Structural and functional characterization of the Redβ recombinase from bacteriophage λ

The Red system of bacteriophage λ is responsible for the genetic rearrangements that contribute to its rapid evolution and has been successfully harnessed as a research tool for genome manipulation. The key recombination component is Redβ, a ring-shaped protein that facilitates annealing of complementary DNA strands. Redβ shares functional similarities with the human Rad52 single-stranded DNA (ssDNA) annealing protein although their evolutionary relatedness is not well established. Alignment of Rad52 and Redβ sequences shows an overall low level of homology, with 15% identity in the N-terminal core domains as well as important similarities with the Rad52 homolog Sak from phage ul36. Key conserved residues were chosen for mutagenesis and their impact on oligomer formation, ssDNA binding and annealing was probed. Two conserved regions were identified as sites important for binding ssDNA; a surface basic cluster and an intersubunit hydrophobic patch, consistent with findings for Rad52. Surprisingly, mutation of Redβ residues in the basic cluster that in Rad52 are involved in ssDNA binding disrupted both oligomer formation and ssDNA binding. Mutations in the equivalent of the intersubunit hydrophobic patch in Rad52 did not affect Redβ oligomerization but did impair DNA binding and annealing. We also identified a single amino acid substitution which had little effect on oligomerization and DNA binding but which inhibited DNA annealing, indicating that these two functions of Redβ can be separated. Taken together, the results provide fresh insights into the structural basis for Redβ function and the important role of quaternary structure

Hemostatic efficacy of pathogen-inactivated vs untreated platelets: a randomized controlled trial

Stemcel biology/Regenerative medicine (incl. bloodtransfusion

Crystal structure of hemoglobin protease, a heme binding autotransporter protein from pathogenic Escherichia coli

The acquisition of iron is essential for the survival of pathogenic bacteria, which have consequently evolved a wide variety of uptake systems to extract iron and heme from host proteins such as hemoglobin. Hemoglobin protease (Hbp) was discovered as a factor involved in the symbiosis of pathogenic Escherichia coli and Bacteroides fragilis, which cause intra-abdominal abscesses. Released from E. coli, this serine protease autotransporter degrades hemoglobin and delivers heme to both bacterial species. The crystal structure of the complete passenger domain of Hbp (110 kDa) is presented, which is the first structure from this class of serine proteases and the largest parallel β-helical structure yet solved. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc

Phage Orf family recombinases: conservation of activities and involvement of the central channel in DNA binding

Genetic and biochemical evidence suggests that λ Orf is a recombination mediator, promoting nucleation of either bacterial RecA or phage Redβ recombinases onto single-stranded DNA (ssDNA) bound by SSB protein. We have identified a diverse family of Orf proteins that includes representatives implicated in DNA base flipping and those fused to an HNH endonuclease domain. To confirm a functional relationship with the Orf family, a distantly-related homolog, YbcN, from Escherichia coli cryptic prophage DLP12 was purified and characterized. As with its λ relative, YbcN showed a preference for binding ssDNA over duplex. Neither Orf nor YbcN displayed a significant preference for duplex DNA containing mismatches or 1-3 nucleotide bulges. YbcN also bound E. coli SSB, although unlike Orf, it failed to associate with an SSB mutant lacking the flexible C-terminal tail involved in coordinating heterologous protein-protein interactions. Residues conserved in the Orf family that flank the central cavity in the λ Orf crystal structure were targeted for mutagenesis to help determine the mode of DNA binding. Several of these mutant proteins showed significant defects in DNA binding consistent with the central aperture being important for substrate recognition. The widespread conservation of Orf-like proteins highlights the importance of targeting SSB coated ssDNA during lambdoid phage recombination

A study protocol for a randomised controlled trial evaluating clinical effects of platelet transfusion products: the Pathogen Reduction Evaluation and Predictive Analytical Rating Score (PREPAReS) trial

Thrombosis and Hemostasi

The productivity and response to inorganic fertilizers of species- rich wetland hay meadows on the Somerset Moors: nitrogen response under hay cutting and aftermath grazing

The productivity and response to fertilizer nitrogen (N) was measured in herb-rich wetland hay meadows within a Site of Special Scientific Interest (SSSI) in Somerset, UK. Output from hay cut after 1 July each year and from beef production at aftermath grazing was measured over a total of 4 years. Total utilized metabolizable energy (UME) output averaged from 40.6 GJ ha-1 year-1 without fertilizers to 61.7 GJ ha-1 at 200 kg N ha-1 (N-200), the highest rate used, with about two-thirds of this output from hay. N response was markedly curvilinear above about 50 kg N ha-1, but data from a concurrent experiment suggested that the comparatively low replacement rates of P and K applied were limiting at higher N rates. When hay cutting was delayed until early August in a wet year, yield response to N was lost because fertilized swards had passed a peak in yield several weeks before harvest. Compared with other published data, the results suggest that output and response to N is not constrained by the diversity of the flora or the damp conditions. The data will help to estimate the financial implications for farmers of restricted or zero fertilizer use within SSSIs and the wider Environmentally Sensitive Areas