The DNA polymerase I from Thermus aquaticus (Taq polymerase) performs lagging-strand DNA synthesis and DNA repair. Taq polymerase contains a polymerase domain for synthesizing a new DNA strand and a 5 -nuclease domain for cleaving RNA primers or damaged DNA strands. The extended crystal structure of Taq polymerase poses a puzzle on how this enzyme coordinates its polymerase and the nuclease activities to generate only a nick. Using contrast variation solution small angle neutron scattering, we have examined the conformational changes that occur in Taq polymerase upon binding "overlap flap" DNA, a structure-specific DNA substrate that mimics the substrate in strand replacement reactions. In solution, apoTaq polymerase has an overall expanded equilibrium conformation similar to that in the crystal structure. Upon binding to the DNA substrate, both the polymerase and the nuclease domains adopt more compact overall conformations, but these changes are not enough to bring the two active sites close enough to generate a nick. Reconstruction of the three-dimensional molecular envelope from small angle neutron scattering data shows that in the DNAbound form, the nuclease domain is lifted up relative to its position in the non-DNA-bound form so as to be in closer contact with the thumb and palm subdomains of the polymerase domain. The results suggest that a form of structure sensing is responsible for the coordination of the polymerase and nuclease activities in nick generation. However, interactions between the polymerase and the nuclease domains can assist in the transfer of the DNA substrate from one active site to the other.In addition to its well known in vitro applications in PCR, in vivo the DNA polymerase I from Thermus aquaticus (Taq polymerase) performs nucleotide replacement reactions in DNA repair and RNA primer removal in Okazaki fragment processing during lagging-strand synthesis (1). Taq polymerase also utilizes both its polymerase and 5Ј-nuclease activities in a nucleotide replacement reaction: it catalyzes the upstream addition of deoxynucleoside 5Ј-triphosphates to the 3Ј-hydroxyl terminus of an RNA primer and also cleaves the downstream, single-stranded 5Ј-nucleotide displaced by the growing upstream strand. On a DNA duplex substrate, upstream primer extension and downstream strand excision must be highly coordinated (2) to leave a ligatable nick, instead of a gap or an overhang, on a DNA duplex that is to be sealed later by a DNA ligase.The structure and functional properties of Taq polymerase are similar to those of the polymerase I of Escherichia coli (3). Taq polymerase has two distinct domains: an N-terminal domain (residues 1-289) that has single-stranded 5Ј-nucleotide cleavage activity (5Ј-nuclease domain), and a C-terminal domain (residues 306 -832) that has DNA polymerase activity (Klentaq domain). Integrated into the Klentaq domain is another domain (residues 306 -423) that is structurally analogous to the proof editing 3Ј 3 5Ј-exonuclease domain of E. coli polymerase I, but this domain has n...

As part of an overall project to characterize the streptomycin phosphotransferase enzyme APH(6)-Id, which confers bacterial resistance to streptomycin, we cloned, expressed, purified and characterized the enzyme. When expressed in E. coli, the recombinant enzyme increased by up to 70-fold the minimum inhibitory concentration (MIC) needed to inhibit cell growth. Size exclusion chromatography gave a molecular mass of 31.4 ± 1.3 kDa for the enzyme, showing that it functions as a monomer. Activity was assayed using three methods: (1) an HPLC-based method that measures the consumption of streptomycin over time; (2) a spectrophotometric method that utilizes a coupled assay; and (3) a radioenzymatic method that detects production of 32P-labeled streptomycin phosphate. Altogether, the three methods demonstrated that streptomycin was consumed in the APH(6)-Id-catalyzed reaction, ATP was hydrolyzed, and streptomycin phosphate was produced in a substrate-dependent manner, demonstrating that APH(6)-Id is a streptomycin phosphotransferase. Steady state kinetic analysis gave the following results: Km(streptomycin) of 0.38 ± 0.13 mM, Km(ATP) of 1.03 ± 0.1 mM, Vmax of 3.2 ± 1.1 μmol/min/mg and kcat of 1.7 ± 0.6 s−1. Our study demonstrates that APH(6)-Id is a bona fide streptomycin phosphotransferase, functions as a monomer, and confers resistance to streptomycin.

Ecological developmental biology (Eco-Devo) involves the study of development in its natural environmental context as opposed to the laboratory setting. Ernest E. Just was an early 20th century African-American embryologist who devoted his career to studying the early development of marine invertebrates in the United States and abroad. Through detailed study of the fertilization process, he came to see the cell cortex as playing a central role in development, inheritance, and evolution. This paper, after presenting some of Just's scientific and philosophical contributions, argues that Just was an Eco-Devo biologist. Three lines of evidence are given. First, Just believed that intimate knowledge of the natural history of the marine animal under study--hence, the natural setting in which fertilization occurs--was essential. Second, he stressed the importance of the egg's "normality"--how well its condition in the laboratory corresponds to the natural, fertilizable state. Finally, Just was an organicist, believing that organisms are holistic systems with emergent properties that arise from their organization and complexity. Although other scientists may stand out more clearly as founding architects of Eco-Devo, E. E. Just, with his unwavering insistence on the normality and holistic integrity of the egg cell, was one of its purest adherents.

In the current study, we developed a HPLC method to quantitatively measure the permeability of the BpT-based chelators, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT) and 2-benzoylpyridine 4-allyl-3-thiosemicarbazone (Bp4aT), across human colorectal adenocarcinoma (Caco-2) monolayers as a model of gut absorption. In aqueous solution, Bp4eT and Bp4aT formed inter-convertible Z and E isomers that were resolved by HPLC. Peak area was linear with respect to chelator concentration. Acceptable within-day and between-day precision (<22%) and accuracy (85–115% of true values) were obtained over a range of 1.0 – 100 µM for Bp4eT and 1.5 – 300 µM for Bp4aT. Limits of detection were 0.3 µM and 1 µM for Bp4eT and Bp4aT, respectively, while corresponding limits of quantification were 1 µM and 5 µM. Both chelators showed significant ability to chelate iron in THP-1 cells using a calcein-based assay and no apparent cytotoxicity was observed within 24 h. Ratios of the apical to basolateral and basolateral to apical transport for Bp4eT were 1.10 and 0.89 at 100 µM and 300 µM respectively, indicating equal bi-directional movement of the compounds. Similarly, ratios were 0.77 and 0.92 for Bp4aT, respectively. This study demonstrates that Bp4eT and Bp4aT can be efficiently transported through Caco-2 cells and can potentially be formulated for oral delivery.

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