If the helix is undertwisted, the edges of the narrow groove move further apart. Notice that changing the twist from the relaxed state requires adding energy and increases the stress along the molecule. If DNA is in the form of a circular molecule, or if the ends are rigidly held so that it forms a loop, then overtwisting or undertwisting leads to the supercoiled state. Supercoiling occurs when the molecule relieves the helical stress by twisting around itself.
An open area is energetically unfavorable. The covalently closed molecule cannot adjust for this by increasing the Linking number. That is, it cannot spontaneously break one or both strands of the duplex, introduce another 25 turns into the duplex increase the Linking number by 25 and re-ligate the duplex.
The DNA has three choices: It can adjust the number of basepairs per turn throughout the molecule from a desired NOTE: an increase in the number of basepairs per turn will decrease the twist value; underwound DNA has a greater number of basepairs per turn.
The DNA can coil up into a "supercoil" topology and maintain the desired twist value The duplex can exist with a twist of This is quite unfavorable due to the geometry required of bond angles. As the replication fork opens up, the region of the duplex in front of the fork becomes overwound - i. The linking number has not changed, but the length of DNA which contains all the turns is effectively shorter.
To maintain This is energetically unfavorable, and one option for the DNA is to adopt a supercoiled configuration to achieve In this case the DNA has adopted The 5' phosphate of the nicked strand is covalently attached to a tyrosine in the protein. The 3' end of the nick then passes once through the duplex.
This can therefore result in the removal of a single negative supercoil. The net result of E. Type II topoisomerases can convert a single positive supercoil into a negative supercoil. Thus the linkage number is reduced by two -2 in a single step. Type II topoisomerases are involved in both decatenation of daughter chromosomes, and relieving the positive supercoiling ahead of the replication fork.
Our ability to keep freely diffusing molecules under observation for long timescales allows observation of unconstrained structural dynamics and interactions. Although this study focuses on indirectly measuring supercoil-induced DNA unwinding, our technique could be used to investigate its dynamics directly. For example, one could devise a plasmid system in which multiple fluorophores are inserted directly into or near the edges of an unwinding site.
Temporal control over the solution and confinement environment is also possible by extensions of the presented techniques. Further, the capability to fabricate custom pit geometries enables us to optimize the single-molecule imaging conditions for assaying different sizes and concentrations of molecules in solution.
Exploring the effects of nanoscale dimensions and molecular crowding is a subject of immense interest as it mimics conditions in the cellular nucleus; these conditions are made accessible by our methods.
Finally, gene editing technologies, such as CRISPR—Cas9, could benefit from a more thorough understanding of nucleotide—nucleotide interactions. Further insight into how these systems function on a molecular level can only help develop these techniques to become more effective. In conclusion, we have used CLiC microscopy and nanopits to observe and characterize the interactions between a series of fluorescently-labeled DNA probes and a target unwinding region on a supercoiled DNA plasmid.
We have shown that by increasing the temperature and level of plasmid supercoiling, we are able to increase the rate of binding of the probes to the target region.
By performing experiments as a function of the probe size and sequence, we have discovered that probe molecules are capable of interacting with and binding to the unwinding region even when the probability of base pair denaturation at the target area is low.
This result—that even a short probe molecule remains bound—means that the global free energy of the plasmid system is lowered when the unwinding site remains denatured, due to the consequent release of global torsional strain in the DNA molecule.
What has enabled these contributions is the new method that we present here—the ability to visualize small ensembles of interacting DNA and probe molecules, in large pit arrays, for long times—without tethering them to surfaces or beads.
We have shown that we can detect rare kinetic events which are topology-dependent; events which are otherwise out of reach to existing TIRF and tweezer technologies. Further, Stephen Michnick contributed important manuscript feedback and scientific discussions.
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Volume Article Contents Abstract. Visualizing structure-mediated interactions in supercoiled DNA molecules. Shane Scott , Shane Scott. Oxford Academic. Zhi Ming Xu. Fedor Kouzine. Daniel J Berard. Cynthia Shaheen. Barbara Gravel. Laura Saunders. Alexander Hofkirchner. Catherine Leroux. Jill Laurin. David Levens , David Levens.
Craig J Benham. Sabrina R Leslie. To whom correspondence should be addressed.
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