Binding Mechanism of Metal⋅NTP Substrates and Stringent-Response Alarmones to Bacterial DnaG-Type Primases

SummaryPrimases are DNA-dependent RNA polymerases found in all cellular organisms. In bacteria, primer synthesis is carried out by DnaG, an essential enzyme that serves as a key component of DNA replication initiation, progression, and restart. How DnaG associates with nucleotide substrates and how certain naturally prevalent nucleotide analogs impair DnaG function are unknown. We have examined one of the earliest stages in primer synthesis and its control by solving crystal structures of the S. aureus DnaG catalytic core bound to metal ion cofactors and either individual nucleoside triphosphates or the nucleotidyl alarmones, pppGpp and ppGpp. These structures, together with both biochemical analyses and comparative studies of enzymes that use the same catalytic fold as DnaG, pinpoint the predominant nucleotide-binding site of DnaG and explain how the induction of the stringent response in bacteria interferes with primer synthesis

Access of energetic particles to Titan's exobase: a study of Cassini's T9 flyby

We study how the local electromagnetic disturbances introduced by Titan affect the ionization rates of the atmosphere. For this, we model the precipitation of energetic particles, specifically hydrogen and oxygen ions with energies between 1 keV and 1 MeV, into Titan's exobase for the specific magnetospheric configuration of the T9 flyby. For the study, a particle tracing software package is used which consists of an integration of the single particle Lorentz force equation using a 4th order Runge-Kutta numerical method. For the electromagnetic disturbances, the output of the A.I.K.E.F. hybrid code (kinetic ions, fluid electrons) is used, allowing the possibility of analyzing the disturbances and asymmetries in the access of energetic particles originated by their large gyroradii. By combining these methods, 2D maps showing the access of each set of particles were produced. We show that the access of different particles is largely dominated by their gyroradii, with the complexity of the maps increasing with decreasing gyroradius, due to the larger effect that local disturbances introduced by the presence of the moon have in the trajectory of the particles with lower energies. We also show that for particles with gyroradii much larger than the moon's radius, simpler descriptions of the electromagnetic environment can reproduce similar results to those obtained when using the full hybrid simulation description, with simple north-south fields being sufficient to reproduce the hybrid code results for O+ ions with energies larger than 10 keV but not enough to reproduce those for H+H+ ions at any of the energies covered in the present study. Finally, by combining the maps created with upstream plasma flow measurements by the MIMI/CHEMS instrument, we are able to estimate normalized fluxes arriving at different selected positions of the moon's exobase. We then use these fluxes to calculate energy deposition and non-dissociative N2 ionization rates for precipitating O+O+ and H+H+ ions and find differences in the ion production rates of up to almost 80% at the selected positions. All these results combined show that the electromagnetic field disturbances present in the vicinity of Titan significantly affect the contribution of energetic ions to local ionization profiles

Mutations in Zebrafish lrp2 Result in Adult-Onset Ocular Pathogenesis That Models Myopia and Other Risk Factors for Glaucoma

The glaucomas comprise a genetically complex group of retinal neuropathies that typically occur late in life and are characterized by progressive pathology of the optic nerve head and degeneration of retinal ganglion cells. In addition to age and family history, other significant risk factors for glaucoma include elevated intraocular pressure (IOP) and myopia. The complexity of glaucoma has made it difficult to model in animals, but also challenging to identify responsible genes. We have used zebrafish to identify a genetically complex, recessive mutant that shows risk factors for glaucoma including adult onset severe myopia, elevated IOP, and progressive retinal ganglion cell pathology. Positional cloning and analysis of a non-complementing allele indicated that non-sense mutations in low density lipoprotein receptor-related protein 2 (lrp2) underlie the mutant phenotype. Lrp2, previously named Megalin, functions as an endocytic receptor for a wide-variety of bioactive molecules including Sonic hedgehog, Bone morphogenic protein 4, retinol-binding protein, vitamin D-binding protein, and apolipoprotein E, among others. Detailed phenotype analyses indicated that as lrp2 mutant fish age, many individuals—but not all—develop high IOP and severe myopia with obviously enlarged eye globes. This results in retinal stretch and prolonged stress to retinal ganglion cells, which ultimately show signs of pathogenesis. Our studies implicate altered Lrp2-mediated homeostasis as important for myopia and other risk factors for glaucoma in humans and establish a new genetic model for further study of phenotypes associated with this disease