Synergistic Coordination of Chromatin Torsional Mechanics and Topoisomerase Activity

Cornell University Background
This technology allows for the in-vitro construction and physical manipulation of biomimetic braided chromatin substrates to screen topoisomerase II (Topo II) activity in the presence of inhibitory drug candidates. Positive candidates will inhibit topoisomerase II chromatin relaxation which is depicted to the researcher using ‘hat’ (extension versus turns) curves (Figure 1.).
Technology Overview
Researchers in the chemotherapeutic space have identified Topo II enzymes as promising targets for drug inhibition. This is because Topo II inhibition in cancer cell lines would lead to excessive genomic torsional stress creating DNA damage and eventual cell death – a promising and effective strategy for cancer chemotherapy. This technology is the first of its kind to allow researchers to screen Topo II enzymatic activity during candidate drug discovery in the presence of a braided chromatin substrate as would occur in an in-vivo eukaryotic environment. In short, synthetic DNA strands consisting of nucleosome position element repeats are tethered between one stable and one mobile surface (streptavidin-coated magnetic bead) (Figure 1). Chromatin reconstruction is then achieved by assembling nucleosomes onto the DNA substrate via purified histone octamer saturation. Following nucleosome assembly, topoisomerase II enzyme in the presence of a chemotherapeutic drug candidate is introduced and the mobile surface is twisted at a rate comparable to the rate that might occur during replication – mimicking the supercoiling introduced during in-vivo eukaryotic replication (+3.6 turns/sec). As torsional stress is introduced, the extension/compression of the chromatin substrate is measured and a ‘hat’ curve comparing extension vs a number of turns introduced is generated. Positive drug candidates will inhibit topoisomerase II chromatin relaxation which is depicted to the researcher using ‘hat’ (extension versus turns) curves (Figure 1.).

Further Details:
Tung T. Le, Xiang Gao, Seong ha Park, Jaeyoon Lee, James T. Inman, Joyce H. Lee, Jessica L. Killian, Ryan P. Badman, James M. Berger, Michelle D. Wang, Synergistic Coordination of Chromatin Torsional Mechanics and Topoisomerase Activity, Cell, Volume 179, Issue 3, 2019, Pages 619-631.e15, ISSN 0092-8674, https://doi.org/10.1016/j.cell.2019.09.034.
Benefits

First technology to use biologically relevant Topo II enzymatic substrate (braided chromatin) to screen enzyme activity/inhibition.
The braided chromatin substrate is supercoiled at +3.6 turns/sec mimicking supercoiling introduced during eukaryotic replication.
Topo II binding affinity and Topo II relaxation efficiency can be calculated in one assay.

Applications

Topo II – targeted chemotherapeutic drug discovery.
Facilitate transcription in nucleosomes.
Develop better in vitro approaches to study chromosome stress in lab settings.

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