scholarly journals Visualizing structure-mediated interactions in supercoiled DNA molecules

2018 ◽  
Author(s):  
Shane Scott ◽  
Zhi Ming Xu ◽  
Fedor Kouzine ◽  
Daniel J. Berard ◽  
Cynthia Shaheen ◽  
...  

ABSTRACTWe directly visualize the topology-mediated interactions between an unwinding site on a supercoiled DNA plasmid and a specific probe molecule designed to bind to this site, as a function of DNA supercoiling and temperature. The visualization relies on containing the DNA molecules within an enclosed array of glass nanopits using the Convex Lens-induced Confinement (CLiC) imaging method. This method traps molecules within the focal plane while excluding signal from out-of-focus probes. Simultaneously, the molecules can freely diffuse within the nanopits, allowing for accurate measurements of exchange rates, unlike other methods which could introduce an artifactual bias in measurements of binding kinetics. We demonstrate that the plasmid’s structure influences the binding of the fluorescent probes to the unwinding site through the presence, or lack, of other secondary structures. With this method, we observe an increase in the binding rate of the fluorescent probe to the unwinding site with increasing temperature and negative supercoiling. This increase in binding is consistent with the results of our numerical simulations of the probability of site-unwinding. The temperature dependence of the binding rate has allowed us to distinguish the effects of competing higher order DNA structures, such as Z-DNA, in modulating local site-unwinding, and therefore binding.

Nanoscale ◽  
2015 ◽  
Vol 7 (7) ◽  
pp. 3205-3216 ◽  
Author(s):  
Rifka Vlijm ◽  
Alireza Mashaghi ◽  
Stéphanie Bernard ◽  
Mauro Modesti ◽  
Cees Dekker

Phase diagram of experimentally determined DNA structures under (extreme) negative supercoiling.


2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Rossitza N. Irobalieva ◽  
Jonathan M. Fogg ◽  
Daniel J. Catanese ◽  
Thana Sutthibutpong ◽  
Muyuan Chen ◽  
...  

Abstract By regulating access to the genetic code, DNA supercoiling strongly affects DNA metabolism. Despite its importance, however, much about supercoiled DNA (positively supercoiled DNA, in particular) remains unknown. Here we use electron cryo-tomography together with biochemical analyses to investigate structures of individual purified DNA minicircle topoisomers with defined degrees of supercoiling. Our results reveal that each topoisomer, negative or positive, adopts a unique and surprisingly wide distribution of three-dimensional conformations. Moreover, we uncover striking differences in how the topoisomers handle torsional stress. As negative supercoiling increases, bases are increasingly exposed. Beyond a sharp supercoiling threshold, we also detect exposed bases in positively supercoiled DNA. Molecular dynamics simulations independently confirm the conformational heterogeneity and provide atomistic insight into the flexibility of supercoiled DNA. Our integrated approach reveals the three-dimensional structures of DNA that are essential for its function.


2020 ◽  
Vol 118 (3) ◽  
pp. 297a-298a
Author(s):  
Zachary Sierzega ◽  
Christopher Prior ◽  
Jeffery M. Wereszczynski

Science ◽  
2013 ◽  
Vol 340 (6140) ◽  
pp. 1580-1583 ◽  
Author(s):  
Jie Ma ◽  
Lu Bai ◽  
Michelle D. Wang

In cells, RNA polymerase (RNAP) must transcribe supercoiled DNA, whose torsional state is constantly changing, but how RNAP deals with DNA supercoiling remains elusive. We report direct measurements of individual Escherichia coli RNAPs as they transcribed supercoiled DNA. We found that a resisting torque slowed RNAP and increased its pause frequency and duration. RNAP was able to generate 11 ± 4 piconewton-nanometers (mean ± standard deviation) of torque before stalling, an amount sufficient to melt DNA of arbitrary sequence and establish RNAP as a more potent torsional motor than previously known. A stalled RNAP was able to resume transcription upon torque relaxation, and transcribing RNAP was resilient to transient torque fluctuations. These results provide a quantitative framework for understanding how dynamic modification of DNA supercoiling regulates transcription.


2003 ◽  
Vol 185 (17) ◽  
pp. 5324-5327 ◽  
Author(s):  
Annie Conter

ABSTRACT The relationship between the survival of Escherichia coli during long-term starvation in rich medium and the supercoiling of a reporter plasmid (pBR322) has been studied. In aerated continuously shaken cultures, E. coli lost the ability to form colonies earlier in rich NaCl-free Luria-Bertani medium than in NaCl-containing medium, and the negative supercoiling of plasmid pBR322 declined more rapidly in the absence of NaCl. Addition of NaCl at the 24th hour restored both viability and negative supercoiling in proportion to the concentration of added NaCl. Addition of ofloxacin, a quinolone inhibitor of gyrase, abolished rescue by added NaCl in proportion to the ofloxacin added. This observation raises the possibility that cells had the ability to recover plasmid supercoiling even if nutrients were not available and could survive during long-term starvation in a manner linked, at least in part, to the topological state of DNA and gyrase activity.


1988 ◽  
Vol 16 (3) ◽  
pp. 1079-1093 ◽  
Author(s):  
Karen M. Sullivan ◽  
David M.J. Lilley

1990 ◽  
Vol 9 (13) ◽  
pp. 4551-4554 ◽  
Author(s):  
M. Adrian ◽  
B. ten Heggeler-Bordier ◽  
W. Wahli ◽  
A. Z. Stasiak ◽  
A. Stasiak ◽  
...  

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