DNA Dynamics and Chromatin Landscapes in the Circulome
Stephen Levene,1,7 Andrew Fire,2,3 William Sandel,4 Thana Sutthibutpong,5 Euan Ashley,2,6 Vicki Parikh,6 Thomas C Bishop,4 Massa Shoura,2,3,7 and Sarah Harris8
1Departments of Bioengineering, Biological Sciences, and Physics, University of Texas at Dallas, USA
2Department of Genetics, Stanford University School of Medicine, USA
3Department of Pathology, Stanford University School of Medicine, USA
4Department of Physics, Louisiana Tech University, USA
5Department of Physics, King Mongkut's University of Technology, Thonburi, Thailand
6Department of Medicine, Stanford University School of Medicine, USA
7Phinomics, Inc., USA
8Department of Physics, Sheffield University, UK
The topological state of a genome, linear versus circular, is generally taken to be an important phylogenetic boundary that separates prokaryotes from eukaryotes. Exceptions to this tenet were revealed beginning in the 1960s, when circular extrachromosomal DNA elements were found in frog oocytes. Since that time circular DNAs have been shown to exist in every eukaryote, from yeast to humans, in which they have been sought. Although their ultimate function and the mechanisms responsible for their biogenesis remain poorly understood, next-generation DNA-sequencing methods have begun to uncover details of this novel molecular population. We have shown that extrachromosomal-circular DNAs (eccDNAs) exist as distinct and partially overlapping populations in both normal and aberrant cell types and have coined the term “circulome” to encompass the general repertoire of circular DNAs present in any genome. Novel methods for circular-DNA enrichment and characterization have facilitated further studies to investigate nucleosome occupancy on eccDNAs in vivo and the consequences of “chromatinization” on eccDNA function. For example, the recent finding that small (< 1000 bp) eccDNAs function as transcriptional templates in the absence of canonical promoter sequences provokes questions about alternative, promoter-independent mechanisms for transcription initiation.
Both experimental and computational data regarding nucleosome stability on small DNA circles are limited; there are presently no data in the literature on nucleosomal eccDNAs. This motivated an extensive molecular-dynamics study of a particular 358-bp eccDNA sequence composed of an exon from the human Titin gene. To assess nucleosome stability on this small DNA template, we simulated three different forms of this circle: the naked DNA circle, the mononucleosomal form, and a dinucleosomal circle. Unexpectedly, the dinucleosomal form showed greater stability than the mononucleosomal circle in explicit-solvent simulations containing either sodium or calcium counterions. Moreover, we show that instantaneous removal of one histone octamer from the dinucleosomal structure promotes strand separation distal from the previously occupied nucleosome binding site. The strong coupling of minicircle-DNA twist to nucleosome occupancy therefore provides an alternative pathway for duplex opening and potentially, transcription initiation.