Tomomi Ishido

Pinophilins A and B, Inhibitors of Mammalian A-, B-, and Y-Family DNA Polymerases and Human Cancer Cell Proliferation

Pinophilins A (1) and B (2), new hydrogenated azaphilones, and Sch 725680 (3) were isolated from cultures of a fungus (Penicillium pinophilum Hedgcok) derived from a seaweed, and their structures were determined using spectroscopic analyses. These compounds selectively inhibited the activities of mammalian DNA polymerases (pols), A (pol γ), B (pols α, δ, and ε), and Y (pols η, ι, and κ) families, but did not influence the activities of the four X-family pols (pols β, λ, μ, and terminal deoxynucleotidyl transferase). Compound 1 was the strongest inhibitor, with IC₅₀ values of 48.6 to 55.6 μM. Kinetic analysis showed that compound 1 is a noncompetitive inhibitor of both pol α and κ activities with the DNA template-primer substrate, and a competitive inhibitor with the nucleotide substrate. In contrast, compounds 1-3 showed no effect on the activities of plant and prokaryotic pols or any other DNA metabolic enzymes tested. The compounds suppressed cell proliferation and growth in five human cancer cell lines, but had no effect on the viability of normal human cell lines.

How environmental solution conditions determine the compaction velocity of single DNA molecules

Understanding the mechanisms of DNA compaction is becoming increasingly important for gene therapy and nanotechnology DNA applications. The kinetics of the compaction velocity of single DNA molecules was studied using two non-protein condensation systems, poly(ethylene glycol) (PEG) with Mg2+ for the polymer-salt-induced condensation system and spermine for the polyamine condensation system. The compaction velocities of single tandem λ-DNA molecules were measured at various PEG and spermine concentrations by video fluorescent microscopy. Single DNA molecules were observed using a molecular stretching technique in the microfluidic flow. The results show that the compaction velocity of a single DNA molecule was proportional to the PEG or spermine concentration to the power of a half. Theoretical considerations indicate that the compaction velocity is related to differences in the free energy of a single DNA molecule between the random coil and compacted states. In the compaction kinetics with PEG, acceleration of the compaction velocity occurred above the overlap concentration while considerable deceleration occurred during the coexistence state of the random coil and the compacted conformation. This study demonstrates the control factors of DNA compaction kinetics and contributes toward the understanding of the compaction mechanisms of non-protein DNA interactions as well as DNA–protein interactions in vivo.

Measuring the length distribution of self-assembled lipid nanotubes by orientation control with a high-frequency alternating current electric field in aqueous solutions.

The present work addresses the length distribution of self-assembled lipid nanotubes (LNTs) by controlling the orientation of the LNTs using an alternating current (ac) electric field in aqueous solutions. The effect of the ac field on the orientation and rotation of individual LNTs was examined to evaluate the optimum orientation frequency by visualizing the individual LNTs in real time. By using the high-frequency ac field, we have successfully measured the length distribution for two different types of LNTs and have quantitatively analyzed the maximum occurrences of the length distribution as well as the extension of the longer length region.

Plasmonic Imaging of Brownian Motion of Single DNA Molecules Spontaneously Binding to Ag Nanoparticles

We find the spontaneous binding of single DNA molecules to uncoated silver nanoparticles (AgNPs) in aqueous solution with Mn(2+) (3 mM). From dark-field optical microscopic imaging of AgNPs bound to DNA molecules, we demonstrate analysis of the Brownian motion of single DNA molecules via plasmon resonance elastic light scattering. Our results provide that the plasmonic imaging technique is free from photobleaching and blinking and thus is useful in long-time observations of single-molecule DNA dynamics.

Sizing of single globular DNA molecules by using a circular acceleration technique with laser trapping.

We describe a method for in situ sizing individual huge DNA molecules by laser trapping. Single DNA molecules are reversibly transformed, without mechanical fragmentation of fragile huge-sized DNA, from their random coil state into their globular state induced by condensing agents poly(ethylene glycol) and Mg(2+). With the use of a globular DNA molecule folded by condensation, the critical velocity of the circularly accelerated single globular DNA molecule by laser trapping was found to be proportional to the size of the DNA. Yeast, Saccharomyces cerevisiae, chromosome III (285 kbp) was successfully sized (281 +/- 40 kbp) from a calibration curve scaled using lambda, T4, and yeast chromosome VI (48.5, 166, and 385 kbp, respectively). The use of critical velocity as a sizing parameter makes it possible to size single DNA molecules without prior conformational information, i.e., the radius of a single globular huge DNA molecule as a nanoparticle. A sized single globular DNA molecule could be trapped again for subsequent manipulation, such as transportation of it anywhere. We also investigated a possibility of reusing the globular DNA molecules condensed by PEG and Mg(2+) for PCR and found that PCR efficiency was not deteriorated in the presence of the condensation agents.

Marked difference in conformational fluctuation between giant DNA molecules in circular and linear forms

We performed monomolecular observations on linear and circular giant DNAs (208 kbp) in an aqueous solution by the use of fluorescence microscopy. The results showed that the degree of conformational fluctuation in circular DNA was ca. 40% less than that in linear DNA, although the long-axis length of circular DNA was only 10% smaller than that of linear DNA. Additionally, the relaxation time of a circular chain was shorter than that of a linear chain by at least one order of magnitude. The essential features of this marked difference between linear and circular DNAs were reproduced by numerical simulations on a ribbon-like macromolecule as a coarse-grained model of a long semiflexible, double-helical DNA molecule. In addition, we calculated the radius of gyration of an interacting chain in a circular form on the basis of the mean field model, which provides a better understanding of the present experimental trend than a traditional theoretical equation.

Characterization of DNA polymerase β from Danio rerio by overexpression in E. coli using the in vivo/in vitro compatible pIVEX plasmid

BackgroundEukaryotic DNA polymerase β (pol β), the polymerase thought to be responsible for DNA repair synthesis, has been extensively characterized in rats and humans. However, pol β has not been purified or enzymatically characterized from the model fish species Danio rerio (zebrafish). We used the in vitro/in vivo dual expression system plasmid, pIVEX, to express Danio rerio pol β (Danio pol β) for biochemical characterization.ResultsDanio pol β encoded by the in vitro/in vivo-compatible pIVEX plasmid was expressed in E. coli BL21(DE3), BL21(DE3)pLysS, and KRX, and in vitro as a C-terminal His-tagged protein. Danio pol β expressed in vitro was subject to proteolysis; therefore, bacterial overexpression was used to produce the protein for kinetic analyses. KRX cells were preferred because of their reduced propensity for leaky expression of pol β. The cDNA of Danio rerio pol β encodes a protein of 337 amino acids, which is 2-3 amino acids longer than other pol β proteins, and contains a P63D amino acid substitution, unlike mammalian pol βs. This substitution lies in a hairpin sequence within an 8-kDa domain, likely to be important in DNA binding. We performed extensive biochemical characterization of Danio pol β in comparison with rat pol β, which revealed its sensitivity to metal ion activators (Mn2+ and Mg2+), its optimum salt concentration (10 mM KCl and 50 mM NaCl), alkaline pH optimum (pH 9.0), and low temperature optimum (30°C). Substituting Mn2+ for Mg2+ resulted in 8.6-fold higher catalytic efficiency (kcat/Km).ConclusionsOur characterization of pol β from a model fish organism contributes to the study of the function and evolution of DNA polymerases, which are emerging as important cellular targets for chemical intervention in the development of anticancer agents.

Consecutive incorporation of fluorophore-labeled nucleotides by mammalian DNA polymerase β

In the present study, we investigated mammalian polymerases that consecutively incorporate various fluorophore-labeled nucleotides. We found that rat DNA polymerase beta (pol beta) consecutively incorporated fluorophore-labeled nucleotides to a greater extent than four bacterial polymerases, Sequenase Version 2.0, Vent(R) (exo-), DNA polymerase IIIalpha and the Klenow fragment, and the mammalian polymerases DNA polymerase alpha and human DNA polymerase delta, under mesophilic conditions. Furthermore, we investigated the kinetics of correct or mismatched incorporation with labeled nucleotides during synthesis by rat pol beta. The kinetic parameters K(m) and k(cat) were measured and used for evaluating: (i) the discrimination against correct pair incorporation of labeled nucleotides relative to unlabeled nucleotides; and (ii) the fidelity for all nucleotide combinations of mismatched pairs in the presence of labeled or unlabeled nucleotides. We also investigated the effect of fluorophore-labeled nucleotides on terminal deoxynucleotidyl transferase activity of rat pol beta. We have demonstrated for the first time that mammalian pol beta can consecutively incorporate various fluorophore-labeled dNTPs. These findings suggest that pol beta is useful for high-density labeling of DNA probes and single-molecule sequencing for high-speed genome analysis.

Rapid sequencing gel electrophoresis using glycerol-tolerant sodium taurine medium

We describe a new glycerol-tolerant sodium taurine (ST) medium for rapid sequencing gel electrophoresis by substituting the standard conductive media of Tris-boric acid-ethylenediaminetetraacetic acid (EDTA) (TBE) and Tris-taurine-EDTA (TTE) and other low-ionic-strength media of sodium boric acid (SB). Low-ionic-strength and cost-effective ST media gave glycerol tolerance up to 50% (v/v) glycerol-containing DNA sample solution, shorter running time, and better resolution to separate small DNA oligonucleotides (20-45 mer) in 12% denaturing sequencing gel electrophoresis.

Analysis of supercoiled DNA by agarose gel electrophoresis using low-conducting sodium threonine medium

We describe a new low-ionic-strength sodium threonine (STh) medium with the advantage of avoiding relative DNA band migration changes following electrophoresis of supercoiled DNA in agarose gel when substituted for the standard conductive medium of TBE (Tris-boric acid-ethylenediaminetetraacetic acid [EDTA]) or TAE (Tris-acetic acid-EDTA) or the low-ionic-strength sodium boric acid medium. Low-ionic-strength STh medium provided better resolution, less heat generation, and prevention of relative migration order changes among linear, covalently closed circular-, and open circular-formed DNA in the range of 2-10 kilobase pairs in 1% agarose gel electrophoresis.