Andrew D. Hirsh

Structural Ensemble and Dynamics of Toroidal-like DNA Shapes in Bacteriophage ϕ29 Exit Cavity

In the bacteriophage ϕ29, DNA is packed into a preassembled capsid from which it ejects under high pressure. A recent cryo-EM reconstruction of ϕ29 revealed a compact toroidal DNA structure (30-40 basepairs) lodged within the exit cavity formed by the connector-lower collar protein complex. Using multiscale models, we compute a detailed structural ensemble of intriguing DNA toroids of various lengths, all highly compatible with experimental observations. In particular, coarse-grained (elastic rod) and atomistic (molecular dynamics) models predict the formation of DNA toroids under significant compression, a largely unexplored state of DNA. Model predictions confirm that a biologically attainable compressive force of 25 pN sustains the toroid and yields DNA electron density maps highly consistent with the experimental reconstruction. The subsequent simulation of dynamic toroid ejection reveals large reactions on the connector that may signal genome release.

Role of microscopic flexibility in tightly curved DNA

The genetic material in living cells is organized into complex structures in which DNA is subjected to substantial contortions. Here we investigate the difference in structure, dynamics, and flexibility between two topological states of a short (107 base pair) DNA sequence in a linear form and a covalently closed, tightly curved circular DNA form. By employing a combination of all-atom molecular dynamics (MD) simulations and elastic rod modeling of DNA, which allows capturing microscopic details while monitoring the global dynamics, we demonstrate that in the highly curved regime the microscopic flexibility of the DNA drastically increases due to the local mobility of the duplex. By analyzing vibrational entropy and Lipari–Szabo NMR order parameters from the simulation data, we propose a novel model for the thermodynamic stability of high-curvature DNA states based on vibrational untightening of the duplex. This novel view of DNA bending provides a fundamental explanation that bridges the gap between classical models of DNA and experimental studies on DNA cyclization, which so far have been in substantial disagreement.

DNA buckling in bacteriophage cavities as a mechanism to aid virus assembly.

While relatively simple biologically, bacteriophages are sophisticated biochemical machines that execute a precise sequence of events during virus assembly, DNA packaging, and ejection. These stages of the viral life cycle require intricate coordination of viral components whose structures are being revealed by single molecule experiments and high resolution (cryo-electron microscopy) reconstructions. For example, during packaging, bacteriophages employ some of the strongest known molecular motors to package DNA against increasing pressure within the viral capsid shell. Located upstream of the motor is an elaborate portal system through which DNA is threaded. A high resolution reconstruction of the portal system for bacteriophage ϕ29 reveals that DNA buckles inside a small cavity under large compressive forces. In this study, we demonstrate that DNA can also buckle in other bacteriophages including T7 and P22. Using a computational rod model for DNA, we demonstrate that a DNA buckle can initiate and grow within the small confines of a cavity under biologically-attainable force levels. The forces of DNA-cavity contact and DNA-DNA electrostatic repulsion ultimately limit cavity filling. Despite conforming to very different cavity geometries, the buckled DNA within T7 and P22 exhibits near equal volumetric energy density (∼1kT/nm(3)) and energetic cost of packaging (∼22kT). We hypothesize that a DNA buckle creates large forces on the cavity interior to signal the conformational changes to end packaging. In addition, a DNA buckle may help retain the genome prior to tail assembly through significantly increased contact area with the portal.

Existence and Possible Function of Buckled DNA in Tailed DSDNA Bacteriophage Portals

Tailed double-stranded DNA (dsDNA) bacteriophages control genome packaging and ejection from their viral capsids using a portal system. The portal vertex serves as a docking site for the viral ATPase packing motor which translocates DNA through the central channel during packaging. In the case of several well-studied bacteriophages including T7 and e15, the central channel extends into the capsids interior by a core assembly of stacked, cylindrical protein subunits. Often possessing different internal diameters, these protein subunits can create large cavities in an otherwise straight channel. The height of these cavities are typically within 10-20% of the DNA persistence length and, in the case of T7, the cavity is 50A tall and 110A wide. Given that these cavities exist upstream of the packing motor, we postulate that they allow DNA to buckle under large packing forces (∼100 pN). A cryo-EM reconstruction of φ29 revealed that DNA buckles in a toroidal supercoil within a cavity only 3.5 times wider and 2.5 times taller than the width of DNA. A recent reconstruction of bacteriophage P22 also revealed DNA density inside its portal cavity that is over twice the width of dsDNA, suggesting that DNA may be compressed into a highly-bent supercoil. using analytic and numerical approaches, we compute the forces required for and during DNA buckling in bacteriophages T7 and P22. We demonstrate that DNA can indeed buckle and that the buckled conformation subsequently pushes outward on the cavity. Thus, the buckle could mechanically initiate a conformational change in the portal protein to provide the head-full signal.

Investigating a Novel Toroid-Shaped DNA Structure Found in Mature Bacteriophage φ29

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.

A Model for Highly Strained DNA in a Cavity

A single DNA molecule is a long and flexible biopolymer that contains the genetic code. Building upon the discovery of the iconic double helix over 50 years ago, subsequent studies have emphasized how its biological function is related to the mechanical properties of the molecule. A remarkable system which high-lights the role of DNA bending and twisting is the packing and ejection of DNA into and from viral capsids. A recent 3D reconstruction of bacteriophage φ29 reveals a novel toroidal structure thought to be 30–40 bp of highly bent/twisted DNA contained in a small cavity below the capsid. Here, we extend an elastic rod model for DNA to enable simulation of the toroid as it is compacted and subsequently ejected from a small volume. We compute biologically-realistic forces required to form the toroid and predict ejection times of several nanoseconds.© 2011 ASME