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Title:Interactions between nucleoid associated proteins and DNA in the presence of mechanical and chemical forces
Author(s):Dahlke, Katelyn B.
Director of Research:Sing, Charles E.
Doctoral Committee Chair(s):Sing, Charles E.
Doctoral Committee Member(s):Aksimentiev, Aleksei; Schroeder, Charles M.; Shukla, Diwakar
Department / Program:Chemical & Biomolecular Engr
Discipline:Chemical Engineering
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Coarse-grained simulation
nucleoid associated proteins
Abstract:The way that DNA is organized within a cell controls its physiological behavior. DNA must be condensed in order to fit into the much smaller cell, but must also be accessible to proteins responsible for biological processes. Architectural proteins assist with this large-scale arrangement of DNA to achieve the correct balance between these two competing requirements. The structural proteins that interact with DNA in prokaryotes are known as nucleoid associated proteins (NAPs). These proteins play a vital role in shaping the DNA and assist in many cell processes, including gene expression, replication, and transcription. NAPs have been studied extensively in order to elucidate how their physical properties (such as binding kinetics or DNA manipulation) aid in regulating cell function. It has been shown in experiment and simulation that NAPs can adopt multiple binding states (i.e., the protein can be partially associated with its DNA substrate), which leads to complex binding and unbinding kinetics. For a simple binary system, where a protein can either be bound or unbound, the kinetics are relatively straightforward: there is a concentration dependent on-rate (kon) and a concentration independent off-rate (koff). When a protein-substrate complex has a non-binary set of bound states (including an intermediate "partially bound" state), other molecules in solution can impact the dissociation behavior. In fact, these competitors compete with the original protein for binding sites, which enhances the dissociation of the protein from its original substrate. This concentration-dependent dissociation is called "facilitated dissociation" (FD). We have developed a coarse-grained model of a typical NAP-DNA system that is built up from local interactions, such as the mutlivalent binding that leads to FD as well as physical deformations of DNA induced by protein binding. This methodical coarse-graining allows us to investigate the effect that these short-range interactions have on the mesoscale behavior of the system. We have investigated the cooperative and competing behavior of NAP-DNA interactions that result in concentration-, force-, and topology-dependent changes to both protein kinetics and physical DNA behavior. Our model qualitatively matches experimental observations, and provides a physical explanation for the observed behavior based on cooperative local interactions. We demonstrate how the competition for binding sites along a DNA strand is affected by the energy barriers between the three possible bound states in the system (bound, partially bound, and unbound). This is the driving force behind facilitated dissociation; thus, changing the level of binding competition changes the dissociation behavior. Our model allows us to manually manipulate the binding energy landscape that other methods are unable to achieve. We can independently change the energy barriers between the three bound states, which in turn changes a protein's preferred bound state. This leads to three different concentration-dependent FD kinetic regimes: a concentration-independent off-rate, a linear dependence on concentration, and a combination of the two. The multivalent binding also leads to multiple dissociation pathways: spontaneous and facilitated. The dissociation pathway a protein undergoes is dependent on a number of factors including force, the local geometric deformation, and protein concentration. We investigate how these factors impact the dissociation kinetics of a system that undergoes FD by expanding our model to account for the physical bends that NAPs induce in DNA upon binding in the DNA model, and also in the energy barrier landscape. At low forces, more proteins will be bound due to the more relaxed nature of the DNA strand that more easily allows local kinks caused by NAPs. As force is increased, there will be fewer bound proteins because of the more extended nature of the DNA strand, which is in a less preferential conformation. This force-enhanced unbinding and force-inhibited binding changes how a protein dissociates from DNA, either through FD at low force or spontaneously at high force. We observe two two classes of dissociation: a classical "slip bond," where a bond weakens with force, and a "catch bond," where a bond is strengthened with force. The physical deformation that NAPs cause affects not only the binding and unbinding kinetics; it also impacts the long-scale equilibrium and dynamic DNA elasticity. As more NAPs are bound to the DNA, there are more local kinks, decreasing the end-to-end distance of the single DNA strand. Because NAPs can adopt two possible binding states, DNA can undergo two different types of deformations. This leads to a non-monotonic effect of concentration on the force-extension behavior of the DNA strand. Our method allows us to study non-equilibrium elastic behaviors as well, such as DNA extending dynamically. The competition between the two characteristic time scales of the system (unbinding time and pulling time) leads to extension-rate dependent effects on both DNA elasticity and binding behavior. We are able to show that NAPs help stabilize DNA supercoils due to these same local, cooperative effects. DNA supercoiling occurs when DNA wraps around itself to relieve torsional stress. Both DNA supercoiling and NAPs are present in prokaryotic cells, but the role of NAPs in supercoiling activity is not fully understood. Our model demonstrates that NAPs are more likely to bind to supercoiled DNA, due to the protein's preference to bind to already-bent DNA, which in turn stabilizes the supercoil. This leads to a concentration-dependent change of the phase transition between extended and supercoiled DNA in the force-torque ensemble. We are able to use a combination of simulation data and theoretical predictions of the various energies of the system, such as the stretching, bending, and excluded volume energies, as a function of both force and concentration. This information can be used to develop a theory that provides a thorough understanding of how NAPs affect DNA supercoiling.
Issue Date:2019-05-28
Rights Information:Copyright 2019 Katelyn Dahlke
Date Available in IDEALS:2019-11-26
Date Deposited:2019-08

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