Replication fork collapse at a protein‐DNA roadblock leads to fork reversal,promoted by the RecQ helicase

Proteins that bind DNA are the cause of the majority of impediments to replication fork progression and can lead to subsequent collapse of the replication fork. Failure to deal with fork collapse efficiently leads to mutation or cell death. Several models have been proposed for how a cell processes a stalled or collapsed replication fork; eukaryotes and bacteria are not dissimilar in terms of the general pathways undertaken to deal with these events. This study shows that replication fork regression, the combination of replication fork reversal leading to formation of a Holliday Junction along with exonuclease digestion, is the preferred pathway for dealing with a collapsed fork in Escherichia coli. Direct endo‐nuclease activity at the replication fork was not observed. The protein that had the greatest effect on these fork processing events was the RecQ helicase, while RecG and RuvABC, which have previously been implicated in this process, were found to play a lesser role. Eukaryotic RecQ homologues, BLM and WRN, have also been implicated in processing events following replication fork collapse and may reflect a conserved mechanism. Finally, the SOS response was not induced by the protein‐DNA roadblock under these conditions, so did not affect fork processing.     

Diversity and distribution of hidden cultivable fungi associated with marine animals of Antarctica

In the present study, we surveyed the distribution and diversity of fungal assemblages associated with 10 species of marine animals from Antarctica. The collections yielded 83 taxa from 27 distinct genera, which were identified using molecular biology methods. The most abundant taxa were Cladosporium sp. 1, Debaryomyces hansenii, Glaciozyma martinii, Metschnikowia australis, Pseudogymnoascus destructans, Thelebolus cf. globosus, Pseudogymnoascus pannorum, Tolypocladium tundrense, Metschnikowia australis, and different Penicillium species. The diversity, richness, and dominance of fungal assemblages ranged among the host; however, in general, the fungal community, which was composed of endemic and cold-adapted cosmopolitan taxa distributed across the different sites of Antarctic Peninsula, displayed high diversity, richness, and dominance indices. Our results contribute to knowledge about fungal diversity in the marine environment across the Antarctic Peninsula and their phylogenetic relationships with species that occur in other cold, temperate, and tropical regions of the World. Additionally, despite their extreme habitats, marine Antarctic animals shelter cryptic and complex fungal assemblages represented by endemic and cosmopolitan cold-adapted taxa, which may represent interesting models to study different symbiotic associations between fungi and their animal hosts in the extreme conditions of Antarctica.     

Altered -3 substrate specificity of Escherichia coli signal peptidase 1 mutants as revealed by screening a combinatorial peptide library

Signal peptidase functions to cleave signal peptides from preproteins at the cell membrane. It has a substrate specificity for small uncharged residues at -1 (P1) and aliphatic residues at the -3 (P3) position. Previously, we have reported that certain alterations of the Ile-144 and Ile-86 residues in Escherichia coli signal peptidase I (SPase) can change the specificity such that signal peptidase is able to cleave pro-OmpA nuclease A in vitro after phenylalanine or asparagine residues at the -1 position (Karla, A., Lively, M. O., Paetzel, M. and Dalbey, R. (2005) J. Biol. Chem. 280, 6731-6741). In this study, screening of a fluorescence resonance energy transfer-based peptide library revealed that the I144A, I144C, and I144C/I86T SPase mutants have a more relaxed substrate specificity at the -3 position, in comparison to the wild-type SPase. The double mutant tolerated arginine, glutamine, and tyrosine residues at the -3 position of the substrate. The altered specificity of the I144C/I86T mutant was confirmed by in vivo processing of pre-beta-lactamase containing non-canonical arginine and glutamine residues at the -3 position. This work establishes Ile-144 and Ile-86 as key P3 substrate specificity determinants for signal peptidase I and demonstrates the power of the fluorescence resonance energy transfer-based peptide library approach in defining the substrate specificity of proteases.     

Acquisition and voxelwise analysis of multi-subject diffusion data with tract-based spatial statistics

There is much interest in using magnetic resonance diffusion imaging to provide information on anatomical connectivity in the brain by measuring the diffusion of water in white matter tracts. Among the measures, the most commonly derived from diffusion data is fractional anisotropy (FA), which quantifies local tract directionality and integrity. Many multi-subject imaging studies are using FA images to localize brain changes related to development, degeneration and disease. In a recent paper, we presented a new approach, tract-based spatial statistics (TBSS), which aims to solve crucial issues of cross-subject data alignment, allowing localized cross-subject statistical analysis. This works by transforming the data from the centers of the tracts that are consistent across a study's subjects into a common space. In this protocol, we describe the MRI data acquisition and analysis protocols required for TBSS studies of localized change in brain connectivity across multiple subjects.     

RPM-1 Uses Both Ubiquitin Ligase and Phosphatase-Based Mechanisms to Regulate DLK-1 during Neuronal Development

The Pam/Highwire/RPM-1 (PHR) proteins are key regulators of neuronal development that function in axon extension and guidance, termination of axon outgrowth, and synapse formation. Outside of development, the PHR proteins also regulate axon regeneration and Wallerian degeneration. The PHR proteins function in part by acting as ubiquitin ligases that degrade the Dual Leucine zipper-bearing Kinase (DLK). Here, we show that the Caenorhabditis elegans PHR protein, Regulator of Presynaptic Morphology 1 (RPM-1), also utilizes a phosphatase-based mechanism to regulate DLK-1. Using mass spectrometry, we identified Protein Phosphatase Magnesium/Manganese dependent 2 (PPM-2) as a novel RPM-1 binding protein. Genetic, transgenic, and biochemical studies indicated that PPM-2 functions coordinately with the ubiquitin ligase activity of RPM-1 and the F-box protein FSN-1 to negatively regulate DLK-1. PPM-2 acts on S874 of DLK-1, a residue implicated in regulation of DLK-1 binding to a short, inhibitory isoform of DLK-1 (DLK-1S). Our study demonstrates that PHR proteins function through both phosphatase and ubiquitin ligase mechanisms to inhibit DLK. Thus, PHR proteins are potentially more accurate and sensitive regulators of DLK than originally thought. Our results also highlight an important and expanding role for the PP2C phosphatase family in neuronal development.     

Addition of proteinase K during the culture alter the physiology of Bacillus thuringiensis culture and the cry1Ac,nprX, nprA,and spo0A gene transcription

Antonie van Leeuwenhoek - Bacillus thuringiensis is the major bioinsecticide worldwide produced due to the Cry protein activity. Several studies have been done to improve the...     

The double-stranded RNA-binding protein,Staufen1, is an IRES-transacting factor regulating HIV-1 cap-independent translation initiation

Translation initiation of the viral genomic mRNA (vRNA) of human immunodeficiency virus-type 1 (HIV-1) can be mediated by a cap- or an internal ribosome entry site (IRES)-dependent mechanism. A previous report shows that Staufen1, a cellular double-stranded (ds) RNA-binding protein (RBP), binds to the 5’untranslated region (5′UTR) of the HIV-1 vRNA and promotes its cap-dependent translation. In this study, we now evaluate the role of Staufen1 as an HIV-1 IRES-transacting factor (ITAF). We first confirm that Staufen1 associates with both the HIV-1 vRNA and the Gag protein during HIV-1 replication. We found that in HIV-1-expressing cells, siRNA-mediated depletion of Staufen1 reduces HIV-1 vRNA translation. Using dual-luciferase bicistronic mRNAs, we show that the siRNA-mediated depletion and cDNA-mediated overexpression of Staufen1 acutely regulates HIV-1 IRES activity. Furthermore, we show that Staufen1-vRNA interaction is required for the enhancement of HIV-1 IRES activity. Interestingly, we find that only Staufen1 harboring an intact dsRNA-binding domain 3 (dsRBD3) rescues HIV-1 IRES activity in Staufen1 CRISPR-Cas9 gene edited cells. Finally, we show that the expression of Staufen1-dsRBD3 alone enhances HIV-1 IRES activity. This study provides evidence of a novel role for Staufen1 as an ITAF promoting HIV-1 vRNA IRES activity.