scholarly journals Emerging Accessibility Patterns in Long Telomeric Overhangs

2021 ◽  
Author(s):  
Sajad Shiekh ◽  
Golam Mustafa ◽  
Mohammed Enamul Hoque ◽  
Eric Yokie ◽  
John J. Portman ◽  
...  
Keyword(s):  
Complex Formation ◽  
Computational Modeling ◽  
Computational Model ◽  
Single Molecule ◽  
Computational Studies ◽  
Telomeric Dna ◽  
Shelterin Complex ◽  
Modeling Studies ◽  
G Quadruplex ◽  
Overhang Length

We present single molecule experimental and computational modeling studies investigating the accessibility and folding landscape of human telomeric overhangs of physiologically relevant lengths. The overhangs contain 4-28 repeats of GGGTTA (G-Tract) sequence and accommodate 1-7 tandem G-quadruplex (GQ) structures. Using FRET-PAINT, we probed the distribution of accessible sites via a short imager strand, which is complementary to a G-Tract and transiently binds to unfolded sites. We report accessibility patterns that periodically change with overhang length and provide insights about the underlying folding frustration. Overhangs that have 4n G-Tracts, (12, 16...), demonstrate maximum frustration, while those with 4n+2 G-Tracts, (14, 18...), have minimal frustration. We also developed a computational model that suggests positive folding cooperativity between neighboring GQs is required for persistence of such patterns. Our experimental and computational studies suggest lower folding stability at the junction between single and double stranded telomeric DNA, which has implications for Shelterin complex formation.

scholarly journals TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres

2013 ◽  
Vol 42 (4) ◽  
pp. 2493-2504 ◽  
Author(s):  
Jiangguo Lin ◽  
Preston Countryman ◽  
Noah Buncher ◽  
Parminder Kaur ◽  
Longjiang E ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Sequences ◽  
Specific Binding ◽  
General Mechanism ◽  
Telomeric Dna ◽  
External Energy ◽  
Telomeric Sequences ◽  
Shelterin Complex ◽  
Protein Partners ◽  
A Genome

Abstract Human telomeres are maintained by the shelterin protein complex in which TRF1 and TRF2 bind directly to duplex telomeric DNA. How these proteins find telomeric sequences among a genome of billions of base pairs and how they find protein partners to form the shelterin complex remains uncertain. Using single-molecule fluorescence imaging of quantum dot-labeled TRF1 and TRF2, we study how these proteins locate TTAGGG repeats on DNA tightropes. By virtue of its basic domain TRF2 performs an extensive 1D search on nontelomeric DNA, whereas TRF1’s 1D search is limited. Unlike the stable and static associations observed for other proteins at specific binding sites, TRF proteins possess reduced binding stability marked by transient binding (∼9–17 s) and slow 1D diffusion on specific telomeric regions. These slow diffusion constants yield activation energy barriers to sliding ∼2.8–3.6 κBT greater than those for nontelomeric DNA. We propose that the TRF proteins use 1D sliding to find protein partners and assemble the shelterin complex, which in turn stabilizes the interaction with specific telomeric DNA. This ‘tag-team proofreading’ represents a more general mechanism to ensure a specific set of proteins interact with each other on long repetitive specific DNA sequences without requiring external energy sources.

scholarly journals Interrogating accessibility of telomeric sequences with FRET-PAINT: evidence for length-dependent telomere compaction

2021 ◽  
Author(s):  
Golam Mustafa ◽  
Sajad Shiekh ◽  
Keshav GC ◽  
Sanjaya Abeysirigunawardena ◽  
Hamza Balci
Keyword(s):  
Dna Damage ◽  
Telomere Length ◽  
Dna Damage Response ◽  
Single Molecule ◽  
Telomeric Repeat ◽  
Dna Molecules ◽  
Telomeric Dna ◽  
Telomeric Sequences ◽  
G Quadruplex ◽  
Damage Response

Abstract Single-stranded telomeric overhangs are ∼200 nucleotides long and can form tandem G-quadruplex (GQ) structures, which reduce their accessibility to nucleases and proteins that activate DNA damage response. Whether these tandem GQs further stack to form compact superstructures, which may provide better protection for longer telomeres, is not known. We report single-molecule measurements where the accessibility of 24–144 nucleotide long human telomeric DNA molecules is interrogated by a short PNA molecule that is complementary to a single GGGTTA repeat, as implemented in the FRET-PAINT method. Binding of the PNA strand to available GGGTTA sequences results in discrete FRET bursts which were analyzed in terms of their dwell times, binding frequencies, and topographic distributions. The binding frequencies were greater for binding to intermediate regions of telomeric DNA compared to 3′- or 5′-ends, suggesting these regions are more accessible. Significantly, the binding frequency per telomeric repeat monotonically decreased with increasing telomere length. These results are consistent with telomeres forming more compact structures at longer lengths, reducing accessibility of these critical genomic sites.

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.

scholarly journals How Computational Modeling Can Force Theory Building in Psychological Science

2021 ◽  
pp. 174569162097058
Author(s):  
Olivia Guest ◽  
Andrea E. Martin
Keyword(s):  
Computational Modeling ◽  
Computational Model ◽  
Process Modeling ◽  
Theory Building ◽  
Formal Modeling ◽  
Inference Process ◽  
Experimental Practice ◽  
Psychological Science ◽  
Scientific Inference ◽  
And Behaviors

Psychology endeavors to develop theories of human capacities and behaviors on the basis of a variety of methodologies and dependent measures. We argue that one of the most divisive factors in psychological science is whether researchers choose to use computational modeling of theories (over and above data) during the scientific-inference process. Modeling is undervalued yet holds promise for advancing psychological science. The inherent demands of computational modeling guide us toward better science by forcing us to conceptually analyze, specify, and formalize intuitions that otherwise remain unexamined—what we dub open theory. Constraining our inference process through modeling enables us to build explanatory and predictive theories. Here, we present scientific inference in psychology as a path function in which each step shapes the next. Computational modeling can constrain these steps, thus advancing scientific inference over and above the stewardship of experimental practice (e.g., preregistration). If psychology continues to eschew computational modeling, we predict more replicability crises and persistent failure at coherent theory building. This is because without formal modeling we lack open and transparent theorizing. We also explain how to formalize, specify, and implement a computational model, emphasizing that the advantages of modeling can be achieved by anyone with benefit to all.

scholarly journals PhenQE8, a Novel Ligand of the Human Telomeric Quadruplex

2021 ◽  
Vol 22 (2) ◽  
pp. 749
Author(s):  
Patricia B. Gratal ◽  
Julia G. Quero ◽  
Adrián Pérez-Redondo ◽  
Zoila Gándara ◽  
Lourdes Gude
Keyword(s):  
Equilibrium Dialysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
The Novel ◽  
X Ray Diffraction ◽  
Telomeric Dna ◽  
Healthy Human ◽  
Quadruplex Dna ◽  
Step Procedure ◽  
G Quadruplex

A novel quadruplex ligand based on 1,10-phenanthroline and incorporating two guanyl hydrazone functionalities, PhenQE8, is reported herein. Synthetic access was gained in a two-step procedure with an overall yield of 61%. X-ray diffraction studies revealed that PhenQE8 can adopt an extended conformation that may be optimal to favor recognition of quadruplex DNA. DNA interactions with polymorphic G-quadruplex telomeric structures were studied by different techniques, such as Fluorescence resonance energy transfer (FRET) DNA melting assays, circular dichroism and equilibrium dialysis. Our results reveal that the novel ligand PhenQE8 can efficiently recognize the hybrid quadruplex structures of the human telomeric DNA, with high binding affinity and quadruplex/duplex selectivity. Moreover, the compound shows significant cytotoxic activity against a selected panel of cultured tumor cells (PC-3, HeLa and MCF-7), whereas its cytotoxicity is considerably lower in healthy human cells (HFF-1 and RPWE-1).

Research Progress in the Typical Structure of Human Telomeric G-Quadruplex

2014 ◽  
Vol 955-959 ◽  
pp. 419-422
Author(s):  
Gui Lin Liu ◽  
Yan Ping Ding ◽  
Yan Ling Wu ◽  
Wen Zhang
Keyword(s):  
Cell Cycle ◽  
Dna Sequences ◽  
Research Progress ◽  
Human Chromosomes ◽  
Telomeric Dna ◽  
Typical Structure ◽  
Special Sequence ◽  
Physiological Processes ◽  
G Quadruplex ◽  
Small Molecule Ligands

Telomeric DNA of human chromosomes plays a significant role in physiological processes such as cell cycle, aging, cancer and genetic stability due to its special sequence and structure. The research on small molecule ligands targeting G-quadruplex formed by such special sequence has attracted considerable attention, and has achieved great breakthrough. In this paper, we summarize the DNA sequences and structures of three kinds of typical human telomeric G-quadruplex, providing an important reference for further research.

scholarly journals Voltammetry and molecular assembly of G-quadruplex DNAzyme on single-crystal Au(111)-electrode surfaces – hemin as an electrochemical intercalator

2016 ◽  
Vol 193 ◽  
pp. 99-112 ◽  
Author(s):  
Ling Zhang ◽  
Jens Ulstrup ◽  
Jingdong Zhang
Keyword(s):  
Single Crystal ◽  
Single Molecule ◽  
Electrode Surface ◽  
Molecular Assembly ◽  
Electrode Surfaces ◽  
Potential Control ◽  
G Quadruplex ◽  
Tunnelling Microscopy ◽  
Dna Quadruplexes

DNA quadruplexes (qs) are a class of “non-canonical” oligonucleotides (OGNs) composed of stacked guanine (G) quartets stabilized by specific cations. Metal porphyrins selectively bind to G-qs complexes to form what is known as DNAzyme, which can exhibit peroxidase and other catalytic activity similar to heme group metalloenzymes. In the present study we investigate the electrochemical properties and the structure of DNAzyme monolayers on single-crystal Au(111)-electrode surfaces using cyclic voltammetry and scanning tunnelling microscopy under electrochemical potential control (in situ STM). The target DNAzyme is formed from a single-strand OGN with 12 guanines and iron(iii) porphyrin IX (hemin), and assembles on Au(111) through a mercapto alkyl linker. The DNAzyme monolayers exhibit a strong pair of redox peaks at 0.0 V (NHE) at pH 7 in acetate buffer, shifted positively by about 50 mV compared to free hemin weakly physisorbed on the Au(111)-electrode surface. The voltammetric hemin signal of DNAzyme is enhanced 15 times compared with that of hemin adsorbed directly on the Au(111)-electrode surface. This is indicative of both the formation of a close to dense DNAzyme monolayer and that hemin is strongly bound to the immobilized 12G-qs in well-defined orientation favorable for interfacial ET with a rate constant of 6.0 ± 0.4 s−1. This is supported by in situ STM which discloses single-molecule G-quartet structures with a size of 1.6 ± 0.2 nm.

Interaction of hnRNP A1 with telomere DNA G-quadruplex structures studied at the single molecule level

2010 ◽  
Vol 39 (9) ◽  
pp. 1343-1350 ◽  
Author(s):  
A. C. Krüger ◽  
M. K. Raarup ◽  
M. M. Nielsen ◽  
M. Kristensen ◽  
F. Besenbacher ◽  
...  
Keyword(s):  
Single Molecule ◽  
Hnrnp A1 ◽  
Single Molecule Level ◽  
G Quadruplex

Lab in a tube: a fast-assembled colorimetric sensor for highly sensitive detection of oligonucleotides based on a hybridization chain reaction

RSC Advances ◽  
2015 ◽  
Vol 5 (55) ◽  
pp. 44714-44721 ◽  
Author(s):  
Siqi Zhang ◽  
Kun Wang ◽  
Zhenyu Li ◽  
Zhongmin Feng ◽  
Ting Sun
Keyword(s):  
Complex Formation ◽  
Dna Sequence ◽  
Self Assembly ◽  
The Self ◽  
Hybridization Chain Reaction ◽  
Chain Reaction ◽  
Highly Sensitive ◽  
G Quadruplex ◽  
Colored Product ◽  
Highly Sensitive Detection

Upon adding THBV, the self-assembly of THBV with H1 allows the rest of the DNA sequence of H1 to accelerate H1–H2 complex formation. The G-quadruplex at the end of the H1–H2 complex could catalyze TMB into a colored product.

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