DNA heats up : Energetics of genome ejection from phage revealed by isothermal titration calorimetry

Most bacteriophages are known to inject their double-stranded DNA into bacteria upon receptor binding in an essentially spontaneous way. This downhill thermodynamic process from the intact virion toward the empty viral capsid plus released DNA is made possible by the energy stored during active packaging of the genome into the capsid. Only indirect measurements of this energy have been available until now using either single-molecule or osmotic suppression techniques. In this paper, we describe for the first time the use of isothermal titration calorimetry to directly measure the heat released (or equivalently the enthalpy) during DNA ejection from phage lambda, triggered in solution by a solubilized receptor. Quantitative analyses of the results lead to the identification of thermodynamic determinants associated with DNA ejection. The values obtained were found to be consistent with those previously predicted by analytical models and numerical simulations. Moreover, the results confirm the role of DNA hydration in the energetics of genome confinement in viral capsids.Comment: 24 pages including figures and tabl

Biophysical and Ultrastructural Characterization of Adeno-Associated Virus Capsid Uncoating and Genome Release

We describe biophysical and ultrastructural differences in genome release from adeno-associated virus (AAV) capsids packaging wild-type DNA, recombinant single-stranded DNA (ssDNA), or dimeric, self-complementary DNA (scDNA) genomes. Atomic force microscopy and electron microscopy (EM) revealed that AAV particles release packaged genomes and undergo marked changes in capsid morphology upon heating in physiological buffer (pH 7.2). When different AAV capsids packaging ss/scDNA varying in length from 72 to 123% of wild-type DNA (3.4 to 5.8 kb) were incrementally heated, the proportion of uncoated AAV capsids decreased with genome length as observed by EM. Genome release was further characterized by a fluorimetric assay, which demonstrated that acidic pH and high osmotic pressure suppress genome release from AAV particles. In addition, fluorimetric analysis corroborated an inverse correlation between packaged genome length and the temperature needed to induce uncoating. Surprisingly, scAAV vectors required significantly higher temperatures to uncoat than their ssDNA-packaging counterparts. However, externalization of VP1 N termini appears to be unaffected by packaged genome length or self-complementarity. Further analysis by tungsten-shadowing EM revealed striking differences in the morphologies of ssDNA and scDNA genomes upon release from intact capsids. Computational modeling and molecular dynamics simulations suggest that the unusual thermal stability of scAAV vectors might arise from partial base pairing and optimal organization of packaged scDNA. Our work further defines the biophysical mechanisms underlying adeno-associated virus uncoating and genome release

Modeling and simulations of single stranded rna viruses

The presented work is the application of recent methodologies on modeling and simulation of single stranded RNA viruses. We first present the methods of modeling RNA molecules using the coarse-grained modeling package, YUP. Coarse-grained models simplify complex structures such as viruses and let us study general behavior of the complex biological systems that otherwise cannot be studied with all-atom details. Second, we modeled the first all-atom T=3, icosahedral, single stranded RNA virus, Pariacoto virus (PaV). The x-ray structure of PaV shows only 35% of the total RNA genome and 88% of the capsid. We modeled both missing portions of RNA and protein. The final model of the PaV demonstrated that the positively charged protein N- terminus was located deep inside the RNA. We propose that the positively charged N- terminal tails make contact with the RNA genome and neutralize the negative charges in RNA and subsequently collapse the RNA/protein complex into an icosahedral virus. Third, we simulated T=1 empty capsids using a coarse-grained model of three capsid proteins as a wedge-shaped triangular capsid unit. We varied the edge angle and the potentials of the capsid units to perform empty capsid assembly simulations. The final model and the potential are further improved for the whole virus assembly simulations. Finally, we performed stability and assembly simulations of the whole virus using coarse-grained models. We tested various strengths of RNA-protein tail and capsid protein-capsid protein attractions in our stability simulations and narrowed our search for optimal potentials for assembly. The assembly simulations were carried out with two different protocols: co-transcriptional and post-transcriptional. The co-transcriptional assembly protocol mimics the assembly occurring during the replication of the new RNA. Proteins bind the partly transcribed RNA in this protocol. The post-transcriptional assembly protocol assumes that the RNA is completely transcribed in the absence of proteins. Proteins later bind to the fully transcribed RNA. We found that both protocols can assemble viruses, when the RNA structure is compact enough to yield a successful virus particle. The post-transcriptional protocol depends more on the compactness of the RNA structure compared to the co-transcriptional assembly protocol. Viruses can exploit both assembly protocols based on the location of RNA replication and the compactness of the final structure of the RNA.PhDCommittee Chair: Stephen C. Harvey; Committee Member: Adegboyega Oyelere; Committee Member: Loren Williams; Committee Member: Rigoberto Hernandez; Committee Member: Roger Wartel

An In Vitro Characterization of Functional Interactions Between Purified Telomere Repeat Binding Factors 1 and 2 and Rad51 Recombinase

A growing body of literature suggests that the homologous recombination/repair (HR) pathway cooperates with components of the shelterin complex to promote both telomere maintenance and non-telomeric HR. This may be due to the ability of both HR and shelterin proteins to promote strand invasion, wherein a single-stranded DNA (ssDNA) substrate base pairs with a homologous double-stranded DNA (dsDNA) template displacing a loop of ssDNA (D-loop). Rad51 recombinase catalyzes D-loop formation during HR, and telomere repeat-binding factor 2 (TRF2) catalyzes the formation of a telomeric D-loop that stabilizes a looped structure in telomeric DNA (t-loop) that may facilitate telomere protection. We have characterized this functional interaction in vitro using a fluorescent D-loop assay measuring the incorporation of Cy3-labeled 90 nucleotide telomeric and non-telomeric substrates into telomeric and non-telomeric plasmid templates. We report that pre-incubation of a telomeric template with TRF2 inhibits the ability of Rad51 to promote telomeric D-loop formation when pre-incubated with a telomeric substrate. This suggests Rad51 does not facilitate t-loop formation, and suggests a mechanism whereby TRF2 can inhibit HR at telomeres. We also report a TRF2 mutant lacking the dsDNA binding domain promotes Rad51-mediated non-telomeric D-loop formation, possibly explaining how TRF2 promotes non-telomeric HR. Finally, we report telomere repeat binding factor 1 (TRF1) promotes Rad51-mediated telomeric D-loop formation, which may facilitate HR-mediated replication fork restart and explain why TRF1 is required for efficient telomere replication.Doctor of Philosoph

Spherically Confined Polymers: Monte Carlo Simulations with Expanded Ensemble Density-of-States Method

In this thesis, the Expanded Ensemble Density-of-States (EXEDOS) method - a combination of the Wang-Landau and Expanded Ensemble Monte Carlo algorithms is employed to investigate spatial conformations of a polymer chain under spherical confinement. The study focuses on flexible chains up to 600 monomers and semi-flexible chains with various stiffnesses up to 300 monomers in length. Spatial conformations of the polymer are studied, using a simple pearl-necklace chain model of varied diameter and stiffness, as well as the model of fused-sphere chain. To test the applicability of the EXEDOS method, the confinement free energy was calculated for ideal and non-ideal flexible chains inside spheres of sizes smaller than their unconfined size. For ideal chains, the power-law dependence of the free energy on a confining radius is in excellent agreement with previous theoretical predictions. For self-avoiding chains at intermediate concentrations, the dependence of free energy on concentration deviates from that predicted by the blob scaling theory, most likely due to the finite size effects. At high concentrations, a stronger dependence of free energy on concentration is observed, compared to that obtained at intermediate concentrations. The density profile of a self-avoiding flexible chain was also studied, showing that at sufficiently high concentrations, excluded volume interactions push the chain close to the confining surface, leading to an oscillation in monomer number density near the surface. In semi-flexible chains, bending energy experiences largest changes at low densities as the polymer folds to conform the confining sphere, and at high density its growth slows down as the chain starts forming ordered layer near the surface. We observe isotropic-nematic (I-N) transition for all considered polymer chains. The I-N transition of more flexible chains happens at higher densities than that of stiff chains. All chains form disordered to imperfect helicoildal structures, and at densities above the I-N transition, the structure with four +1/2 defects is observed in all considered chains. However, the polymer spatial arrangement is far from an ideal tetrahedral and tennis ball structure. The EXEDOS algorithm is further extended to investigate the effects of steric hindrance on the structure in a semi-flexible chain, spherically confined at various concentrations. Semi-flexible chains modeled as pearl-necklace chains with ratio of diameter to bond length d/a less than or equal to 0.5 did not develop ordered structures at any considered concentrations, while chains with d/a = 0.8 and d/a = 1, formed imperfect helicoildal structures. On the contrary, a semi-flexible fused-sphere chain with monomer overlap (d/a = 2) forms distinct helicoildal structures, when confined in- side a small sphere of the same size as the pearl-necklace chains with d/a = 0.8 and 1. The evolution of ordered parameters with concentration suggests that during the transition from disordered to ordered configuration, the fused-sphere chain with d/a = 2 and pearl-necklace chains with d/a = 1 and 0.8 may approach tetrahedral configuration before shifting to a helicoildal arrangement. Four +1/2 defects are observed in these chains confined at concentrations above the I-N transition, forming in places, where horizontal and vertical stands of polymer intersect. In the fused-sphere chain, the two +1/2 defects nearly merge in each pair to form a +1 defect at each pole

Sažimanje nukleinskih kiselina : fizikalni mehanizmi i biološka relevantnost

Packing of nucleic acids inside (naturally occurring) confined spaces presents an intriguing problem of compacting a long and highly charged polymer into a small space possibly crowded with other particles (proteins). For example, viruses have a large amount of genomic information that is encoded in nucleic acids packed in small spaces resulting in high densities of matter. The arising interactions are coupled to the confinement giving a more complex phase diagram than expected in bulk. In this work we study the problem of packing nucleic acids in confined spaces in the context of physical virology. First, we study compacted states of DNA including condensed DNA in cells and confined DNA in bacteriophage capsids. We apply polymer and liquid crystal theory along with mean field approximations for the bending energy to characterize the state of DNA. The resulting framework is used to explain in vivo ejection of DNA from a bacteriophage into a Gram-positive bacteria based only on thermodynamic considerations, without invoking any active cellular mechanisms. The packing mechanism for DNA with condensing proteins in adenoviruses is studied by comparing Langevin dynamics simulations of effective particle models, representing condensing proteins, with experimental data. The DNA is found to act as an effective medium for condensing core protein interactions. A backbone of DNA linking the condensing proteins is not needed to explain the experimental results. To further explain such systems, we construct a full model of packed polymer and condensing proteins inside spherical confinement using Langevin dynamics. Internal organization of condensing particles shows that they tend to cover themselves with the DNA polymer which provides an effective medium for interactions with other condensers, confirming the applicability of our effective model for core particle organization in adenoviruses. Crowding of the viral interior and confinement influences the conformation of the DNA and protein, facilitating more direct contacts between the DNA polymer and the condensing particles, and modifying the interactions between them. Our model is able to explain the general internal organization of adenovirus cores, and provide insight into packing of genetic material in similar systems.Rad ne sadrži sažetak na drugom jeziku