A comparative analysis of Dmc1 and Rad51 nucleoprotein filaments

filament

Rad51 Paralogs Remodel Pre-synaptic Rad51 Filaments to Stimulate Homologous Recombination

SummaryRepair of DNA double strand breaks by homologous recombination (HR) is initiated by Rad51 filament nucleation on single-stranded DNA (ssDNA), which catalyzes strand exchange with homologous duplex DNA. BRCA2 and the Rad51 paralogs are tumor suppressors and critical mediators of Rad51. To gain insight into Rad51 paralog function, we investigated a heterodimeric Rad51 paralog complex, RFS-1/RIP-1, and uncovered the molecular basis by which Rad51 paralogs promote HR. Unlike BRCA2, which nucleates RAD-51-ssDNA filaments, RFS-1/RIP-1 binds and remodels pre-synaptic filaments to a stabilized, “open,” and flexible conformation, in which the ssDNA is more accessible to nuclease digestion and RAD-51 dissociation rate is reduced. Walker box mutations in RFS-1, which abolish filament remodeling, fail to stimulate RAD-51 strand exchange activity, demonstrating that remodeling is essential for RFS-1/RIP-1 function. We propose that Rad51 paralogs stimulate HR by remodeling the Rad51 filament, priming it for strand exchange with the template duplex

The Bipolar Filaments Formed by Herpes Simplex Virus Type 1 SSB/Recombination Protein (ICP8) Suggest a Mechanism for DNA Annealing

Herpes simplex virus type 1 encodes a multifunctional protein, ICP8, which serves both as a single strand binding protein and recombinase, catalyzing reactions involved in replication and recombination of the viral genome. In the presence of divalent ions and at low temperature, previous electron microscopic (EM) studies showed that ICP8 will form long left-handed helical filaments. Here EM image reconstruction reveals that the filaments are bipolar, with an asymmetric unit containing two subunits of ICP8 that constitute a symmetrical dimer. This organization of the filament has been confirmed using Scanning Transmission Electron Microscopy. The pitch of the filaments is ~ 250 Å, with ~ 6.2 dimers per turn. Docking of a crystal structure of ICP8 into the reconstructed filament shows that the C-terminal domain of ICP8, attached to the body of the subunit by a flexible linker containing ~ 10 residues, is packed into a pocket in the body of a neighboring subunit in the crystal in a similar manner as in the filament. However, the interactions between the large N-terminal domains are quite different in the filament from that observed in the crystal. A previously proposed model for ICP8 binding single-stranded DNA, based upon the crystal structure, leads to a model for a continuous strand of ssDNA near the filament axis. The bipolar nature of the ICP8 filaments means that a second strand of ssDNA would be running through this filament in the opposite orientation, and this provides a potential mechanism for how ICP8 anneals complementary single stranded DNA into double-stranded DNA, where each strand runs in opposite directions

Cryo-Electron Tomography of Marburg Virus Particles and Their Morphogenesis within Infected Cells

Ultrastructural analysis of a filovirus assembling within infected eukaryotic cells reveals differences in structure and assembly mechanisms between related RNA viruses

The elementary events underlying force generation in neuronal lamellipodia

We have used optical tweezers to identify the elementary events underlying force generation in neuronal lamellipodia. When an optically trapped bead seals on the lamellipodium membrane, Brownian fluctuations decrease revealing the underlying elementary events. The distribution of bead velocities has long tails with frequent large positive and negative values associated to forward and backward jumps occurring in 0.1–0.2 ms with varying amplitudes up to 20 nm. Jump frequency and amplitude are reduced when actin turnover is slowed down by the addition of 25 nM Jasplakinolide. When myosin II is inhibited by the addition of 20 μM Blebbistatin, jump frequency is reduced but to a lesser extent than by Jasplainolide. These jumps constitute the elementary events underlying force generation

In vitro assembly of Ebola virus nucleocapsid-like complex expressed in E. coli

Ebola virus (EBOV) harbors an RNA genome encapsidated by nucleoprotein (NP) along with other viral proteins to form a nucleocapsid complex. Previous Cryo-eletron tomography and biochemical studies have shown the helical structure of EBOV nucleocapsid at nanometer resolution and the first 450 amino-acid of NP (NPΔ451–739) alone is capable of forming a helical nucleocapsid-like complex (NLC). However, the structural basis for NP-NP interaction and the dynamic procedure of the nucleocapsid assembly is yet poorly understood. In this work, we, by using an E. coli expression system, captured a series of images of NPΔ451–739 conformers at different stages of NLC assembly by negative-stain electron microscopy, which allowed us to picture the dynamic procedure of EBOV nucleocapsid assembly. Along with further biochemical studies, we showed the assembly of NLC is salt-sensitive, and also established an indispensible role of RNA in this process. We propose the diverse modes of NLC elongation might be the key determinants shaping the plasticity of EBOV virions. Our findings provide a new model for characterizing the self-oligomerization of viral nucleoproteins and studying the dynamic assembly process of viral nucleocapsid in vitro

The Organisation of Ebola Virus Reveals a Capacity for Extensive, Modular Polyploidy

BACKGROUND: Filoviruses, including Ebola virus, are unusual in being filamentous animal viruses. Structural data on the arrangement, stoichiometry and organisation of the component molecules of filoviruses has until now been lacking, partially due to the need to work under level 4 biological containment. The present study provides unique insights into the structure of this deadly pathogen. METHODOLOGY AND PRINCIPAL FINDINGS: We have investigated the structure of Ebola virus using a combination of cryo-electron microscopy, cryo-electron tomography, sub-tomogram averaging, and single particle image processing. Here we report the three-dimensional structure and architecture of Ebola virus and establish that multiple copies of the RNA genome can be packaged to produce polyploid virus particles, through an extreme degree of length polymorphism. We show that the helical Ebola virus inner nucleocapsid containing RNA and nucleoprotein is stabilized by an outer layer of VP24-VP35 bridges. Elucidation of the structure of the membrane-associated glycoprotein in its native state indicates that the putative receptor-binding site is occluded within the molecule, while a major neutralizing epitope is exposed on its surface proximal to the viral envelope. The matrix protein VP40 forms a regular lattice within the envelope, although its contacts with the nucleocapsid are irregular. CONCLUSIONS: The results of this study demonstrate a modular organization in Ebola virus that accommodates a well-ordered, symmetrical nucleocapsid within a flexible, tubular membrane envelope

Eisosome proteins assemble into a membrane scaffold

Membrane organization by eisosomes is mediated by self-assembly of its main components into a membrane-bound protein scaffold with lipid-binding specificity

Heteropolymeric Triplex-Based Genomic Assay® to Detect Pathogens or Single-Nucleotide Polymorphisms in Human Genomic Samples

Human genomic samples are complex and are considered difficult to assay directly without denaturation or PCR amplification. We report the use of a base-specific heteropolymeric triplex, formed by native duplex genomic target and an oligonucleotide third strand probe, to assay for low copy pathogen genomes present in a sample also containing human genomic duplex DNA, or to assay human genomic duplex DNA for Single Nucleotide Polymorphisms (SNP), without PCR amplification. Wild-type and mutant probes are used to identify triplexes containing FVL G1691A, MTHFR C677T and CFTR mutations. The specific triplex structure forms rapidly at room temperature in solution and may be detected without a separation step. YOYO-1, a fluorescent bis-intercalator, promotes and signals the formation of the specific triplex. Genomic duplexes may be assayed homogeneously with single base pair resolution. The specific triple-stranded structures of the assay may approximate homologous recombination intermediates, which various models suggest may form in either the major or minor groove of the duplex. The bases of the stable duplex target are rendered specifically reactive to the bases of the probe because of the activity of intercalated YOYO-1, which is known to decondense duplex locally 1.3 fold. This may approximate the local decondensation effected by recombination proteins such as RecA in vivo. Our assay, while involving triplex formation, is sui generis, as it is not homopurine sequence-dependent, as are “canonical triplexes”. Rather, the base pair-specific heteropolymeric triplex of the assay is conformation-dependent. The highly sensitive diagnostic assay we present allows for the direct detection of base sequence in genomic duplex samples, including those containing human genomic duplex DNA, thereby bypassing the inherent problems and cost associated with conventional PCR based diagnostic assays

Unique Properties of Eukaryote-Type Actin and Profilin Horizontally Transferred to Cyanobacteria

A eukaryote-type actin and its binding protein profilin encoded on a genomic island in the cyanobacterium Microcystis aeruginosa PCC 7806 co-localize to form a hollow, spherical enclosure occupying a considerable intracellular space as shown by in vivo fluorescence microscopy. Biochemical and biophysical characterization reveals key differences between these proteins and their eukaryotic homologs. Small-angle X-ray scattering shows that the actin assembles into elongated, filamentous polymers which can be visualized microscopically with fluorescent phalloidin. Whereas rabbit actin forms thin cylindrical filaments about 100 µm in length, cyanobacterial actin polymers resemble a ribbon, arrest polymerization at 5-10 µm and tend to form irregular multi-strand assemblies. While eukaryotic profilin is a specific actin monomer binding protein, cyanobacterial profilin shows the unprecedented property of decorating actin filaments. Electron micrographs show that cyanobacterial profilin stimulates actin filament bundling and stabilizes their lateral alignment into heteropolymeric sheets from which the observed hollow enclosure may be formed. We hypothesize that adaptation to the confined space of a bacterial cell devoid of binding proteins usually regulating actin polymerization in eukaryotes has driven the co-evolution of cyanobacterial actin and profilin, giving rise to an intracellular entity