological structures, stability, intermediates, unwinding, and functions of pTG4.

scholarly journals TIN2 facilitates TRF1-mediated trans- and cis-interactions on physiologically relevant long telomeric DNA

2020 ◽  
Author(s):  
Hai Pan ◽  
Parminder Kaur ◽  
Ming Liu ◽  
Pengning Xu ◽  
Chelsea Mahn ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Complexes ◽  
Molecular Structures ◽  
Telomeric Sequence ◽  
Dependent Manner ◽  
Dna Breaks ◽  
Telomeric Dna ◽  
Double Strand Dna Breaks ◽  
Regulator Protein ◽  
Tankyrase 1

ABSTRACTThe shelterin complex consisting of TRF1, TRF2, RAP1, TIN2, TPP1, and POT1, functions to prevent false recognition of telomeres as double-strand DNA breaks, and to regulate telomerase and DNA repair protein access. TIN2 is a core component linking double-stranded telomeric DNA binding proteins (TRF1 and TRF2) and proteins at the 3’ overhang (TPP1-POT1). Since knockdown of TIN2 also removes TRF1 and TRF2 from telomeres, determining TIN2’s unique mechanistic function has been elusive. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 using complementary single-molecule imaging platforms, including atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that TIN2S and TIN2L isoforms facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (6 TTAGGG repeats), the majority of TRF1 mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG), TIN2 forms multi-protein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last for at least minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 significantly reduces TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protects the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Our study provides evidence that TIN2 functions to promote TRF1 mediated trans-interactions of telomeric DNA, leading to new mechanistic insight into sister telomere cohesion.

scholarly journals Targeting G-quadruplex Forming Sequences with Cas9

2020 ◽  
Author(s):  
Hamza Balci ◽  
Viktorija Globyte ◽  
Chirlmin Joo
Keyword(s):  
Single Molecule ◽  
Dna Sequences ◽  
Structural Characteristics ◽  
Structural Heterogeneity ◽  
Loop Formation ◽  
Conformational States ◽  
Single Molecule Fret ◽  
G Quadruplex ◽  
The Stability ◽  
The Impact

ABSTRACTClustered Regularly Interspaced Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins, particularly Cas9, have provided unprecedented control on targeting and editing specific DNA sequences. If the target sequences are prone to folding into non-canonical secondary structures, such as G-quadruplex (GQ), the conformational states and activity of CRISPR-Cas9 complex would be influenced, but the impact has not been assessed. Using single molecule FRET, we investigated structural characteristics of the complex formed by CRISPR-Cas9 and target DNA, which contains a potentially GQ forming sequence (PQS) in either the target or the non-target strand (TS or NTS). We observed different conformational states and dynamics depending on the stability of the GQ and the position of PQS. When PQS was in NTS, we observed evidence for GQ formation for both weak and stable GQs. This is consistent with R-loop formation between TS and crRNA releasing NTS from Watson-Crick pairing and facilitating secondary structure formation in it. When PQS was in TS, R-loop formation was adequate to maintain a weak GQ in the unfolded state but not a GQ with moderate or high stability. The observed structural heterogeneity within the target dsDNA and the R-loop strongly depended on whether the PQS was in TS or NTS. We propose these variations in the complex structures to have functional implications for Cas9 activity.

scholarly journals Mesoscopic model for DNA G-quadruplex unfolding

2017 ◽  
Author(s):  
A. E. Bergues-Pupo ◽  
I. Gutiérrez ◽  
J. R. Arias-Gonzalez ◽  
F. Falo ◽  
A. Fiasconaro
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Protein Structures ◽  
Telomeric Sequence ◽  
Correct Prediction ◽  
Helical Structures ◽  
Mesoscopic Model ◽  
G Quadruplex ◽  
Single Bead ◽  
Physical Interactions

Genomes contain rare guanine-rich sequences capable of assembling into four-stranded helical structures, termed G-quadruplexes, with potential roles in gene regulation and chromosome stability. Their mechanical unfolding has only been reported to date by all-atom simulations, which cannot dissect the major physical interactions responsible for their cohesion. Here, we propose a mesoscopic model to describe both the mechanical and thermal stability of DNA G-quadruplexes, where each nucleotide of the structure, as well as each central cation located at the inner channel, is mapped onto a single bead. In this framework we are able to simulate loading rates similar to the experimental ones, which are not reachable in simulations with atomistic resolution. In this regard, we present single-molecule force-induced unfolding experiments by a high-resolution optical tweezers on a DNA telomeric sequence capable of forming a G-quadruplex conformation. Fitting the parameters of the model to the experiments we find a correct prediction of the rupture-force kinetics and a good agreement with previous near equilibrium measurements. Since G-quadruplex unfolding dynamics is halfway in complexity between secondary nucleic acids and tertiary protein structures, our model entails a nanoscale paradigm for non-equilibrium processes in the cell.

scholarly journals The ribosome modulates folding inside the ribosomal exit tunnel

2020 ◽  
Author(s):  
Florian Wruck ◽  
Pengfei Tian ◽  
Renuka Kudva ◽  
Robert B. Best ◽  
Gunnar von Heijne ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Electrostatic Interactions ◽  
Zinc Finger Domain ◽  
Single Molecule Fret ◽  
Ribosomal Tunnel ◽  
Nascent Chain ◽  
Folding Dynamics ◽  
Exit Tunnel ◽  
Dynamics Simulations

Proteins commonly fold cotranslationally on the ribosome, while the nascent chain emerges from the ribosomal tunnel. Protein domains that are sufficiently small can even fold while still located inside the tunnel. However, the effect of the tunnel on the folding dynamics of these domains is still not well understood. Here, we combine optical tweezers with single-molecule FRET and molecular dynamics simulations to investigate folding of the small zinc-finger domain ADR1a inside and at the vestibule of the ribosomal tunnel. The tunnel is found to accelerate folding and stabilize the folded state, reminiscent of the effects of chaperonins. However, a simple mechanism involving stabilization by confinement does not reproduce the results. Instead, it appears that electrostatic interactions between the protein and ribosome contribute to the observed folding acceleration and stabilization of ADR1a.

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

2019 ◽  
Author(s):  
Eric M. Patrick ◽  
Joseph Slivka ◽  
Bramyn Payne ◽  
Matthew J. Comstock ◽  
Jens C. Schmidt
Keyword(s):  
High Resolution ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Telomerase Activity ◽  
Telomere Maintenance ◽  
Telomeric Dna ◽  
Telomeric Repeats ◽  
G Quadruplex ◽  
Stable Substrate ◽  
Template Region

Telomere 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. To compensate for telomeric DNA lost during DNA replication, telomerase processively adds GGTTAG repeats to chromosome ends by copying the template region within its RNA subunit. Between repeat additions, the RNA template must be recycled. How telomerase remains associated with substrate DNA during this critical translocation step remains unknown. Using a newly developed single-molecule telomerase activity assay utilizing high-resolution optical tweezers, we demonstrate that stable substrate DNA binding at an anchor site within telomerase facilitates the processive synthesis of telomeric repeats. After release of multiple telomeric repeats from telomerase, we observed folding of product DNA into G-quadruplex structures. Our results provide detailed mechanistic insights into telomerase catalysis, a process of critical importance in aging and cancer.

scholarly journals TIN2 is an architectural protein that facilitates TRF2-mediated trans- and cis-interactions on telomeric DNA

2021 ◽  
Author(s):  
Parminder Kaur ◽  
Ryan Barnes ◽  
Hai Pan ◽  
Ariana C Detwiler ◽  
Ming Liu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Molecular Model ◽  
Loop Formation ◽  
Dna Breaks ◽  
Telomeric Dna ◽  
Dna Structures ◽  
Shelterin Complex ◽  
Double Strand Dna Breaks ◽  
Architectural Protein

Abstract The telomere specific shelterin complex, which includes TRF1, TRF2, RAP1, TIN2, TPP1 and POT1, prevents spurious recognition of telomeres as double-strand DNA breaks and regulates telomerase and DNA repair activities at telomeres. TIN2 is a key component of the shelterin complex that directly interacts with TRF1, TRF2 and TPP1. In vivo, the large majority of TRF1 and TRF2 are in complex with TIN2 but without TPP1 and POT1. Since knockdown of TIN2 also removes TRF1 and TRF2 from telomeres, previous cell-based assays only provide information on downstream effects after the loss of TRF1/TRF2 and TIN2. Here, we investigated DNA structures promoted by TRF2–TIN2 using single-molecule imaging platforms, including tracking of compaction of long mouse telomeric DNA using fluorescence imaging, atomic force microscopy (AFM) imaging of protein–DNA structures, and monitoring of DNA–DNA and DNA–RNA bridging using the DNA tightrope assay. These techniques enabled us to uncover previously unknown unique activities of TIN2. TIN2S and TIN2L isoforms facilitate TRF2-mediated telomeric DNA compaction (cis-interactions), dsDNA–dsDNA, dsDNA–ssDNA and dsDNA–ssRNA bridging (trans-interactions). Furthermore, TIN2 facilitates TRF2-mediated T-loop formation. We propose a molecular model in which TIN2 functions as an architectural protein to promote TRF2-mediated trans and cis higher-order nucleic acid structures at telomeres.

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