Bulk and Single-Molecule Fluorescence Studies of the Saturation of the DNA Double Helix Using YOYO-3 Intercalator Dye

2012 ◽  
Vol 116 (38) ◽  
pp. 11561-11569 ◽  
Author(s):  
Sergio G. Lopez ◽  
Maria J. Ruedas-Rama ◽  
Salvador Casares ◽  
Jose M. Alvarez-Pez ◽  
Angel Orte
Keyword(s):  
Single Molecule ◽  
Double Helix ◽  
Single Molecule Fluorescence ◽  
Fluorescence Studies ◽  
Dna Double Helix

Direct Single-Molecule Observations of Local Denaturation of a DNA Double Helix under a Negative Supercoil State

2015 ◽  
Vol 87 (6) ◽  
pp. 3490-3497 ◽  
Author(s):  
Shunsuke Takahashi ◽  
Shinya Motooka ◽  
Tomohiro Usui ◽  
Shohei Kawasaki ◽  
Hidefumi Miyata ◽  
...  
Keyword(s):  
Single Molecule ◽  
Double Helix ◽  
Dna Double Helix

scholarly journals Mechanism of RPA-Facilitated Processive DNA Unwinding by the Eukaryotic CMG Helicase

2019 ◽  
Author(s):  
Hazal B. Kose ◽  
Sherry Xie ◽  
George Cameron ◽  
Melania S. Strycharska ◽  
Hasan Yardimci
Keyword(s):  
Single Molecule ◽  
Replication Fork ◽  
Double Helix ◽  
Dna Unwinding ◽  
Helicase Activity ◽  
Replicative Helicase ◽  
Double Stranded Dna ◽  
Order Of Magnitude ◽  
Dna Strand ◽  
Dna Double Helix

AbstractThe DNA double helix is unwound by the Cdc45/Mcm2-7/GINS (CMG) complex at the eukaryotic replication fork. While isolated CMG unwinds duplex DNA very slowly, its fork unwinding rate is stimulated by an order of magnitude by single-stranded DNA binding protein, RPA. However, the molecular mechanism by which RPA enhances CMG helicase activity remained elusive. Here, we demonstrate that engagement of CMG with parental double-stranded DNA (dsDNA) at the replication fork impairs its helicase activity, explaining the slow DNA unwinding by isolated CMG. Using single-molecule and ensemble biochemistry, we show that binding of RPA to the excluded DNA strand prevents duplex engagement by the helicase and speeds up CMG-mediated DNA unwinding. When stalled due to dsDNA interaction, DNA rezipping-induced helicase backtracking re-establishes productive helicase-fork engagement underscoring the significance of plasticity in helicase action. Together, our results elucidate the dynamics of CMG at the replication fork and reveal how other replisome components can mediate proper DNA engagement by the replicative helicase to achieve efficient fork progression.

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.

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