Dynamic transitions of initiator binding coordinate the replication of the two chromosomes in Vibrio cholerae.

The replication of the two chromosomes in the pathogenic bacterium Vibrio cholerae is coordinated by the binding of initiator protein RctB to a checkpoint sequence, crtS. Replication of crtS on the primary chromosome (Chr1) triggers replication of the secondary chromosome (Chr2), but the details are poorly understood. Here, we analyze RctB binding patterns in the V.

Homologous Recombination and DNA Intermediates Analyzed by Electron Microscopy.

Homologous recombination (HR) is a high-fidelity DNA repair pathway that uses a homologous DNA sequence as a template. Recombinase proteins are the central HR players in the three kingdoms of life. RecA/RadA/Rad51 assemble on ssDNA, generated after the processing of double-strand breaks or stalled replication forks into an active and dynamic presynaptic helical nucleofilament.

In vivo reduction of RAD51-mediated homologous recombination triggers aging but impairs oncogenesis.

Homologous recombination (HR) is a prominent DNA repair pathway maintaining genome integrity. Mutations in many HR genes lead to cancer predisposition. Paradoxically, the implication of the pivotal HR factor RAD51 on cancer development remains puzzling.

Amyloid-like Hfq interaction with single-stranded DNA: involvement in recombination and replication in .

Interactions between proteins and single-stranded DNA (ssDNA) are crucial for many fundamental biological processes, including DNA replication and genetic recombination. Thus, understanding detailed mechanisms of these interactions is necessary to uncover regulatory rules occurring in all living cells. The RNA-binding Hfq is a pleiotropic bacterial regulator that mediates many aspects of nucleic acid metabolism.

Assembly mechanism and cryoEM structure of RecA recombination nucleofilaments from Streptococcus pneumoniae.

RecA-mediated homologous recombination (HR) is a key mechanism for genome maintenance and plasticity in bacteria. It proceeds through RecA assembly into a dynamic filament on ssDNA, the presynaptic filament, which mediates DNA homology search and ordered DNA strand exchange. Here, we combined structural, single molecule and biochemical approaches to characterize the ATP-dependent assembly mechanism of the presynaptic filament of RecA from Streptococcus pneumoniae (SpRecA), in comparison to the Escherichia coli RecA (EcRecA) paradigm.

Structural Insights of the DciA Helicase Loader in Its Relationship with DNA.

DciA is the ancestral bacterial replicative helicase loader, punctually replaced during evolution by the DnaC/I loaders of phage origin. DnaC helps the helicase to load onto DNA by cracking open the hexameric ring, but the mechanism of loading by DciA remains unknown. We demonstrate by electron microscopy, nuclear magnetic resonance (NMR) spectroscopy, and biochemistry experiments that DciA, which folds into a KH-like domain, interacts with not only single-stranded but also double-stranded DNA, in an atypical mode.

Changes in the architecture and abundance of replication intermediates delineate the chronology of DNA damage tolerance pathways at UV-stalled replication forks in human cells.

DNA lesions in S phase threaten genome stability. The DNA damage tolerance (DDT) pathways overcome these obstacles and allow completion of DNA synthesis by the use of specialised translesion (TLS) DNA polymerases or through recombination-related processes. However, how these mechanisms coordinate with each other and with bulk replication remains elusive.

RAD51 protects against nonconservative DNA double-strand break repair through a nonenzymatic function.

Selection of the appropriate DNA double-strand break (DSB) repair pathway is decisive for genetic stability. It is proposed to act according to two steps: 1-canonical nonhomologous end-joining (C-NHEJ) versus resection that generates single-stranded DNA (ssDNA) stretches; 2-on ssDNA, gene conversion (GC) versus nonconservative single-strand annealing (SSA) or alternative end-joining (A-EJ). Here, we addressed the mechanisms by which RAD51 regulates this second step, preventing nonconservative repair in human cells.

Study of the DnaB:DciA interplay reveals insights into the primary mode of loading of the bacterial replicative helicase.

Replicative helicases are essential proteins that unwind DNA in front of replication forks. Their loading depends on accessory proteins and in bacteria, DnaC and DnaI are well characterized loaders. However, most bacteria do not express either of these two proteins.

Modular Imaging Scaffold for Single-Particle Electron Microscopy.

Technological breakthroughs in electron microscopy (EM) have made it possible to solve structures of biological macromolecular complexes and to raise novel challenges, specifically related to sample preparation and heterogeneous macromolecular assemblies such as DNA-protein, protein-protein, and membrane protein assemblies. Here, we built a V-shaped DNA origami as a scaffolding molecular system to template proteins at user-defined positions in space. This template positions macromolecular assemblies of various sizes, juxtaposes combinations of biomolecules into complex arrangements, isolates biomolecules in their active state, and stabilizes membrane proteins in solution.

The Role of Musculoskeletal Disorders in Chronic Disease: A Narrative Review.

Chronic diseases and musculoskeletal conditions are responsible for a significant portion of the global disease burden and are frequently comorbid, such as with low back pain in patients who also have chronic organ disease. Low back pain is the leading cause of long-term disability and is the most common reason adults seek adjunctive treatment, including osteopathic manipulative treatment (OMT). OMT has been shown to be effective in relieving low back pain and improving back-specific functioning.

Method combining BAC film and positive staining for the characterization of DNA intermediates by dark-field electron microscopy.

DNA intermediate structures are formed in all major pathways of DNA metabolism. Transmission electron microscopy (TEM) is a tool of choice to study their choreography and has led to major advances in the understanding of these mechanisms, particularly those of homologous recombination (HR) and replication. In this article, we describe specific TEM procedures dedicated to the structural characterization of DNA intermediates formed during these processes.

Verotoxin-1-Induced ER Stress Triggers Apoptotic or Survival Pathways in Burkitt Lymphoma Cells.

Shiga toxins (Stxs) expressed by the enterohaemorrhagic and enteric pathogens are protein synthesis inhibitors. Stxs have been shown to induce apoptosis via the activation of extrinsic and intrinsic pathways in many cell types (epithelial, endothelial, and B cells) but the link between the protein synthesis inhibition and caspase activation is still unclear. Endoplasmic reticulum (ER) stress induced by the inhibition of protein synthesis may be this missing link.

Mechanism of stimulation of DNA binding of the transcription factors by human apurinic/apyrimidinic endonuclease 1, APE1.

Aerobic respiration generates reactive oxygen species (ROS), which can damage nucleic acids, proteins and lipids. A number of transcription factors (TFs) contain redox-sensitive cysteine residues at their DNA-binding sites, hence ROS-induced thiol oxidation strongly inhibits their recognition of the cognate DNA sequences. Major human apurinic/apyrimidinic (AP) endonuclease 1 (APE1/APEX1/HAP-1), referred also as a redox factor 1 (Ref-1), stimulates the DNA binding activities of the oxidized TFs such as AP-1 and NF-κB.

In vitro role of Rad54 in Rad51-ssDNA filament-dependent homology search and synaptic complexes formation.

Homologous recombination (HR) uses a homologous template to accurately repair DNA double-strand breaks and stalled replication forks to maintain genome stability. During homology search, Rad51 nucleoprotein filaments probe and interact with dsDNA, forming the synaptic complex that is stabilized on a homologous sequence. Strand intertwining leads to the formation of a displacement-loop (D-loop).

XLF and APLF bind Ku80 at two remote sites to ensure DNA repair by non-homologous end joining.

The Ku70-Ku80 (Ku) heterodimer binds rapidly and tightly to the ends of DNA double-strand breaks and recruits factors of the non-homologous end-joining (NHEJ) repair pathway through molecular interactions that remain unclear. We have determined crystal structures of the Ku-binding motifs (KBM) of the NHEJ proteins APLF (A-KBM) and XLF (X-KBM) bound to a Ku-DNA complex. The two KBM motifs bind remote sites of the Ku80 α/β domain.

Rad52-Rad51 association is essential to protect Rad51 filaments against Srs2, but facultative for filament formation.

Homology search and strand exchange mediated by Rad51 nucleoprotein filaments are key steps of the homologous recombination process. In budding yeast, Rad52 is the main mediator of Rad51 filament formation, thereby playing an essential role. The current model assumes that Rad51 filament formation requires the interaction between Rad52 and Rad51.

Recombinases and Related Proteins in the Context of Homologous Recombination Analyzed by Molecular Microscopy.

Transmission electron microscopy (TEM) and atomic force microscopy (AFM) are powerful tools to study the behavior of various actors in homologous recombination including molecular motors such as recombinases and helicases/translocases. Here we present specific approaches developed in terms of sample preparation and imaging methods to contribute to the understanding of homologous recombination process and its regulation focusing on the interplay between recombinases and other related proteins such as mediators or antirecombinase actors.Homologous recombination (HR) is a high-fidelity DNA repair pathway since it uses a homologous DNA as template.

The role of the N-terminal domain of human apurinic/apyrimidinic endonuclease 1, APE1, in DNA glycosylase stimulation.

Unlabelled: The base excision repair (BER) pathway consists of sequential action of DNA glycosylase and apurinic/apyrimidinic (AP) endonuclease necessary to remove a damaged base and generate a single-strand break in duplex DNA. Human multifunctional AP endonuclease 1 (APE1, a.k.

Sepiolite as a New Nanocarrier for DNA Transfer into Mammalian Cells: Proof of Concept, Issues and Perspectives.

Sepiolite is a nanofibrous natural silicate that can be used as a nanocarrier for DNA transfer thanks to its strong interaction with DNA molecules and its ability to be naturally internalized into mammalian cells through both non-endocytic and endocytic pathways. Sepiolite, due to its ability to bind various biomolecules, could be a good candidate for use as a nanocarrier for the simultaneous vectorization of diverse biological molecules. In this paper, we review our recent work, issued from a starting collaboration with Prof.