Protein-Assisted Room-Temperature Assembly of Rigid, Immobile Holliday Junctions and Hierarchical DNA Nanostructures.

Immobile Holliday junctions represent not only the most fundamental building block of structural DNA nanotechnology but are also of tremendous importance for the in vitro investigation of genetic recombination and epigenetics. Here, we present a detailed study on the room-temperature assembly of immobile Holliday junctions with the help of the single-strand annealing protein Redß. Individual DNA single strands are initially coated with protein monomers and subsequently hybridized to form a rigid blunt-ended four-arm junction. We investigate the efficiency of this approach for different DNA/protein ratios, as well as for different DNA sequence lengths. Furthermore, we also evaluate the potential of Redß to anneal sticky-end modified Holliday junctions into hierarchical assemblies. We demonstrate the Redß-mediated annealing of Holliday junction dimers, multimers, and extended networks several microns in size. While these hybrid DNA-protein nanostructures may find applications in the crystallization of DNA-protein complexes, our work shows the great potential of Redß to aid in the synthesis of functional DNA nanostructures under mild reaction conditions.

Proteomic navigation using proximity-labeling.

The identification of bona fide protein-protein interactions and the mapping of proteomes was greatly enhanced by protein tagging for generic affinity purification methods and analysis by mass spectrometry (AP-MS). The high quality of AP-MS data permitted the development of proteomic navigation by sequential tagging of identified interactions. However AP-MS is laborious and limited to relatively high affinity protein-protein interactions. Proximity labeling, first with the biotin ligase BirA, termed BioID, and then with ascorbate peroxidase, termed APEX, permits a greater reach into the proteome than AP-MS enabling both the identification of a wider field and weaker protein-protein interactions. This additional reach comes with the need for stringent controls. Proximity labeling also permits experiments in living cells allowing spatiotemporal investigations of the proteome. Here we discuss proximity labeling with accompanying methodological descriptions for E. coli and mammalian cells.

Correction: Contribution of the Cpx envelope stress system to metabolism and virulence regulation in Salmonella enterica serovar Typhimurium.

[This corrects the article DOI: 10.1371/journal.pone.0211584.].

Contribution of the Cpx envelope stress system to metabolism and virulence regulation in Salmonella enterica serovar Typhimurium.

The Cpx-envelope stress system regulates the expression of virulence factors in many Gram-negative pathogens. In Salmonella enterica serovar Typhimurium deletion of the sensor kinase CpxA but not of the response regulator CpxR results in the down regulation of the key regulator for invasion, HilA encoded by the Salmonella pathogenicity island 1 (SPI-1). Here, we provide evidence that cpxA deletion interferes with dephosphorylation of CpxR resulting in increased levels of active CpxR and consequently in misregulation of target genes. 14 potential operons were identified to be under direct control of CpxR. These include the virulence determinants ecotin, the omptin PgtE, and the SPI-2 regulator SsrB. The Tat-system and the PocR regulator that together promote anaerobic respiration of tetrathionate on 1,2-propanediol are also under direct CpxR control. Notably, 1,2-propanediol represses hilA expression. Thus, our work demonstrates for the first time the involvement of the Cpx system in a complex network mediating metabolism and virulence function.

Regular Nanoscale Protein Patterns via Directed Adsorption through Self-Assembled DNA Origami Masks.

DNA origami has become a widely used method for synthesizing well-defined nanostructures with promising applications in various areas of nanotechnology, biophysics, and medicine. Recently, the possibility to transfer the shape of single DNA origami nanostructures into different materials via molecular lithography approaches has received growing interest due to the great structural control provided by the DNA origami technique. Here, we use ordered monolayers of DNA origami nanostructures with internal cavities on mica surfaces as molecular lithography masks for the fabrication of regular protein patterns over large surface areas. Exposure of the masked sample surface to negatively charged proteins results in the directed adsorption of the proteins onto the exposed surface areas in the holes of the mask. By controlling the buffer and adsorption conditions, the protein coverage of the exposed areas can be varied from single proteins to densely packed monolayers. To demonstrate the versatility of this approach, regular nanopatterns of four different proteins are fabricated: the single-strand annealing proteins Redß and Sak, the iron-storage protein ferritin, and the blood protein bovine serum albumin (BSA). We furthermore demonstrate the desorption of the DNA origami mask after directed protein adsorption, which may enable the fabrication of hierarchical patterns composed of different protein species. Because selectivity in adsorption is achieved by electrostatic interactions between the proteins and the exposed surface areas, this approach may enable also the large-scale patterning of other charged molecular species or even nanoparticles.

DNA annealing by Redß is insufficient for homologous recombination and the additional requirements involve intra- and inter-molecular interactions.

Single strand annealing proteins (SSAPs) like Redß initiate homologous recombination by annealing complementary DNA strands. We show that C-terminally truncated Redß, whilst still able to promote annealing and nucleoprotein filament formation, is unable to mediate homologous recombination. Mutations of the C-terminal domain were evaluated using both single- and double stranded (ss and ds) substrates in recombination assays. Mutations of critical amino acids affected either dsDNA recombination or both ssDNA and dsDNA recombination indicating two separable functions, one of which is critical for dsDNA recombination and the second for recombination per se. As evaluated by co-immunoprecipitation experiments, the dsDNA recombination function relates to the Redα-Redß protein-protein interaction, which requires not only contacts in the C-terminal domain but also a region near the N-terminus. Because the nucleoprotein filament formed with C-terminally truncated Redß has altered properties, the second C-terminal function could be due to an interaction required for functional filaments. Alternatively the second C-terminal function could indicate a requirement for a Redß-host factor interaction. These data further advance the model for Red recombination and the proposition that Redß and RAD52 SSAPs share ancestral and mechanistic roots.

A Single-Strand Annealing Protein Clamps DNA to Detect and Secure Homology.

Repair of DNA breaks by single-strand annealing (SSA) is a major mechanism for the maintenance of genomic integrity. SSA is promoted by proteins (single-strand-annealing proteins [SSAPs]), such as eukaryotic RAD52 and λ phage Redß. These proteins use a short single-stranded region to find sequence identity and initiate homologous recombination. However, it is unclear how SSAPs detect homology and catalyze annealing. Using single-molecule experiments, we provide evidence that homology is recognized by Redß monomers that weakly hold single DNA strands together. Once annealing begins, dimerization of Redß clamps the double-stranded region and nucleates nucleoprotein filament growth. In this manner, DNA clamping ensures and secures a successful detection for DNA sequence homology. The clamp is characterized by a structural change of Redß and a remarkable stability against force up to 200 pN. Our findings not only present a detailed explanation for SSAP action but also identify the DNA clamp as a very stable, noncovalent, DNA-protein interaction.