Abstract
Genomic DNA is vastly longer than the space allotted to it in a cell. The molecule must fold with a level of organization that satisfies the imposed spatial constraints as well as allow for the processing of genetic information. Key players in this organization include the negative supercoiling of DNA, which facilitates the unwinding of the double-helical molecule, and the associations of DNA with proteins, which partition the DNA into isolated loops, or domains. In order to gain insight into the principles of genome organization and to visualize the folding of spatially constrained DNA, we have developed new computational methods to identify the preferred three-dimensional pathways of protein-mediated DNA loops and to characterize the topological properties of these structures. Here, we focus on the levels of supercoiling and the spatial arrangements of DNA in model nucleoprotein systems with two topological domains. We construct these systems by anchoring DNA loops in opposing orientations on a common protein–DNA assembly, namely the Lac repressor protein with two bound DNA operators. The linked pieces of DNA form a covalently closed circle such that the protein attaches to two widely spaced sites along the DNA. We examine the effects of operator spacing, loop orientation, and long-range contacts on overall chain configuration and topology, and discuss our findings in the context of classic experiments on the effects of supercoiling and operator spacing on Lac repressor-mediated looping and recent work on the role of proteins as barriers that divide genomes into independent topological domains.
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Notes
If the 5′-ends of the DNA are anchored to a common operator, the former pair of loops initially points toward the inside of the repressor assembly and the latter pair toward the outside.
Concatenation of opposing loops entails removal of a common base pair from each of the bound operators, thereby reducing the number of base-pair steps in the resultant minicircle by two compared to the total number of steps in the two loops.
The choice of loop lengths is based on the difficulty of anchoring very short (<73 bp) loops to the assumed V-shaped Lac repressor model (Czapla et al. 2013) and the restraints of the minicircle on maximum loop size, i.e., 182 + 2 = 73 + 111.
Estimation of the twist from the rigid-body parameter of the same name exaggerates the unwinding at the CG step and incorrectly suggests that the repressor unwinds DNA by ~15°. Such treatment ignores the contribution to supercoiling from the large shear of base pairs at the CG step (Britton et al. 2009).
Our work to date has focused on the energies and topological properties of short DNA loops of 73–143 bp, where one can omit consideration of the thermal fluctuations of the closed structures and the accompanying effects of these changes on long-range inter-loop interactions.
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Acknowledgments
This work was generously supported by the U.S. Public Health Service under research grant GM34809. P.J.P. gratefully acknowledges support from a U.S. Department of Education Graduate Assistance in Areas of National Need Fellowship.
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Pamela J. Perez declares that she has no conflict of interest.
Wilma K. Olson declares that she has no conflict of interest.
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This article is part of a Special Issue on “DNA supercoiling, protein interactions and genetic function” edited by Laura Finzi and Wilma Olson.
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Perez, P.J., Olson, W.K. Insights into genome architecture deduced from the properties of short Lac repressor-mediated DNA loops. Biophys Rev 8 (Suppl 1), 135–144 (2016). https://doi.org/10.1007/s12551-016-0209-7
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DOI: https://doi.org/10.1007/s12551-016-0209-7