Reconstitution of a Minimal DNA Replicase From Pseudomonas Aeruginosa and Stimulation by Non-Cognate Auxiliary Factors

DNA polymerase III holoenzyme is responsible for chromosomal replication in bacteria. The components and functions of Escherichia coli DNA polymerase III holoenzyme have been studied extensively. Here, we report the reconstitution of replicase activity by essential components of DNA polymerase holoenzyme from the pathogen Pseudomonas aeruginosa. We have expressed and purified the processivity factor (β), single-stranded DNA-binding protein, a complex containing the polymerase (α) and exonuclease (ϵ) subunits, and the essential components of the DnaX complex (τ3δδ′). Efficient primer elongation requires the presence of αϵ, β, and τ3δδ′. Pseudomonas aeruginosa αϵ can substitute completely for E. coli polymerase III in E. coli holoenzyme reconstitution assays. Pseudomonas β and τ3δδ′ exhibit a 10-fold lower activity relative to their E. coli counterparts in E. coli holoenzyme reconstitution assays. Although the Pseudomonas counterpart to the E. coli ψ subunit was not apparent in sequence similarity searches, addition of purified E. coli χ and ψ (components of the DnaX complex) increases the apparent specific activity of the Pseudomonas τ3δδ′ complex ∼10-fold and enables the reconstituted enzyme to function better under physiological salt conditions

DNA Polymerase III Holoenzyme From Thermus Thermophilus Identification Expression Purification of Components and use to Reconstitute a Processive Replicase

DNA replication in bacteria is performed by a specialized multicomponent replicase, the DNA polymerase III holoenzyme, that consist of three essential components: a polymerase, the β sliding clamp processivity factor, and the DnaX complex clamp-loader. We report here the assembly of the minimal functional holoenzyme from Thermus thermophilus (Tth), an extreme thermophile. The minimal holoenzyme consists of α (pol III catalytic subunit), β (sliding clamp processivity factor), and the essential DnaX (τ/γ), δ and δ′ components of the DnaX complex. We show with purified recombinant proteins that these five components are required for rapid and processive DNA synthesis on long single-stranded DNA templates. Subunit interactions known to occur in DNA polymerase III holoenzyme from mesophilic bacteria including δ-δ′ interaction, δδ′-τ/γ complex formation, and α-τ interaction, also occur within the Tth enzyme. As in mesophilic holoenzymes, in the presence of a primed DNA template, these subunits assemble into a stable initiation complex in an ATP-dependent manner. However, in contrast to replicative polymerases from mesophilic bacteria, Tth holoenzyme is efficient only at temperatures above 50 °C, both with regard to initiation complex formation and processive DNA synthesis. The minimalTth DNA polymerase III holoenzyme displays an elongation rate of 350 bp/s at 72 °C and a processivity of greater than 8.6 kilobases, the length of the template that is fully replicated after a single association event

A Three-Domain Structure for the delta Subunit of the DNA Polymerase III Holoenzyme delta Domain III Binds delta\u27 and Assembles Into the DnaX Complex

Using ψ-BLAST, we have developed a method for identifying the poorly conserved δ subunit of the DNA polymerase III holoenzyme from all sequenced bacteria. This approach, starting withEscherichia coli δ, leads not only to the identification of δ but also to the DnaX and δ′ subunits of the DnaX complex and other AAA+-class ATPases. This suggests that, although not an ATPase, δ is related structurally to the other subunits of the DnaX complex that loads the β sliding clamp processivity factor onto DNA. To test this prediction, we aligned δ sequences with those of δ′ and, using the start of δ′ Domain III established from its x-ray crystal structure, predicted the juncture between Domains II and III of δ. This putative δ Domain III could be expressed to high levels, consistent with the prediction that it folds independently. δ Domain III, like Domain III of DnaX and δ′, assembles by itself into a complex with the other DnaX complex components. Cross-linking studies indicated a contact of δ with the DnaX subunits. These observations are consistent with a model where two τ subunits and one each of the γ, δ′, and δ subunits mutually interact to form a pentameric functional core for the DnaX complex

Aptamer-based multiplexed proteomic technology for biomarker discovery

Interrogation of the human proteome in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology. We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 [mu]L of serum or plasma). Our current assay allows us to measure ~800 proteins with very low limits of detection (1 pM average), 7 logs of overall dynamic range, and 5% average coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding DNA aptamer concentration signature, which is then quantified with a DNA microarray. In essence, our assay takes advantage of the dual nature of aptamers as both folded binding entities with defined shapes and unique sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to discover unique protein signatures characteristic of various disease states. More generally, we describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine

Protein Signature of Lung Cancer Tissues

Lung cancer remains the most common cause of cancer-related mortality. We applied a highly multiplexed proteomic technology (SOMAscan) to compare protein expression signatures of non small-cell lung cancer (NSCLC) tissues with healthy adjacent and distant tissues from surgical resections. In this first report of SOMAscan applied to tissues, we highlight 36 proteins that exhibit the largest expression differences between matched tumor and non-tumor tissues. The concentrations of twenty proteins increased and sixteen decreased in tumor tissue, thirteen of which are novel for NSCLC. NSCLC tissue biomarkers identified here overlap with a core set identified in a large serum-based NSCLC study with SOMAscan. We show that large-scale comparative analysis of protein expression can be used to develop novel histochemical probes. As expected, relative differences in protein expression are greater in tissues than in serum. The combined results from tissue and serum present the most extensive view to date of the complex changes in NSCLC protein expression and provide important implications for diagnosis and treatment

Elucidating mechanisms of genetic cross-disease associations at the PROCR vascular disease locus

AbstractMany individual genetic risk loci have been associated with multiple common human diseases. However, the molecular basis of this pleiotropy often remains unclear. We present an integrative approach to reveal the molecular mechanism underlying the PROCR locus, associated with lower coronary artery disease (CAD) risk but higher venous thromboembolism (VTE) risk. We identify PROCR-p.Ser219Gly as the likely causal variant at the locus and protein C as a causal factor. Using genetic analyses, human recall-by-genotype and in vitro experimentation, we demonstrate that PROCR-219Gly increases plasma levels of (activated) protein C through endothelial protein C receptor (EPCR) ectodomain shedding in endothelial cells, attenuating leukocyte–endothelial cell adhesion and vascular inflammation. We also associate PROCR-219Gly with an increased pro-thrombotic state via coagulation factor VII, a ligand of EPCR. Our study, which links PROCR-219Gly to CAD through anti-inflammatory mechanisms and to VTE through pro-thrombotic mechanisms, provides a framework to reveal the mechanisms underlying similar cross-phenotype associations.</jats:p

Mode of Action and Biochemical Characterization of REP8839, a Novel Inhibitor of Methionyl-tRNA Synthetase

Aminoacyl-tRNA synthetases have attracted interest as essential and novel targets involved in bacterial protein synthesis. REP8839 is a potent inhibitor of MetS, the methionyl-tRNA synthetase in Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), and in Streptococcus pyogenes. The biochemical activity of REP8839 was shown by specific inhibition of purified S. aureus MetS (50% inhibitory concentration, <1.9 nM). Target specificity was confirmed by overexpression of the metS gene in S. aureus, resulting in an eightfold increase in the MIC for REP8839. Macromolecular synthesis assays in the presence of REP8839 demonstrated a dose-dependent inhibition of protein synthesis and RNA synthesis in S. pneumoniae R6, but only protein synthesis was affected in an isogenic rel mutant deficient in the stringent response. Strains with reduced susceptibility to REP8839 were generated by selection of strains with spontaneous mutations and through serial passages. Point mutations within the metS gene were mapped, leading to a total of 23 different amino acid substitutions within MetS that were located around the modeled active site. The most frequent MetS mutations were I57N, leading to a shift in the MIC from 0.06 μg/ml to 4 μg/ml, and G54S, resulting in a MIC of 32 μg/ml that was associated with a reduced growth rate. The mutation prevention concentration was 32 μg/ml in four S. aureus strains (methicillin-sensitive S. aureus and MRSA), which is well below the drug concentration of 2% (20,000 μg/ml) in a topical formulation. In conclusion, we demonstrate by biochemical, physiologic, and genetic mode-of-action studies that REP8839 exerts its antibacterial activity through specific inhibition of MetS, a novel target