scholarly journals Reconstructing double-stranded DNA fragments on a single-molecule level reveals patterns of degradation in ancient samples

2020 ◽  
Vol 30 (10) ◽  
pp. 1449-1457
Author(s):  
Lukas Bokelmann ◽  
Isabelle Glocke ◽  
Matthias Meyer
Keyword(s):  
Single Molecule ◽  
Dna Fragments ◽  
Single Molecule Level ◽  
Double Stranded Dna

scholarly journals γ-Hemolysin Nanopore Is Sensitive to Guanine-to-Inosine Substitutions in Double-Stranded DNA at the Single-Molecule Level

2018 ◽  
Vol 140 (43) ◽  
pp. 14224-14234 ◽  
Author(s):  
Cherie S. Tan ◽  
Aaron M. Fleming ◽  
Hang Ren ◽  
Cynthia J. Burrows ◽  
Henry S. White
Keyword(s):  
Single Molecule ◽  
Single Molecule Level ◽  
Double Stranded Dna

In-gel multiple displacement amplification of long DNA fragments diluted to the single molecule level

2008 ◽  
Vol 383 (2) ◽  
pp. 151-158 ◽  
Author(s):  
Yuichi Michikawa ◽  
Keisuke Sugahara ◽  
Tomo Suga ◽  
Yoshimi Ohtsuka ◽  
Kenichi Ishikawa ◽  
...  
Keyword(s):  
Single Molecule ◽  
Multiple Displacement Amplification ◽  
Dna Fragments ◽  
Single Molecule Level

scholarly journals Ring-shaped replicative helicase encircles double-stranded DNA during unwinding

2019 ◽  
Vol 47 (21) ◽  
pp. 11344-11354 ◽  
Author(s):  
Sihwa Joo ◽  
Bong H Chung ◽  
Mina Lee ◽  
Tai H Ha
Keyword(s):  
Single Molecule ◽  
Magnetic Tweezers ◽  
Model Systems ◽  
Bovine Papillomavirus ◽  
Viral Origin ◽  
Replicative Helicase ◽  
Single Molecule Level ◽  
Double Stranded Dna ◽  
Dna Strands ◽  
Cellular Dna

Abstract Ring-shaped replicative helicases are hexameric and play a key role in cellular DNA replication. Despite their importance, our understanding of the unwinding mechanism of replicative helicases is far from perfect. Bovine papillomavirus E1 is one of the best-known model systems for replicative helicases. E1 is a multifunctional initiator that senses and melts the viral origin and unwinds DNA. Here, we study the unwinding mechanism of E1 at the single-molecule level using magnetic tweezers. The result reveals that E1 as a single hexamer is a poorly processive helicase with a low unwinding rate. Tension on the DNA strands impedes unwinding, indicating that the helicase interacts strongly with both DNA strands at the junction. While investigating the interaction at a high force (26–30 pN), we discovered that E1 encircles dsDNA. By comparing with the E1 construct without a DNA binding domain, we propose two possible encircling modes of E1 during active unwinding.

scholarly journals Energetics of base flipping at a DNA mismatch site confined at the latch constriction of α-hemolysin

2016 ◽  
Vol 193 ◽  
pp. 471-485 ◽  
Author(s):  
Robert P. Johnson ◽  
Rukshan T. Perera ◽  
Aaron M. Fleming ◽  
Cynthia J. Burrows ◽  
Henry S. White
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Dna Duplexes ◽  
Base Flipping ◽  
Single Molecule Level ◽  
Dna Mismatch ◽  
Double Stranded Dna ◽  
Measured Activation Energy ◽  
Measured Activation ◽  
Significant Mechanism

Unique, two-state modulating current signatures are observed when a cytosine–cytosine mismatch pair is confined at the 2.4 nm latch constriction of the α-hemolysin (αHL) nanopore. We have previously speculated that the modulation is due to base flipping at the mismatch site. Base flipping is a biologically significant mechanism in which a single base is rotated out of the DNA helical stack by 180°. It is the mechanism by which enzymes are able to access bases for repair operations without disturbing the global structure of the helix. Here, temperature dependent ion channel recordings of individual double-stranded DNA duplexes inside αHL are used to derive thermodynamic (ΔH, ΔS) and kinetic (EA) parameters for base flipping of a cytosine at an unstable cytosine–cytosine mismatch site. The measured activation energy for flipping a cytosine located at the latch of αHL out of the helix (18 ± 1 kcal mol−1) is comparable to that previously reported for base flipping at mismatch sites from NMR measurements and potential mean force calculations. We propose that the αHL nanopore is a useful tool for measuring conformational changes in dsDNA at the single molecule level.

scholarly journals Double-stranded flanking ends affect the folding kinetics and conformational equilibrium of G-quadruplexes forming sequences within the promoter of KIT oncogene

2021 ◽  
Author(s):  
Guglielmo Vesco ◽  
Marco Lamperti ◽  
Domenico Salerno ◽  
Claudia Adriana Marrano ◽  
Valeria Cassina ◽  
...  
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fine Tuning ◽  
Folding Kinetics ◽  
Single Molecule Level ◽  
Double Stranded Dna ◽  
G Quadruplex ◽  
Final Equilibrium State ◽  
Complex Relationships

Abstract G-quadruplexes embedded within promoters play a crucial role in regulating the gene expression. KIT is a widely studied oncogene, whose promoter contains three G-quadruplex forming sequences, c-kit1, c-kit2 and c-kit*. For these sequences available studies cover ensemble and single-molecule analyses, although for kit* the latter were limited to a study on a promoter domain comprising all of them. Recently, c-kit2 has been reported to fold according to a multi-step process involving folding intermediates. Here, by exploiting fluorescence resonance energy transfer, both in ensemble and at the single molecule level, we investigated the folding of expressly designed constructs in which, alike in the physiological context, either c-kit2 or c-kit* are flanked by double stranded DNA segments. To assess whether the presence of flanking ends at the borders of the G-quadruplex affects the folding, we studied under the same protocols oligonucleotides corresponding to the minimal G-quadruplex forming sequences. Data suggest that addition of flanking ends results in biasing both the final equilibrium state and the folding kinetics. A previously unconsidered aspect is thereby unravelled, which ought to be taken into account to achieve a deeper insight of the complex relationships underlying the fine tuning of the gene-regulatory properties of these fascinating DNA structures.

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