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Ioan Andricioaei - One of the best experts on this subject based on the ideXlab platform.

  • Conformational Transitions of Nucleic Acids under External Forces: Computer Simulations and a Stochastic Theory for their Kinetics
    Biophysical Journal, 2013
    Co-Authors: Ioan Andricioaei
    Abstract:

    I will present molecular dynamics simulations of several examples of conformational transitions that nucleic acids and their complexes undergo upon the application of external forces and/or torques:(1) DNA Supercoil relaxation by topoisomerases,(2) the condensation of DNA by dendrimers and,(3) RNA unfolding.Then I will showcase the use of the formalism of stochastic path integrals to deduce the kinetics of these transitions, from simulation trajectories or experimental single molecule recordings of the transition, under other conditions that those that are actually simulated or recorded.

  • A multiscale dynamic model of DNA Supercoil relaxation by topoisomerase IB.
    Biophysical journal, 2011
    Co-Authors: Todd D. Lillian, Maryna Taranova, Jeff Wereszczynski, Ioan Andricioaei, Noel C. Perkins
    Abstract:

    In this study, we report what we believe to be the first multiscale simulation of the dynamic relaxation of DNA Supercoils by human topoisomerase IB (topo IB). We leverage our previous molecular dynamics calculations of the free energy landscape describing the interaction between a short DNA fragment and topo IB. Herein, this landscape is used to prescribe boundary conditions for a computational, elastodynamic continuum rod model of a long length of Supercoiled DNA. The rod model, which accounts for the nonlinear bending, twisting, and electrostatic interaction of the (negatively charged) DNA backbone, is extended to include the hydrodynamic drag induced by the surrounding physiological buffer. Simulations for a 200-bp-long DNA Supercoil in complex with topo IB reveal a relaxation timescale of ∼0.1–1.0 μs. The relaxation follows a sequence of cascading reductions in the Supercoil linking number (Lk), twist (Tw), and writhe (Wr) that follow companion cascading reductions in the Supercoil elastic and electrostatic energies. The novel (to our knowledge) multiscale modeling method may enable simulations of the entire experimental setup that measures DNA Supercoiling and relaxation via single molecule magnetic trapping.

  • Investigating a Novel Toroid-Shaped DNA Structure Found in Mature Bacteriophage φ29
    Biophysical Journal, 2011
    Co-Authors: Andrew D. Hirsh, Todd D. Lillian, Maryna Taranova, Ioan Andricioaei, Troy A. Lionberger, Noel C. Perkins
    Abstract:

    While the typical viral genome is several kilobases long, it is packed to near crystalline density within a viral capsid only tens of nanometers in diameter. In the case of bacteriophage φ29, this enormous compaction results from strong molecular motors that generate forces of approximately 100 pN. Recently, a three-dimensional cryo-electron microscopy reconstruction of mature φ29 was published. An intriguing feature in the reconstruction is a 60A diameter toroidal DNA Supercoil, estimated to be only 30-40 base pairs in length, within the cavity formed by the connector and the lower collar. The function of this highly-bent DNA, remains unknown. In this study, we use an elastic rod model to simulate the DNA inside the cavity. We learn that as more DNA is pushed into the capsid, compressive forces build until a critical load is reached and the DNA ‘buckles’ to fill the cavity thereby forming the toroidal Supercoil observed in the reconstruction. We estimate the energy, forces, and torques required to form this toroidal DNA inside the cavity. Based on these results, we propose possible biological functions of this intriguing structure.

  • Free Energy Calculations Reveal Rotating-Ratchet Mechanism for DNA Supercoil Relaxation by Topoisomerase IB and its Inhibition
    Biophysical journal, 2010
    Co-Authors: Jeff Wereszczynski, Ioan Andricioaei
    Abstract:

    Topoisomerases maintain the proper topological state of DNA. Human topoisomerase I removes DNA Supercoils by clamping a duplex DNA segment, nicking one strand at a phosphodiester bond, covalently attaching to the 3′ end of the nick, and allowing the DNA downstream of the cut to rotate around the intact strand. Using molecular dynamics simulations and umbrella sampling free energy calculations, we show that the rotation of downstream DNA in the grip of the enzyme that brings about release of positive or negative Supercoils occurs by thermally assisted diffusion on ratchet energy profiles. The ratchetlike free-energy-versus-rotation profile that we compute provides a model for the function of topoisomerase in which the periodic maxima along the profile modulate the rate of Supercoil relaxation, while the minima provide metastable conformational states for DNA religation. The results confirm previous experimental and computational work, and suggest that relaxation of the two types of Supercoils involves distinct protein pathways. Additionally, simulations performed with the ternary complex of topoisomerase, DNA, and the chemotherapeutic drug topotecan show important differences in the mechanisms for Supercoil relaxation when the drug is present, accounting for the relative values of relaxation rates measured in single-molecule experiments. Good agreement is found between rate constants from tweezer experiments and those calculated from simulations. Evidence is presented for the existence of semiopen states of the protein, which facilitate rotations after the initial one, as a result of biasing the protein into a conformation more favorable to strand rotation than the closed state required for nicking of the DNA.

Todd D. Lillian - One of the best experts on this subject based on the ideXlab platform.

  • Simulation of DNA Supercoil Relaxation
    Biophysical journal, 2016
    Co-Authors: Ikenna D. Ivenso, Todd D. Lillian
    Abstract:

    Several recent single-molecule experiments observe the response of Supercoiled DNA to nicking endonucleases and topoisomerases. Typically in these experiments, indirect measurements of Supercoil relaxation are obtained by observing the motion of a large micron-sized bead. The bead, which also serves to manipulate DNA, experiences significant drag and thereby obscures Supercoil dynamics. Here we employ our discrete wormlike chain model to bypass experimental limitations and simulate the dynamic response of Supercoiled DNA to a single strand nick. From our simulations, we make three major observations. First, extension is a poor dynamic measure of Supercoil relaxation; in fact, the linking number relaxes so fast that it cannot have much impact on extension. Second, the rate of linking number relaxation depends upon its initial partitioning into twist and writhe as determined by tension. Third, the extensional response strongly depends upon the initial position of plectonemes.

  • Brownian Dynamics Study of DNA Supercoil Relaxation
    Biophysical Journal, 2015
    Co-Authors: Ikenna D. Ivenso, Todd D. Lillian
    Abstract:

    The stresses induced in DNA during cell processes such as replication and transcription lead to the formation of plectonemic Supercoils. Supercoiling in turn affects these processes. As a result of this relationship between Supercoiling and these processes, the degree of Supercoiling in DNA needs to be controlled closely in order to optimize these cell processes. This control is facilitated by enzymes such as type 1b topoisomerases and nicking endonucleases which relax Supercoiling in DNA.The dynamics of DNA Supercoil relaxation have been studied in recent experiments by means of single-molecule magnetic and/or optical tweezer experiments. Novel as these experiments are, they do not permit a direct observation of the structural changes that occur in DNA during Supercoil relaxation. They depend on components (e.g. a paramagnetic bead) attached to the DNA to indirectly obtain information about these dynamics.We studied the dynamics of Supercoil relaxation by means of Brownian Dynamics simulations of a discrete wormlike-chain (dWLC) model of DNA. These simulations parallel the single-molecule experiments in which a single DNA molecule is held under constant tension so that its end-to-end extension increases as Supercoils are relaxed by a nicking endonuclease. The dWLC model accounts for elasticity, electrostatics and entropic forces as well as for hydrodynamic interactions.From the simulation results, we more directly extract the rate at which Supercoiling in DNA is relaxed by nicking endonucleases. We also determine the dependence of the relaxation timescales on the tension applied to the DNA molecule.

  • A multiscale dynamic model of DNA Supercoil relaxation by topoisomerase IB.
    Biophysical journal, 2011
    Co-Authors: Todd D. Lillian, Maryna Taranova, Jeff Wereszczynski, Ioan Andricioaei, Noel C. Perkins
    Abstract:

    In this study, we report what we believe to be the first multiscale simulation of the dynamic relaxation of DNA Supercoils by human topoisomerase IB (topo IB). We leverage our previous molecular dynamics calculations of the free energy landscape describing the interaction between a short DNA fragment and topo IB. Herein, this landscape is used to prescribe boundary conditions for a computational, elastodynamic continuum rod model of a long length of Supercoiled DNA. The rod model, which accounts for the nonlinear bending, twisting, and electrostatic interaction of the (negatively charged) DNA backbone, is extended to include the hydrodynamic drag induced by the surrounding physiological buffer. Simulations for a 200-bp-long DNA Supercoil in complex with topo IB reveal a relaxation timescale of ∼0.1–1.0 μs. The relaxation follows a sequence of cascading reductions in the Supercoil linking number (Lk), twist (Tw), and writhe (Wr) that follow companion cascading reductions in the Supercoil elastic and electrostatic energies. The novel (to our knowledge) multiscale modeling method may enable simulations of the entire experimental setup that measures DNA Supercoiling and relaxation via single molecule magnetic trapping.

  • Investigating a Novel Toroid-Shaped DNA Structure Found in Mature Bacteriophage φ29
    Biophysical Journal, 2011
    Co-Authors: Andrew D. Hirsh, Todd D. Lillian, Maryna Taranova, Ioan Andricioaei, Troy A. Lionberger, Noel C. Perkins
    Abstract:

    While the typical viral genome is several kilobases long, it is packed to near crystalline density within a viral capsid only tens of nanometers in diameter. In the case of bacteriophage φ29, this enormous compaction results from strong molecular motors that generate forces of approximately 100 pN. Recently, a three-dimensional cryo-electron microscopy reconstruction of mature φ29 was published. An intriguing feature in the reconstruction is a 60A diameter toroidal DNA Supercoil, estimated to be only 30-40 base pairs in length, within the cavity formed by the connector and the lower collar. The function of this highly-bent DNA, remains unknown. In this study, we use an elastic rod model to simulate the DNA inside the cavity. We learn that as more DNA is pushed into the capsid, compressive forces build until a critical load is reached and the DNA ‘buckles’ to fill the cavity thereby forming the toroidal Supercoil observed in the reconstruction. We estimate the energy, forces, and torques required to form this toroidal DNA inside the cavity. Based on these results, we propose possible biological functions of this intriguing structure.

Noel C. Perkins - One of the best experts on this subject based on the ideXlab platform.

  • A multiscale dynamic model of DNA Supercoil relaxation by topoisomerase IB.
    Biophysical journal, 2011
    Co-Authors: Todd D. Lillian, Maryna Taranova, Jeff Wereszczynski, Ioan Andricioaei, Noel C. Perkins
    Abstract:

    In this study, we report what we believe to be the first multiscale simulation of the dynamic relaxation of DNA Supercoils by human topoisomerase IB (topo IB). We leverage our previous molecular dynamics calculations of the free energy landscape describing the interaction between a short DNA fragment and topo IB. Herein, this landscape is used to prescribe boundary conditions for a computational, elastodynamic continuum rod model of a long length of Supercoiled DNA. The rod model, which accounts for the nonlinear bending, twisting, and electrostatic interaction of the (negatively charged) DNA backbone, is extended to include the hydrodynamic drag induced by the surrounding physiological buffer. Simulations for a 200-bp-long DNA Supercoil in complex with topo IB reveal a relaxation timescale of ∼0.1–1.0 μs. The relaxation follows a sequence of cascading reductions in the Supercoil linking number (Lk), twist (Tw), and writhe (Wr) that follow companion cascading reductions in the Supercoil elastic and electrostatic energies. The novel (to our knowledge) multiscale modeling method may enable simulations of the entire experimental setup that measures DNA Supercoiling and relaxation via single molecule magnetic trapping.

  • Investigating a Novel Toroid-Shaped DNA Structure Found in Mature Bacteriophage φ29
    Biophysical Journal, 2011
    Co-Authors: Andrew D. Hirsh, Todd D. Lillian, Maryna Taranova, Ioan Andricioaei, Troy A. Lionberger, Noel C. Perkins
    Abstract:

    While the typical viral genome is several kilobases long, it is packed to near crystalline density within a viral capsid only tens of nanometers in diameter. In the case of bacteriophage φ29, this enormous compaction results from strong molecular motors that generate forces of approximately 100 pN. Recently, a three-dimensional cryo-electron microscopy reconstruction of mature φ29 was published. An intriguing feature in the reconstruction is a 60A diameter toroidal DNA Supercoil, estimated to be only 30-40 base pairs in length, within the cavity formed by the connector and the lower collar. The function of this highly-bent DNA, remains unknown. In this study, we use an elastic rod model to simulate the DNA inside the cavity. We learn that as more DNA is pushed into the capsid, compressive forces build until a critical load is reached and the DNA ‘buckles’ to fill the cavity thereby forming the toroidal Supercoil observed in the reconstruction. We estimate the energy, forces, and torques required to form this toroidal DNA inside the cavity. Based on these results, we propose possible biological functions of this intriguing structure.

Jeff Wereszczynski - One of the best experts on this subject based on the ideXlab platform.

  • A multiscale dynamic model of DNA Supercoil relaxation by topoisomerase IB.
    Biophysical journal, 2011
    Co-Authors: Todd D. Lillian, Maryna Taranova, Jeff Wereszczynski, Ioan Andricioaei, Noel C. Perkins
    Abstract:

    In this study, we report what we believe to be the first multiscale simulation of the dynamic relaxation of DNA Supercoils by human topoisomerase IB (topo IB). We leverage our previous molecular dynamics calculations of the free energy landscape describing the interaction between a short DNA fragment and topo IB. Herein, this landscape is used to prescribe boundary conditions for a computational, elastodynamic continuum rod model of a long length of Supercoiled DNA. The rod model, which accounts for the nonlinear bending, twisting, and electrostatic interaction of the (negatively charged) DNA backbone, is extended to include the hydrodynamic drag induced by the surrounding physiological buffer. Simulations for a 200-bp-long DNA Supercoil in complex with topo IB reveal a relaxation timescale of ∼0.1–1.0 μs. The relaxation follows a sequence of cascading reductions in the Supercoil linking number (Lk), twist (Tw), and writhe (Wr) that follow companion cascading reductions in the Supercoil elastic and electrostatic energies. The novel (to our knowledge) multiscale modeling method may enable simulations of the entire experimental setup that measures DNA Supercoiling and relaxation via single molecule magnetic trapping.

  • Free Energy Calculations Reveal Rotating-Ratchet Mechanism for DNA Supercoil Relaxation by Topoisomerase IB and its Inhibition
    Biophysical journal, 2010
    Co-Authors: Jeff Wereszczynski, Ioan Andricioaei
    Abstract:

    Topoisomerases maintain the proper topological state of DNA. Human topoisomerase I removes DNA Supercoils by clamping a duplex DNA segment, nicking one strand at a phosphodiester bond, covalently attaching to the 3′ end of the nick, and allowing the DNA downstream of the cut to rotate around the intact strand. Using molecular dynamics simulations and umbrella sampling free energy calculations, we show that the rotation of downstream DNA in the grip of the enzyme that brings about release of positive or negative Supercoils occurs by thermally assisted diffusion on ratchet energy profiles. The ratchetlike free-energy-versus-rotation profile that we compute provides a model for the function of topoisomerase in which the periodic maxima along the profile modulate the rate of Supercoil relaxation, while the minima provide metastable conformational states for DNA religation. The results confirm previous experimental and computational work, and suggest that relaxation of the two types of Supercoils involves distinct protein pathways. Additionally, simulations performed with the ternary complex of topoisomerase, DNA, and the chemotherapeutic drug topotecan show important differences in the mechanisms for Supercoil relaxation when the drug is present, accounting for the relative values of relaxation rates measured in single-molecule experiments. Good agreement is found between rate constants from tweezer experiments and those calculated from simulations. Evidence is presented for the existence of semiopen states of the protein, which facilitate rotations after the initial one, as a result of biasing the protein into a conformation more favorable to strand rotation than the closed state required for nicking of the DNA.

James T. Stivers - One of the best experts on this subject based on the ideXlab platform.

  • Novel and specific inhibitors of a poxvirus type I topoisomerase.
    Molecular pharmacology, 2005
    Co-Authors: Alexis Bond, Zachary Reichert, James T. Stivers
    Abstract:

    Vaccinia DNA topoisomerase (vTopo) is a prototypic pox virus family topoisomerase that shares extensive structural and mechanistic properties with the human type IB enzyme (hTopo) and is important for viral replication. Despite their far-reaching similarities, vTopo and hTopo have surprisingly distinct pharmacological properties. To further exploit these differences, we have developed recently the first high-throughput screen for vTopo, which has allowed rapid screening of a 1990-member small-molecule library for inhibitors. Using this approach, 21 compounds were identified with IC90 values less than 10 μM, and 19 of these were also found to inhibit DNA Supercoil relaxation by vTopo. Four of the most potent compounds were completely characterized and are structurally novel topo I inhibitors with efficacies at nanomolar concentrations. These inhibitors were highly specific for vTopo, showing no inhibition of the human enzyme even at 500- to 2000-fold greater concentrations. We describe a battery of efficient experiments to characterize the unique mechanisms of these vTopo inhibitors and discuss the surprising promiscuity of this enzyme to inhibition by structurally diverse small molecules.

  • Vaccinia DNA topoisomerase I: evidence supporting a free rotation mechanism for DNA Supercoil relaxation.
    Biochemistry, 1997
    Co-Authors: James T. Stivers, Thomas K. Harris, Albert S. Mildvan
    Abstract:

    The Vaccinia type I topoisomerase catalyzes site-specific DNA strand cleavage and religation by forming a transient phosphotyrosyl linkage between the DNA and Tyr-274, resulting in the release of DNA Supercoils. For type I topoisomerases, two mechanisms have been proposed for Supercoil release: (I) a coupled mechanism termed strand passage, in which a single Supercoil is removed per cleavage/religation cycle, resulting in multiple topoisomer intermediates and late product formation, or (2) an uncoupled mechanism termed free rotation, where multiple Supercoils are removed per cleavage/religation cycle, resulting in few intermediates and early product formation. To determine the mechanism, single-turnover experiments were done with Supercoiled plasmid DNA under conditions in which the topoisomerase cleaves predominantly at a single site per DNA molecule. The concentrations of Supercoiled substrate, intermediate topoisomers, and relaxed product vs time were measured by fluorescence imaging, and the rate constants for their interconversion were determined by kinetic simulation. Few intermediates and early product formation were observed. From these data, the rate constants for cleavage (0.3 s(-1)), religation (4 s(-1)), and the cleavage equilibrium constant on the enzyme (0.075) at 22 degrees C are in reasonable agreement with those obtained with small oligonucleotide substrates, while the rotation rate of the cleaved DNA strand is fast (approximately 20 rotations/s). Thus, the average number of Supercoils removed for each cleavage event greatly exceeds unity (delta n = 5) and depends on kinetic competition between religation and Supercoil release, establishing a free rotation mechanism. This free rotation mechanism for a type I topoisomerase differs from the strand passage mechanism proposed for the type II enzymes.