Topological Behavior of Plasmid DNA

Abstract

The discovery of the B-form structure of DNA by Watson and Crick led to an explosion of research on nucleic acids in the fields of biochemistry, biophysics, and genetics. Powerful techniques were developed to reveal a myriad of different structural conformations that change B-DNA as it is transcribed, replicated, and recombined and as sister chromosomes are moved into new daughter cell compartments during cell division. This article links the original discoveries of superhelical structure and molecular topology to non-B form DNA structure and contemporary biochemical and biophysical techniques. The emphasis is on the power of plasmids for studying DNA structure and function. The conditions that trigger the formation of alternative DNA structures such as left-handed Z-DNA, inter- and intra-molecular triplexes, triple-stranded DNA, and linked catenanes and hemicatenanes are explained. The DNA dynamics and topological issues are detailed for stalled replication forks and for torsional and structural changes on DNA in front of and behind a transcription complex and a replisome. The complex and interconnected roles of topoisomerases and abundant small nucleoid association proteins are explained. And methods are described for comparing in vivo and in vitro reactions to probe and understand the temporal pathways of DNA and chromosome chemistry that occur inside living cells.

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

“…A growing body of work reveals that regulation of DNA supercoiling in bacterial chromosomes entails an interplay between topoisomerases and transcription by RNA polymerase (6,10,(31)(32)(33). According to the twin supercoiled domain model (64), actively transcribing RNA polymerase must locally unwind the DNA double helix to provide access to the antisense strand.…”

“…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).…”

“…Despite decades of theoretical and computational investigation on supercoiled DNA (10)(11)(12)(13)(14)(15)(16)(17)(18)(34)(35)(36), the behavior of supercoiled DNA rings in free solution at the length scale of topological domains in bacteria with supercoiling <s~À0.07 has remained underexplored. However, these larger linking deficits represent an important biological regime, because DNA gyrase is capable of generating supercoiling densities up to s~À0.12, and RNA polymerase has been observed to generate similar or greater linking deficits (28)(29)(30).…”

DNA supercoiling is a fundamental regulatory principle in the control of bacterial gene expression

“…The physical manifestation of its underwound state is seen in the adoption by DNA of a minimal energy conformation in which the helical axis writhes about itself; it can also be expressed through the opening of part of the DNA helix through a loss of base-pairing or by a combination of the two (Bauer et al 1980). The writhing of the underwound duplex is described as negative supercoiling (Higgins and Vologodskii 2015). In a bacterium such as Escherichia coli, about half of the DNA supercoils are constrained by interaction with proteins (Bliska and Cozzarelli 1987;Pettijohn and Pfenninger 1980), but the energy in the unconstrained portion is available to drive DNA transactions (Booker et al 2010).…”

Species-specific supercoil dynamics of the bacterial nucleoid

“…1). Supercoil diffusion is critical for many biochemical reactions of the chromosome, including sitespecific recombination, transcription, transposition of mobile elements, and initiation of DNA replication (Higgins and Vologodskii 2015). Yet this DNA movement is not discernable in real time, even with the highest resolution techniques currently available.…”