Convergent evolution in two bacterial replicative helicase loaders.

Dedicated loader proteins play essential roles in bacterial DNA replication by opening ring-shaped DnaB-family helicases and chaperoning single-stranded (ss)DNA into a central motor chamber as a prelude to DNA unwinding. Although unrelated in sequence, the Escherichia coli DnaC and bacteriophage λ P loaders feature a similar overall architecture: a globular domain linked to an extended lasso/grappling hook element, located at their N and C termini, respectively. Both loaders remodel a closed DnaB ring into nearly identical right-handed open conformations. The sole element shared by the loaders is a single alpha helix, which binds to the same site on the helicase. Physical features of the loaders establish that DnaC and λ P evolved independently to converge, through molecular mimicry, on a common helicase-opening mechanism.

Ponatinib is a potential therapeutic approach for malignant pleural mesothelioma.

PURPOSE: Malignant pleural mesothelioma (MPM) is a rare and deadly malignancy. Current MPM therapies remain inadequate, and outcomes are often disappointing. New meaningful therapeutic approaches are urgently needed. Accumulating evidence indicates that the cAbl pathway promotes various tumor-stimulating processes in MPM. In this study, we sought to determine ponatinib's potential utility, a clinically approved and potent cAbl inhibitor, in MPM treatment. MATERIAL AND METHODS: Four MPM lines (MSTO211H, H28, H2452, H2052) were treated with ponatinib in vitro, and their growth was assessed. Scratch wound assay was used to investigate the ponatinib effect on cell migration. The expression levels of pAbl and its downstream effectors pCrkL, pAKT, and pSTAT5 were characterized. The in vivo ponatinib effect was evaluated in human MPM cells derived tumor model. RESULTS: In all four MPM lines, significant expression levels of phosphorylated cAbl/Arg and pCrkl were observed. Differentially but strongly, ponatinib inhibited the in vitro cell growth and migration of all four MPM line. Western blot analysis showed that the activation of Abl signaling was blocked in the ponatinib-treated MMP lines. In keeping, the cellular levels of pAbl and its downstream effector pCrkL, pAKT, and pSTAT5 were markedly decrease following ponatinib treatment. Moreover, ponatinib treatment amplified the levels of γH2AX in cells denoting increased double-strand DNA breaks levels. Notably, ponatinib treatment reduced in vivo tumor growth and reduced pCrkl and pSTAT5 levels in tumor samples. CONCLUSION: Ponatinib may offer a new therapeutic strategy for MPM patients based on cAbl signaling pathway inhibition.

Mechanisms of opening and closing of the bacterial replicative helicase.

Assembly of bacterial ring-shaped hexameric replicative helicases on single-stranded (ss) DNA requires specialized loading factors. However, mechanisms implemented by these factors during opening and closing of the helicase, which enable and restrict access to an internal chamber, are not known. Here, we investigate these mechanisms in the Escherichia coli DnaB helicase•bacteriophage λ helicase loader (λP) complex. We show that five copies of λP bind at DnaB subunit interfaces and reconfigure the helicase into an open spiral conformation that is intermediate to previously observed closed ring and closed spiral forms; reconfiguration also produces openings large enough to admit ssDNA into the inner chamber. The helicase is also observed in a restrained inactive configuration that poises it to close on activating signal, and transition to the translocation state. Our findings provide insights into helicase opening, delivery to the origin and ssDNA entry, and closing in preparation for translocation.

Routine single particle CryoEM sample and grid characterization by tomography.

Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are not adsorbed to the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.

DNA-Based Genetic Markers for Rapid Cycling Brassica Rapa (Fast Plants Type) Designed for the Teaching Laboratory.

We have developed DNA-based genetic markers for rapid cycling Brassica rapa (RCBr), also known as Fast Plants. Although markers for B. rapa already exist, ours were intentionally designed for use in a teaching laboratory environment. The qualities we selected for were robust amplification in PCR, polymorphism in RCBr strains, and alleles that can be easily resolved in simple agarose slab gels. We have developed two single nucleotide polymorphism (SNP) based markers and 14 variable number tandem repeat (VNTR)-type markers spread over four chromosomes. The DNA sequences of these markers represent variation in a wide range of genomic features. Among the VNTR-type markers, there are examples of variation in a non-genic region, variation within an intron, and variation in the coding sequence of a gene. Among the SNP-based markers there are examples of polymorphism in intronic DNA and synonymous substitution in a coding sequence. Thus these markers can serve laboratory exercises in both transmission genetics and molecular biology.