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. By integrating super-resolved single-molecule fluorescence microscopy with biochemical analysis, we find that UvrD self-associates into tetrameric assemblies and, unlike Rep, is not recruited to a specific replisome protein despite being found at approximately 80% of replication forks. Instead, its colocation with forks is likely due to the very high frequency of replication blocks composed of DNA-bound proteins, including RNA polymerase and factors involved in repairing DNA damage. Deleting rep and DNA repair factor genes mutS and uvrA, and inhibiting transcription through RNA polymerase mutation and antibiotic inhibition, indicates that the level of UvrD at the fork is dependent on UvrD's function. Our findings show that UvrD is recruited to sites of nucleoprotein blocks via different mechanisms to Rep and plays a multi-faceted role in ensuring successful DNA replication.

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. DNA content can be estimated using this approach and here we describe how flow cytometry can be used to assess the DNA content of Escherichia coli cells.

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. We demonstrate that Rep colocalizes with 70% of replication forks, with a hexameric stoichiometry, indicating maximal occupancy of the single DnaB hexamer. Rep associates dynamically with the replisome with an average dwell time of 6.5 ms dependent on ATP hydrolysis, indicating rapid binding then translocation away from the fork. We also imaged PriC replication restart factor and observe Rep-replisome association is also dependent on PriC. Our findings suggest two Rep-replisome populations in vivo: one continually associating with DnaB then translocating away to aid nucleoprotein barrier removal ahead of the fork, another assisting PriC-dependent reloading of DnaB if replisome progression fails. These findings reveal how a single helicase at the replisome provides two independent ways of underpinning replication of protein-bound DNA, a problem all organisms face as they replicate their genomes.

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. Accessory helicases promote fork movement through nucleoprotein barriers and exist in viruses, bacteria and eukaryotes. Here, we show that stalled Escherichia coli transcription elongation complexes block reconstituted replisomes. This physiologically relevant block can be alleviated by the accessory helicase Rep or UvrD, resulting in the formation of full-length replication products. Accessory helicase action during replication-transcription collisions therefore promotes continued replication without leaving gaps in the DNA. In contrast, DinG does not promote replisome movement through stalled transcription complexes in vitro. However, our data demonstrate that DinG operates indirectly in vivo to reduce conflicts between replication and transcription. These results suggest that Rep and UvrD helicases operate on DNA at the replication fork whereas DinG helicase acts via a different mechanism.

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. DNA capture processes are unclear but may involve RecG helicase, which stimulates adaptation during its role responding to genome instability. We show that Cascade is a potential source of genome instability because it blocks DNA replication and that RecG helicase alleviates this by dissociating Cascade. This highlights how integrating in vitro CRISPR-Cas interference and adaptation reactions with DNA replication and repair reactions will help to determine precise mechanisms underpinning prokaryotic adaptive immunity.

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. How helicases catalyse DNA unwinding is increasingly well understood but how protein displacement is achieved is unclear. Escherichia coli Rep accessory replicative helicase lacking one of its four subdomains, 2B, has been shown to be hyperactivated for DNA unwinding in vitro but we show here that RepΔ2B is, in contrast, deficient in displacing proteins from DNA. This defect correlates with an inability to promote replication of protein-bound DNA in vitro and lack of accessory helicase function in vivo. Defective protein displacement is manifested on double-stranded and single-stranded DNA. Thus binding and distortion of duplex DNA by the 2B subdomain ahead of the helicase is not the missing function responsible for this deficiency. These data demonstrate that protein displacement from DNA is not simply achieved by helicase translocation alone. They also imply that helicases may have evolved different specific features to optimise DNA unwinding and protein displacement, both of which are now recognised as key functions in all aspects of nucleic acid metabolism.

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. The Vibrio cholerae RctB protein is essential for replication initiation at the origin of chromosome II, oriCII. Using a plasmid-based system in E. coli we have identified the replication origin of chromosome II from P. profundum SS9 and have shown that the putative rctB gene, disrupted in FL31, is essential for oriCII function. Moreover, we found that a region corresponding to the V. cholerae oriCII incompatibility region (incII) exerts an inhibitory effect on P. profundum oriCII. The truncated rctB gene in FL31 confers insensitivity to incII inhibition, indicating that the C-terminus of RctB is important for the negative regulation of replication. The RctB proteins of V. cholerae and P. profundum are partially interchangeable, but full functionality is achieved only with the cognate origin. Our findings provide the first characterization of the replication origin of chromosome II in a deep-sea bacterium.

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. METHODS AND STUDY DESIGN: Six electronic databases and additional grey literature were searched for articles describing the nutritional status by wasting, stunting and underweight, of PNG children under five years of age, published between 1990 and April 2015. Prevalence data using different scales of measurement and reference populations were standardized according to WHO protocols. RESULTS: The search yielded 566 articles, of which, 31 studies met the inclusion criteria. The prevalence of child undernutrition varied from 1% to 76% for wasting (median 11%), 5% to 92% for stunting (median 51%), and 14% to 59% for underweight (median 32%). Wide variations exist according to the index used for measurement, the population characteristics and the geographical region in which they live. Prevalence estimates increase significantly when data using different scales of measurement and population references are standardized to the WHO protocols. CONCLUSIONS: Child undernutrition in PNG is regionally variable due to a complex interplay of poverty, disease, food-security, cultural, environmental and sociopolitical issues requiring a complex mix of solutions by governments, health systems and local communities. Area- specific surveys using multiple measures are necessary to inform local solutions for this important problem.

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 19F 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. A 3WJ with a GC-rich branchpoint adopts an open conformation with unpaired bases at the branch and at least one additional conformation with an increased number of base interactions at the branchpoint. This structural diversity has implications for branch interactions and processing in vivo and for technological applications.

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 aim of the microheater is to take the role of conventional incubators for cell culture for facilitating microscopic observation and/or other online monitoring activities during cell culture and provides portability of cell culture operation. Microheaters (5mm×5mm) have been successfully fabricated on soda-lime glass substrates covered with aluminium layer of thickness 120nm. Experimental results show that the microheaters exhibit good performance in temperature rise and decay characteristics, with localized heating at targeted spatial domains. These microheaters were suitable for a maximum long-term operation temperature of 120°C and validated for operation at 37°C for 48h. Results demonstrated that the microheaters are suitable for the culture of immortalised cell lines. The growth and viability of SW480 colon adenocarcinoma cells cultured the developed microheater chip were comparable to the results obtained in a conventional 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 1.5 Å resolution. The domain adopts a Tudor-like fold that is similar to other RNA polymerase interaction domains, including that of the prototype transcription-repair coupling factor Mfd. Removal or mutation of the interaction domain reduces the ability of PcrA/UvrD to interact with and to remodel RNA polymerase complexes in vitro. The implications of this work for our understanding of the role of PcrA/UvrD at the interface of DNA replication, transcription and repair are discussed.

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. Thus replication-transcription conflicts can be alleviated by the partial sacrifice of a mechanism that reduces replicative barriers, namely translating ribosomes that reduce RNA polymerase backtracking. Suppression depends on RelA-directed synthesis of (p)ppGpp, a signalling molecule that reduces replication-transcription conflicts, with RelA activation requiring ribosomal pausing. Levels of (p)ppGpp in these suppressors also correlate inversely with the need for Rho activity, an RNA translocase that can bind to emerging transcripts and displace transcription complexes. These data illustrate the fine balance between different mechanisms in facilitating gene expression and genome duplication and demonstrate that accessory helicases are a major determinant of this balance. This balance is also critical for other aspects of bacterial survival: the mutations identified here increase persistence indicating that similar mutations could arise in naturally occurring bacterial populations facing antibiotic challenge.

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. Due to limited data availability, meta-analysis was conducted only for diagnostic performance of Pima at a threshold of 350 cells/µl, applying a bivariate multi-level random-effects modelling approach. A covariate extended model was also explored to test for difference in diagnostic performance between capillary and venous blood. RESULTS: Twenty seven studies were included. Published field study results were found for three of the six POC CD4 tests, 24 of which used Pima. For Pima, test failure rates varied from 2 to 23 % across study settings. Pooled sensitivity and specificity were 0.92 (95 % CI = 0.88-0.95) and 0.87 (95 % CI = 0.85-0.88) respectively. Diagnostic performance by blood sample type (venous vs. capillary) revealed non-significant differences in sensitivity (0.94 vs 0.89) and specificity (0.86 vs 0.87), respectively in the extended model (Wald χ2(2) = 4.77, p = 0.09). CONCLUSIONS: POC CD4 testing can provides reliable results for making treatment decision under field conditions in low-resource settings. The Pima test shows a good diagnostic performance at CD4 cut-off of 350 cells/µl. More data are required to evaluate performance of POC CD4 testing using venous versus capillary blood in LMICs which might otherwise influence clinical practice.

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. Here, we demonstrate that the DNA content defects of Escherichia coli cells lacking the strand exchange protein RecA are driven primarily by conflicts between replication and transcription, as is the case in cells lacking the accessory helicase Rep. However, in contrast to Rep, neither RecA nor RecBCD, the helicase/exonuclease that loads RecA onto dsDNA ends, is important for maintaining rapid chromosome duplication. Furthermore, RecA and RecBCD together can sustain viability in the absence of accessory replicative helicases but only when transcriptional barriers to replication are suppressed by an RNA polymerase mutation. Our data indicate that the minimisation of replisome pausing by accessory helicases has a more significant impact on successful completion of chromosome duplication than recombination-directed fork repair.

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. Studies conducted in LMICs and under field conditions outside a laboratory environment were eligible. Qualitative and descriptive data analysis was used to present the findings. RESULTS: Twelve studies were included, 11 of which were conducted in sub-Saharan countries and used one POC CD4 test (The Alere Pima CD4). Patients reported positively regarding the implementation of POC CD4 testing at primary health care and community level with ≥90 % of patients accepting the test across various study settings. Health service providers expressed preference toward POC CD4 testing as it is easy-to-use, efficient and satisfied patients' needs to a greater extent as compared to conventional methods. However, operational challenges including preference toward venous blood rather than finger-prick sampling, frequent device failures and operator errors, quality of training for test operators and supervisors, and increased staff workload were also identified. CONCLUSIONS: POC CD4 testing seems acceptable and feasible in LIMCs under field conditions. Further studies using different POC CD4 tests available on the market are required to provide critical data to support countries in selection and implementation of appropriate POC CD4 technologies.

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. DNA content can be estimated using this approach and here we describe how flow cytometry can be used to assess the DNA content of Escherichia coli cells.

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. coli. We have used dual-colour, alternating-laser, single-molecule narrowfield microscopy to quantify the amount of operator at the block and simultaneously image fluorescently labelled DNA polymerase. We anticipate use of this system as a quantitative platform to study replication stalling and repair proteins.

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. However, many different types of proteins form non-covalent interactions with nucleic acids in vivo and so the true substrates of helicases are protein-nucleic acid complexes rather than naked DNA and RNA. Studies over the last decade have revealed that bound proteins can have substantial inhibitory effects on the ability of helicases to unwind nucleic acids. Any analysis of helicase mechanisms in vitro must therefore consider helicase function within the context of nucleoprotein substrates rather than just DNA or RNA. Here we discuss how to analyse the impact of bound proteins on the ability of helicases to unwind DNA substrates in vitro.

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. Here we show that overexpression of dnaB also inhibits growth and chromosome duplication. This inhibition is countered by co-overexpression of wild-type DnaC but not of a DnaC mutant that cannot interact with DnaB, indicating that a reduction in DnaB6-DnaC6 concentration is responsible for the phenotypes associated with elevated DnaB concentration. Partial defects in the oriC-specific initiator DnaA and in PriA-specific initiation away from oriC during replication repair sensitise cells to dnaB overexpression. Absence of the accessory replicative helicase Rep, resulting in increased replication blockage and thus increased reinitiation away from oriC, also exacerbates DnaB-induced defects. These findings indicate that elevated levels of helicase perturb replication initiation not only at origins of replication but also during fork repair at other sites on the chromosome. Thus, imbalances in levels of the replicative helicase and helicase loader can inhibit replication both via inhibition of DnaB6-DnaC6 complex formation with excess DnaB, as shown here, and promotion of formation of DnaB6-DnaC6 complexes with excess DnaC [Allen GC, Jr., Kornberg A. Fine balance in the regulation of DnaB helicase by DnaC protein in replication in Escherichia coli. J. Biol. Chem. 1991;266:22096-22101; Skarstad K, Wold S. The speed of the Escherichia coli fork in vivo depends on the DnaB:DnaC ratio. Mol. Microbiol. 1995;17:825-831]. Thus, there are two mechanisms by which an imbalance in the replicative helicase and its associated loader protein can inhibit genome duplication.

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. Removal of these barriers demands energy input not only to separate the DNA strands but also to disrupt multiple bonds between the protein and DNA. Replicative helicases that unwind the template DNA for polymerases at the fork can displace proteins bound to the template. However, even occasional failures in protein displacement by the replicative helicase could spell disaster. In such circumstances, failure to restart replication could result in incomplete genome duplication. Avoiding incomplete genome duplication via the repair and restart of blocked replication forks also challenges viability since the involvement of recombination enzymes is associated with the risk of genome rearrangements. Organisms have therefore evolved accessory replicative helicases that aid replication fork movement along protein-bound DNA. These helicases reduce the dangers associated with replication blockage by protein-DNA complexes, aiding clearance of blocks and resumption of replication by the same replisome thus circumventing the need for replication repair and restart. This review summarises recent work in bacteria and eukaryotes that has begun to delineate features of accessory replicative helicases and their importance in genome stability.