A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and the associated proteins (Cas) comprise a system of adaptive immunity against viruses and plasmids in prokaryotes. Cas1 is a CRISPR-associated protein that is common to all CRISPR-containing prokaryotes but its function remains obscure. Here we show that the purified Cas1 protein of Escherichia coli (YgbT) exhibits nuclease activity against single-stranded and branched DNAs including Holliday junctions, replication forks and 5'-flaps. The crystal structure of YgbT and site-directed mutagenesis have revealed the potential active site. Genome-wide screens show that YgbT physically and genetically interacts with key components of DNA repair systems, including recB, recC and ruvB. Consistent with these findings, the ygbT deletion strain showed increased sensitivity to DNA damage and impaired chromosomal segregation. Similar phenotypes were observed in strains with deletion of CRISPR clusters, suggesting that the function of YgbT in repair involves interaction with the CRISPRs. These results show that YgbT belongs to a novel, structurally distinct family of nucleases acting on branched DNAs and suggest that, in addition to antiviral immunity, at least some components of the CRISPR-Cas system have a function in DNA repair.     

The configuration of the sesquiterpenoid 4-hydroxy-myoporone (athanagrandione)

The conversion of eremoacetal to (?)-1-(furan-3-yl)-4-hydroxy-4,8-dimethylnonane-1,6-dione establishes the configuration of (?)-4-hydroxymyoporone (athanagrandione) as R.     

Structural consequences of metallothionein dimerization: solution structure of the isolated Cd4-alpha-domain and comparison with the holoprotein dimer

The NMR determination of the structure of Cd(7)-metallothionein was done previously using a relatively large protein concentration that favors dimer formation. The reactivity of the protein is also affected under this condition. To examine the influence of protein concentration on metallothionein conformation, the isolated Cd(4)-alpha-domain was prepared from rabbit metallothionein-2 (MT 2), and its three-dimensional structure was determined by heteronuclear, (1)H-(111)Cd, and homonuclear, (1)H-(1)H NMR, correlation experiments. The three-dimensional structure was refined using distance and angle constraints derived from these two-dimensional NMR data sets and a distance geometry/simulated annealing protocol. The backbone superposition of the alpha-domain from rabbit holoprotein Cd(7)-MT 2 and the isolated rabbit Cd(4)-alpha was measured at a RMSD of 2.0 A. Nevertheless, the conformations of the two Cd-thiolate clusters were distinctly different at two of the cadmium centers. In addition, solvent access to the sulfhydryl ligands of the isolated Cd(4)-alpha cluster was 130% larger due to this small change in cluster geometry. To probe whether these differences were an artifact of the structure calculation, the Cd(4)-alpha-domain structure in rabbit Cd(7)-MT 2 was redetermined, using the previously defined set of NOEs and the present calculation protocol. All calculations employed the same ionic radius for Cd(2+) and same cadmium-thiolate bond distance. The newly calculated structure matched the original with an RMSD of 1.24 A. It is hypothesized that differences in the two alpha-domain structures result from a perturbation of the holoprotein structure because of head-to-tail dimerization under the conditions of the NMR experiments.     

Hydroxylated analogues of the orally active broad spectrum antifungal, Sch 51048 (1), and the discovery of posaconazole [Sch 56592; 2 or (S,S)-5

As part of a detailed study, the syntheses, biological activities, and pharmacokinetic properties of hydroxylated analogues of the previously described broad spectrum antifungal agents, Sch 51048 (1), Sch 50001 (3), and Sch 50002 (4), are described. Based on an overall superior profile, one of the alcohols, Sch 56592 (2), was selected for clinical studies.     

Gradation in Nutrient Composition and Photosynthetic Pathways Across the Restinga Vegetation of Brazil

The restinga comprises coastal vegetation formations which dominate the Atlantic seaboard of Brazil. Exposed sand ridges and associated lagoon systems have poorly developed soils subject to pronounced water deficits. Distinct vegetation zones support a high diversity of life forms, and a comparative study has been undertaken to investigate interactions between degree of exposure, nutrient supply and photosynthetic pathway (C₃, or CAM) in selected species across the restinga. A number of species occurring throughout the restinga were chosen as representative species of different life forms, comprising C₃ pioneer shrubs (Eugenia rotundifolia and Erythroxylum ovalifolium), impounding (tank) terrestrial bromeliad (Neoregelia cruenta: CAM) and the atmospheric epiphyte (Tillandsia stricta: CAM). Comparisons of plant and soil nutrient composition, and airborne deposition were conducted for each zone. Soil nutrient content and organic matter were closely related, reaching a maximum in zone 4, the seaward face of the inner dune. Salt concentration in leaves was independent of atmospheric deposition for the terrestrial species, in contrast to the atmospheric epiphyte T. stricta. In the slack area, vegetation formed characteristic “islands” with the soil beneath enriched in nutrients, suggesting a complex interplay between plants and soil during the development of vegetation succession. Here, two additional trees were investigated, C₃ and CAM members of the Clusiaceae, respectively Clusia lanceolata and C. fluminensis. Stable isotope composition of nitrogen (δ¹⁵N) was generally more negative (depleted in ¹⁵N) in plants with low total nitrogen content. This was exemplified by the atmospheric bromeliad, T. stricta, with an N content of 2.91 g/kg and δ¹⁵N of ?12.3 per mil. Stable isotopes of carbon (δ¹³C) were used to identify the distribution of photosynthetic pathways, and while the majority of bromeliads and orchids were CAM, analysis of the soil organic matter suggested that C₃ plants made the major contribution in each zone of the restinga. Since δ¹³C of plant material also suggested that water supply was optimal in zone 4, we conclude that succession and high diversity in the restinga is dependent on exposure, edaphic factors, and perhaps a critical mass of vegetation required to stabilize nutrient relations of the system.