Displacement of a DNA binding protein by Dda helicase

Bacteriophage T4 Dda helicase has recently been shown to be active as a monomer for unwinding of short duplex oligonucleotides and for displacing streptavidin from 3′-biotinylated oligonucleotides. However, its activity for streptavidin displacement and DNA unwinding has been shown to increase as the number of Dda molecules bound to the substrate molecule increases. A substrate was designed to address the ability of Dda to displace DNA binding proteins. A DNA binding site for the Escherichia coli trp repressor was introduced into an oligonucleotide substrate for Dda helicase containing single-stranded overhang. Here we show that a Dda monomer is insufficient to displace the E.coli trp repressor from dsDNA under single turnover conditions, although the substrate is unwound and the repressor displaced when the single-stranded overhang is long enough to accommodate two Dda molecules. The quantity of product formed increases when the substrate is able to accommodate more than two Dda molecules. These results indicate that multiple Dda molecules act to displace DNA binding proteins in a manner that correlates with the DNA unwinding activity and streptavidin displacement activity. We suggest a cooperative inchworm model to describe the activities of Dda helicase

Investigation of translocation, DNA unwinding, and protein displacement by NS3h, the helicase domain from the Hepatitis C virus helicase

Helicases are motor proteins that are involved in DNA and RNA metabolism, replication, recombination, transcription and repair. The motors are powered by ATP binding and hydrolysis. Hepatitis C virus encodes a helicase called non-structural protein (NS3). NS3 possesses protease and helicase activities on its N-terminal and C-terminal domains respectively. The helicase domain of NS3 protein is referred as NS3h. In vitro, NS3h catalyzes RNA and DNA unwinding in a 3’ to -5’ direction. The directionality for unwinding is thought to arise in part from the enzyme's ability to translocate along DNA, but translocation has not been shown explicitly. We examined the DNA translocase activity of NS3h by using single-stranded oligonucleotide substrates containing a fluorescent probe on the 5’ end. NS3h can bind to the ssDNA and in the presence of ATP, move towards the 5’-end. When the enzyme encounters the fluorescent probe, a fluorescence change is observed that allows translocation to be characterized. Under conditions that favor binding of one NS3h per DNA substrate (100 nM NS3h, 200 nM oligonucleotide) we find that NS3h translocates on ssDNA at a rate of 46 ± 5 nt s−1 and that it can move for 230 ± 60 nt before dissociating from the DNA. The translocase activity of some helicases is responsible for displacing proteins that are bound to DNA. We studied protein displacement by using a ssDNA oligonucleotide covalently linked to biotin on the 5’-end. Upon addition of streptavidin, a ‘protein-block’ was placed in the pathway of the helicase. Interestingly, NS3h was unable to displace streptavidin from the end of the oligonucleotide, despite its ability to translocate along the DNA. The DNA unwinding activity of NS3h was examined using a 22 bp duplex DNA substrate under conditions that were identical to those used to study translocation. NS3h exhibited little or no DNA unwinding under single cycle conditions, supporting the conclusion that NS3h is a relatively poor helicase in its monomeric form, as has been reported. In summary, NS3h translocates on ssDNA as a monomer, but the translocase activity does not correspond to comparable DNA unwinding activity or protein-displacement activity under identical conditions

Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points

Role and Regulation of Pif1 Family Helicases at the Replication Fork

Pif1 helicases are a multifunctional family of DNA helicases that are important for many aspects of genomic stability in the nucleus and mitochondria. Pif1 helicases are conserved from bacteria to humans. Pif1 helicases play multiple roles at the replication fork, including promoting replication through many barriers such as G-quadruplex DNA, the rDNA replication fork barrier, tRNA genes, and R-loops. Pif1 helicases also regulate telomerase and promote replication termination, Okazaki fragment maturation, and break-induced replication. This review highlights many of the roles and regulations of Pif1 at the replication fork that promote cellular health and viability

proteiNorm – A User-Friendly Tool for Normalization and Analysis of TMT and Label-Free Protein Quantification

The technological advances in mass spectrometry allow us to collect more comprehensive data with higher quality and increasing speed. With the rapidly increasing amount of data generated, the need for streamlining analyses becomes more apparent. Proteomics data is known to be often affected by systemic bias from unknown sources, and failing to adequately normalize the data can lead to erroneous conclusions. To allow researchers to easily evaluate and compare different normalization methods via a user-friendly interface, we have developed “proteiNorm”. The current implementation of proteiNorm accommodates preliminary filters on peptide and sample levels followed by an evaluation of several popular normalization methods and visualization of the missing value. The user then selects an adequate normalization method and one of the several imputation methods used for the subsequent comparison of different differential expression methods and estimation of statistical power. The application of proteiNorm and interpretation of its results are demonstrated on two tandem mass tag multiplex (TMT6plex and TMT10plex) and one label-free spike-in mass spectrometry example data set. The three data sets reveal how the normalization methods perform differently on different experimental designs and the need for evaluation of normalization methods for each mass spectrometry experiment. With proteiNorm, we provide a user-friendly tool to identify an adequate normalization method and to select an appropriate method for differential expression analysis

Yeast Pif1 Accelerates Annealing of Complementary DNA Strands

Pif1 is a helicase involved in the maintenance of nuclear and mitochondrial genomes in eukaryotes. Here we report a new activity of <i>Saccharomyces cerevisiae</i> Pif1, annealing of complementary DNA strands. We identified preferred substrates for annealing as those that generate a duplex product with a single-stranded overhang relative to a blunt end duplex. Importantly, we show that Pif1 can anneal DNA in the presence of ATP and Mg²⁺. Pif1-mediated annealing also occurs in the presence of single-stranded DNA binding proteins. Additionally, we show that partial duplex substrates with 3′-single-stranded overhangs such as those generated during double-strand break repair can be annealed by Pif1