Structural diversity of supercoiled DNA

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.

Large-Scale Conformational Transitions in Supercoiled DNA Revealed by Coarse-Grained Simulation

“…A number of studies have investigated the conformations of supercoiled plasmids using atomic force microscopy (20) or electron microscopy (19,25), which reveal conformations that are qualitatively in agreement with the configurations realized in our simulations.…”

“…In addition, recent electron microscopy and molecular dynamics studies demonstrated that in very small DNA rings (336 bp), where DNA supercoiling requires the presence of sharply bent loops at the apices of the plectoneme, local denaturation may occur to accommodate kinks in the DNA (25). In this case, the short length of the DNA minicircles imposes a geometric constraint for writhing that likely enhances the formation of denatured regions that enable sharp bending.…”

“…Although supercoiled DNA has been extensively studied theoretically (10)(11)(12)(13)(14)(15)(16)(17)(18) as well as experimentally (19)(20)(21)(22)(23)(24)(25)(26), both simulation and experimental studies that directly investigate the large-scale organization of supercoiled DNA are typically limited to supercoiling densities up to the average supercoiling density in E. coli (s~À0.06). However, the topological state of the bacterial chromosome is highly dynamic (27), and both DNA gyrase and RNA polymerase are capable of generating negative supercoiling densities far in excess of average supercoiling levels (28)(29)(30).…”

“…Although simulation work on the scale of topological domains of E. coli has been previously reported, these studies were limited to supercoiling densities with jsj < 0.07 (11). Previous simulation work at larger degrees of unwinding has been restricted to smaller rings (25,37), where branching is limited, or to the presence of extensional forces that suppress plectonemic supercoil formation (38). In contrast, our simulations explore the behavior of DNA rings in solution, without extensional forces, and extend predictions for s < À0.06 up to the length scale of topological domains in E. coli, where we observe previously unreported structural transitions associated with branching statistics.…”

The regulatory role of DNA supercoiling in nucleoprotein complex assembly and genetic activity

“…Again, the DNA in small circles has higher configurational entropy than in larger circles, which accommodate writhing by adopting a plectonemic form (Mitchell and Harris 2013). DNA supercoiling may thus affect the number of configurations adopted by the DNA (Irobalieva et al 2015), or the volume of the configurational space (Bednar et al 1994;Travers et al 2012). In other words, the repertoire of possible local structures will depend on the superhelical state of the DNA and the sign (either negative or positive) of coiling.…”