Biology subjects

Benham, C. J.

Publications and source records attributed to Benham, C. J..

3 recordsLinked to original sources

bioRxiv2025

TORCphysics: A physical model of DNA-topology-controlled gene expression

DNA superhelicity and transcription are intimately related because changes to DNA topology can influence gene expression and vice versa. Information is transferred through the modulation of local DNA torsional stress, where the expression of one gene may influence the superhelical level of neighbouring genes, either promoting or repressing their expression. In this work, we introduce a one-dimensional physical model that simulates supercoiling-mediated regulation. This TORCphysics model takes as input a genome architecture represented either by a plasmid or by a linear DNA sequence with ends constrained under specific biological conditions, and computes the molecules output. Our findings demonstrate that the expression profiles of genes are directly influenced by the gene circuit design, including gene location, the positions of topological barriers, promoter sequences, and topoisomerase activity. The novelty that TORCphysics offers is versatility, where users can define distinct activity models for different types of proteins and protein binding sites. The aim of this research is to establish a flexible framework for developing physical simulations of gene circuits to deepen our comprehension of the intricate mechanisms involved in gene regulation.

biophysics↗

bioRxiv2019

Single-molecule visualization of the effects of ionic strength and crowding on structure-mediated interactions in supercoiled DNA molecules

DNA unwinding is an important cellular process involved in DNA replication, transcription and repair. In cells, molecular crowding caused by the presence of organelles, proteins, and other molecules affects numerous internal cellular structures. Here, we visualize plasmid DNA unwinding and binding dynamics to an oligonucleotide probe as functions of ionic strength, crowding agent concentration, and crowding agent species using single-molecule CLiC microscopy. We demonstrate increased probe-plasmid interaction over time with increasing concentration of 8 kDa polyethylene glycol (PEG), a crowding agent. We show decreased probe-plasmid interactions as ionic strength is increased without crowding. However, when crowding is introduced via 10% 8 kDa PEG, interactions between plasmids and oligos are enhanced. This is beyond what is expected for normal in vitro conditions, and may be a critically important, but as of yet unknown, factor in DNAs proper biological function in vivo. Our results show that crowding has a strong effect on the initial concentration of unwound plasmids. In the dilute conditions used in these experiments, crowding does not impact probe-plasmid interactions once the site is unwound.

biophysics↗

bioRxiv2018

Visualizing structure-mediated interactions in supercoiled DNA molecules

We directly visualize the topology-mediated interactions between an unwinding site on a supercoiled DNA plasmid and a specific probe molecule designed to bind to this site, as a function of DNA supercoiling and temperature. The visualization relies on containing the DNA molecules within an enclosed array of glass nanopits using the Convex Lens-induced Confinement (CLiC) imaging method. This method traps molecules within the focal plane while excluding signal from out-of-focus probes. Simultaneously, the molecules can freely diffuse within the nanopits, allowing for accurate measurements of exchange rates, unlike other methods which could introduce an artifactual bias in measurements of binding kinetics. We demonstrate that the plasmids structure influences the binding of the fluorescent probes to the unwinding site through the presence, or lack, of other secondary structures. With this method, we observe an increase in the binding rate of the fluorescent probe to the unwinding site with increasing temperature and negative supercoiling. This increase in binding is consistent with the results of our numerical simulations of the probability of site-unwinding. The temperature dependence of the binding rate has allowed us to distinguish the effects of competing higher order DNA structures, such as Z-DNA, in modulating local site-unwinding, and therefore binding.

biophysics↗