scholarly journals Kinetics of DNA looping by Anabaena sensory rhodopsin transducer (ASRT) by using DNA cyclization assay

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jae Jin Lee ◽  
Sung Hyun Kim ◽  
Keon Ah Lee ◽  
Kimleng Chuon ◽  
Kwang-Hwan Jung ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Single Molecule ◽  
Essential Role ◽  
Molecular Structures ◽  
Dna Looping ◽  
Loop Formation ◽  
Sensory Rhodopsin ◽  
Single Molecule Fret ◽  
Dna Cyclization ◽  
Kinetics Of

AbstractDNA cyclization assay together with single-molecule FRET was employed to monitor protein-mediated bending of a short dsDNA (~ 100 bp). This method provides a simple and easy way to monitor the structural change of DNA in real-time without necessitating prior knowledge of the molecular structures for the optimal dye-labeling. This assay was applied to study how Anabaena sensory rhodopsin transducer (ASRT) facilitates loop formation of DNA as a possible mechanism for gene regulation. The ASRT-induced DNA looping was maximized at 50 mM of Na+, while Mg2+ also played an essential role in the loop formation.

scholarly journals Single-molecule fluorescence studies on cotranscriptional G-quadruplex formation coupled with R-loop formation

2020 ◽  
Vol 48 (16) ◽  
pp. 9195-9203
Author(s):  
Gunhyoung Lim ◽  
Sungchul Hohng
Keyword(s):  
Gene Regulation ◽  
Single Molecule ◽  
Genome Instability ◽  
Feedback Mechanism ◽  
Rnase H ◽  
Formation Mechanisms ◽  
Loop Formation ◽  
Single Molecule Fluorescence ◽  
Fluorescence Studies ◽  
G Quadruplex

Abstract G-quadruplex (GQ) is formed at various regions of DNA, including telomeres of chromosomes and regulatory regions of oncogenes. Since GQ is important in both gene regulation and genome instability, the biological and medical implications of this abnormal DNA structure have been intensively studied. Its formation mechanisms, however, are not clearly understood yet. We report single-molecule fluorescence experiments to monitor the cotranscriptional GQ formation coupled with R-loop formation using T7 RNA polymerase. The GQ is formed very rarely per single-round transcription. R-loop formation precedes and facilitates GQ formation. Once formed, some GQs are extremely stable, resistant even to RNase H treatment, and accumulate in multiple-round transcription conditions. On the other hand, GQ existing in the non-template strand promotes the R-loop formation in the next rounds of transcription. Our study clearly shows the existence of a positive feedback mechanism of GQ and R-loop formations, which may possibly contribute to gene regulation and genome instability.

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.

Restriction Enzyme Ecl18kI-Induced DNA Looping Dynamics by Single-Molecule FRET

2014 ◽  
Vol 118 (29) ◽  
pp. 8575-8582 ◽  
Author(s):  
Danielis Rutkauskas ◽  
Milda Petkelyte ◽  
Paulius Naujalis ◽  
Giedrius Sasnauskas ◽  
Gintautas Tamulaitis ◽  
...  
Keyword(s):  
Single Molecule ◽  
Restriction Enzyme ◽  
Dna Looping ◽  
Single Molecule Fret

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.

Domain-Specific Folding Kinetics of Staphylococcal Nuclease Observed through Single-Molecule FRET in a Microfluidic Mixer

ChemPhysChem ◽  
2011 ◽  
Vol 12 (18) ◽  
pp. 3515-3518 ◽  
Author(s):  
Zeyong Zhi ◽  
Pengcheng Liu ◽  
Peng Wang ◽  
Yanyi Huang ◽  
Xin Sheng Zhao
Keyword(s):  
Single Molecule ◽  
Staphylococcal Nuclease ◽  
Folding Kinetics ◽  
Microfluidic Mixer ◽  
Domain Specific ◽  
Single Molecule Fret ◽  
Kinetics Of
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