Tetrameric UvrD Helicase Is Located at the E. Coli Replisome due to Frequent Replication Blocks.

DNA replication in all organisms must overcome nucleoprotein blocks to complete genome duplication. Accessory replicative helicases in Escherichia coli, Rep and UvrD, help remove these blocks and aid the re-initiation of replication. Mechanistic details of Rep function have emerged from recent live cell studies; however, the division of UvrD functions between its activities in DNA repair and role as an accessory helicase remain unclear in live cells.

Escherichia coli Chromosome Copy Number Measurement Using Flow Cytometry Analysis.

Flow cytometry is a high-throughput technique that analyzes individual particles as they pass through a laser beam. These particles can be individual cells and by detecting cell-scattered light their number and relative size can be measured as they pass through the beam. Labeling of molecules, usually via a fluorescent reporter, allows the amount of these molecules per cell to be quantified.

Single-molecule live cell imaging of Rep reveals the dynamic interplay between an accessory replicative helicase and the replisome.

DNA replication must cope with nucleoprotein barriers that impair efficient replisome translocation. Biochemical and genetic studies indicate accessory helicases play essential roles in replication in the presence of nucleoprotein barriers, but how they operate inside the cell is unclear. With high-speed single-molecule microscopy we observed genomically-encoded fluorescent constructs of the accessory helicase Rep and core replisome protein DnaQ in live Escherichia coli cells.

Direct removal of RNA polymerase barriers to replication by accessory replicative helicases.

Bacterial genome duplication and transcription require simultaneous access to the same DNA template. Conflicts between the replisome and transcription machinery can lead to interruption of DNA replication and loss of genome stability. Pausing, stalling and backtracking of transcribing RNA polymerases add to this problem and present barriers to replisomes.

DNA replication roadblocks caused by Cascade interference complexes are alleviated by RecG DNA repair helicase.

Cascade complexes underpin E. coli CRISPR-Cas immunity systems by stimulating 'adaptation' reactions that update immunity and by initiating 'interference' reactions that destroy invader DNA. Recognition of invader DNA in Cascade catalysed R-loops provokes DNA capture and its subsequent integration into CRISPR loci by Cas1 and Cas2.

The 2B subdomain of Rep helicase links translocation along DNA with protein displacement.

Helicases catalyse DNA and RNA strand separation. Proteins bound to the nucleic acid must also be displaced in order to unwind DNA. This is exemplified by accessory helicases that clear protein barriers from DNA ahead of advancing replication forks.

Insights into the initiation of chromosome II replication of the pressure-loving deep-sea bacterium Photobacterium profundum SS9.

How DNA metabolism is adapted to survival of organisms such as the bacterium Photobacterium profundum SS9 at high pressure is unknown. Previously, a high pressure-sensitive P. profundum SS9 transposon mutant (FL31) was identified, with an insertion in a putative rctB gene.

Critical examination of evidence for the nutritional status of children in Papua New Guinea - a systematic review.

Background And Objectives: Undernutrition remains a significant cause of childhood illness, poor growth, development, and death in Papua New Guinea (PNG). Studies on child nutritional outcomes in PNG vary by design, measurement protocols and quality. We conducted a systematic review to assess the evidence for the prevalence of child undernutrition across different study populations, geographical locations and time periods.

Conformational Heterogeneity in a Fully Complementary DNA Three-Way Junction with a GC-Rich Branchpoint.

DNA three-way junctions (3WJs) are branched structures that serve as important biological intermediates and as components in DNA nanostructures. We recently derived the global structure of a fully complementary 3WJ and found that it contained unpaired bases at the branchpoint, which is consistent with previous observations of branch flexibility and branchpoint reactivity. By combining high-resolution single-molecule Förster resonance energy transfer, molecular modeling, time-resolved ensemble fluorescence spectroscopy, and the first F nuclear magnetic resonance observations of fully complementary 3WJs, we now show that the 3WJ structure can adopt multiple distinct conformations depending upon the sequence at the branchpoint.

Laser microfabrication of a microheater chip for cell culture outside a cell incubator.

Microfluidic chips have demonstrated their significant application potentials in microbiological processing and chemical reactions, with the goal of developing monolithic and compact chip-sized multifunctional systems. Heat generation and thermal control are critical in some of the biochemical processes. The paper presents a laser direct-write technique for rapid prototyping and manufacturing of microheater chips and its applicability for lab-on-a-chip cell culture outside a cell incubator.

The structure and function of an RNA polymerase interaction domain in the PcrA/UvrD helicase.

The PcrA/UvrD helicase functions in multiple pathways that promote bacterial genome stability including the suppression of conflicts between replication and transcription and facilitating the repair of transcribed DNA. The reported ability of PcrA/UvrD to bind and backtrack RNA polymerase (1,2) might be relevant to these functions, but the structural basis for this activity is poorly understood. In this work, we define a minimal RNA polymerase interaction domain in PcrA, and report its crystal structure at 1.

Inhibiting translation elongation can aid genome duplication in Escherichia coli.

Conflicts between replication and transcription challenge chromosome duplication. Escherichia coli replisome movement along transcribed DNA is promoted by Rep and UvrD accessory helicases with Δrep ΔuvrD cells being inviable under rapid growth conditions. We have discovered that mutations in a tRNA gene, aspT, in an aminoacyl tRNA synthetase, AspRS, and in a translation factor needed for efficient proline-proline bond formation, EF-P, suppress Δrep ΔuvrD lethality.

Performance of point-of-care CD4 testing technologies in resource-constrained settings: a systematic review and meta-analysis.

Background: Point-of-care (POC) CD4 testing increases patient accessibility to assessment of antiretroviral therapy eligibility. This review evaluates field performance in low and middle-income countries (LMICs) of currently available POC CD4 technologies.

Methods: Eight electronic databases were searched for field studies published between January 2005 and January 2015 of six POC CD4 platforms: PointCare NOW™, Alere Pima™ CD4, Daktari™ CD4 Counter, CyFlow® CD4 miniPOC, BD FACSPresto™, and MyT4™ CD4.

Acceptability and feasibility of point-of-care CD4 testing on HIV continuum of care in low and middle income countries: a systematic review.

Background: CD4 testing is, and will remain an important part of HIV treatment and care in low and middle income countries (LMICs). We report the findings of a systematic review assessing acceptability and feasibility of POC CD4 testing in field settings.

Methods: Electronic databases were searched for studies published in English between 2005 and 2015 that describe POC CD4 platforms.

The Balance between Recombination Enzymes and Accessory Replicative Helicases in Facilitating Genome Duplication.

Accessory replicative helicases aid the primary replicative helicase in duplicating protein-bound DNA, especially transcribed DNA. Recombination enzymes also aid genome duplication by facilitating the repair of DNA lesions via strand exchange and also processing of blocked fork DNA to generate structures onto which the replisome can be reloaded. There is significant interplay between accessory helicases and recombination enzymes in both bacteria and lower eukaryotes but how these replication repair systems interact to ensure efficient genome duplication remains unclear.

Escherichia coli Chromosome Copy Number Measurement Using Flow Cytometry Analysis.

Flow cytometry is a high-throughput technique that analyzes individual particles as they pass through a laser beam. These particles can be individual cells and by detecting cell-scattered light their number and relative size can be measured as they pass through the beam. Labeling of molecules, usually via a fluorescent reporter, allows the amount of these molecules per cell to be quantified.

Single-Molecule Observation of DNA Replication Repair Pathways in E. coli.

The method of action of many antibiotics is to interfere with DNA replication-quinolones trap DNA gyrase and topoisomerase proteins onto DNA while metronidazole causes single- and double-stranded breaks in DNA. To understand how bacteria respond to these drugs, it is important to understand the repair processes utilised when DNA replication is blocked. We have used tandem lac operators inserted into the chromosome bound by fluorescently labelled lac repressors as a model protein block to replication in E.

Use of streptavidin bound to biotinylated DNA structures as model substrates for analysis of nucleoprotein complex disruption by helicases.

Helicases are a subfamily of translocases that couple the directional translocation along a nucleic acid lattice to the separation of nucleic acid duplexes using the energy derived from nucleoside triphosphate hydrolysis. These enzymes perform essential functions in all aspects of nucleic acid metabolism by unwinding and remodelling DNA or RNA in DNA replication, repair, recombination, transcription and translation. Most classical biochemical studies assay the ability of these enzymes to separate naked nucleic acids.

Overexpression of the Replicative Helicase in Escherichia coli Inhibits Replication Initiation and Replication Fork Reloading.

Replicative helicases play central roles in chromosome duplication and their assembly onto DNA is regulated via initiators and helicase loader proteins. The Escherichia coli replicative helicase DnaB and the helicase loader DnaC form a DnaB6-DnaC6 complex that is required for loading DnaB onto single-stranded DNA. Overexpression of dnaC inhibits replication by promoting continual rebinding of DnaC to DnaB and consequent prevention of helicase translocation.

Recombination and replication.

The links between recombination and replication have been appreciated for decades and it is now generally accepted that these two fundamental aspects of DNA metabolism are inseparable: Homologous recombination is essential for completion of DNA replication and vice versa. This review focuses on the roles that recombination enzymes play in underpinning genome duplication, aiding replication fork movement in the face of the many replisome barriers that challenge genome stability. These links have many conserved features across all domains of life, reflecting the conserved nature of the substrate for these reactions, DNA.

Accessory replicative helicases and the replication of protein-bound DNA.

Complete, accurate duplication of the genetic material is a prerequisite for successful cell division. Achieving this accuracy is challenging since there are many barriers to replication forks that may cause failure to complete genome duplication or result in possibly catastrophic corruption of the genetic code. One of the most important types of replicative barriers are proteins bound to the template DNA, especially transcription complexes.

The conserved C-terminus of the PcrA/UvrD helicase interacts directly with RNA polymerase.

UvrD-like helicases play diverse roles in DNA replication, repair and recombination pathways. An emerging body of evidence suggests that their different cellular functions are directed by interactions with partner proteins that target unwinding activity to appropriate substrates. Recent studies in E.

Protein-DNA complexes are the primary sources of replication fork pausing in Escherichia coli.

Replication fork pausing drives genome instability, because any loss of paused replisome activity creates a requirement for reloading of the replication machinery, a potentially mutagenic process. Despite this importance, the relative contributions to fork pausing of different replicative barriers remain unknown. We show here that Deinococcus radiodurans RecD2 helicase inactivates Escherichia coli replisomes that are paused but still functional in vitro, preventing continued fork movement upon barrier removal or bypass, but does not inactivate elongating forks.

Helicases at the replication fork.

Helicases are fundamental components of all replication complexes since unwinding of the double-stranded template to generate single-stranded DNA is essential to direct DNA synthesis by polymerases. However, helicases are also required in many other steps of DNA replication. Replicative helicases not only unwind the template DNA but also play key roles in regulating priming of DNA synthesis and coordination of leading and lagging strand DNA polymerases.

The conflict between DNA replication and transcription.

There is mounting evidence that there are frequent conflicts between complexes that replicate DNA and those that transcribe the same template, and that these conflicts lead to blockage of replication and genome instability. Such problems are perhaps best understood in bacteria, but it is becoming apparent that replicative barriers associated with transcription are a universal feature of life. This review summarizes what is currently known about how collisions between replisomes and transcription complexes are minimized and the mechanisms that help to resolve such collisions when they do occur.