Yuko Yoshikawa

Diaminoalkanes with an odd number of carbon atoms induce compaction of a single double-stranded DNA chain

We have performed direct observations on the conformational change of a single double‐stranded T4 DNA molecule induced by diaminoalkanes, NH3 +(CH2)nNH3 + (n = 1 ∼ 6), by use of fluorescence microscopy. It was found that diamines with three and five methylene groups show a significant effect on the compaction of individual single DNA molecules. On the other hand, diamines with two, four and six methylene groups are not effective in the compaction of DNA. Such a drastic difference in the action of diamines suggests that not only the number of charge but also the length between the amino groups in the diamines plays an essential role in their interactions with DNA.

Daunomycin unfolds compactly packed DNA

Daunomycin is an antitumor antibiotic known to inhibit DNA replication and transcription. Although the inhibition is assumed to be caused by a direct interaction of the drug with DNA, the exact effect of daunomycin on the higher order DNA structure remains uncertain. We studied the effect of daunomycin on DNA compacted states using fluorescence and electron microscopies. Structural changes in individual DNA molecules were examined under the following conditions. T4 phage DNA (166 kbp) was first compacted by spermidine followed by the addition of daunomycin to the compacted DNA. A direct observation of individual single duplex DNAs by fluorescence microscopy indicated that daunomycin induced unfolding of the compacted DNA. Electron microscopic observation of the morphological changes of the higher order DNA structure supported the results obtained by fluorescence microscopy. We discuss here the mechanisms of the unfolding of the compacted structure following intercalation of daunomycin into DNA particularly in terms of the free energy.

Folding and aggregation of DNA chains induced by complexation with lipospermine: formation of a nucleosome-like structure and network assembly.

Dioctadecylamidoglycylspermine (DOGS) is a cationic lipid vector capable of efficiently introducing DNA into various eukaryotic cells. We investigated the higher‐order structure of the DNA/DOGS complex using fluorescence and electron microscopy. Our results show that the DNA/DOGS complex exhibits a nucleosome‐like structure in which DNA wraps around an aggregate of DOGS molecules. In addition, DNA/DOGS complexes tend to associate with each other to form network structures. The resulting network assembly may play a role in effective gene transfection.

A single-molecule assessment of the protective effect of DMSO against DNA double-strand breaks induced by photo-and γ-ray-irradiation, and freezing

Dimethyl sulfoxide (DMSO) is widely used as a cryoprotectant for organs, tissues, and cell suspension in storage. In addition, DMSO is known to be a useful free radical scavenger and a radio-protectant. To date, many in vitro assays using cultured cells have been performed for analysing the protective effect of DMSO against genomic DNA damage; however, currently it has been rather difficult to detect DNA double strand breaks (DSBs) in a quantitative manner. In the present study, we aimed to observe the extent of DNA damage by use of single molecular observation with a fluorescence microscope to evaluate DSBs induced by photo- and γ-ray-irradiation, or freeze/thawing in variable concentrations of DMSO. As a result, we found that 2% DMSO conferred the maximum protective effect against all of the injury sources tested, and these effects were maintained at higher concentrations. Further, DMSO showed a significantly higher protective effect against freezing-induced damage than against photo- and γ-ray-irradiation-induced damage. Our study provides significant data for the optimization of DNA cryopreservation with DMSO, as well as for the usage of DNA as the protective agent against the injuries caused by active oxygen and radiations.

Double‐Strand Breaks in Genome‐Sized DNA Caused by Ultrasound

Abstract DNA double‐strand breaks (DSBs) caused by ultrasound were evaluated in a quantitative manner by single‐molecule fluorescence microscopy. We compared the effect of time‐interval (or pulse) sonication to that of continuous wave (CW) sonication at a fixed frequency of 30u2005kHz. Pulses caused fewer DSBs than CW sonication under the same total input ultrasound energy when the pulse repetition period was above the order of a second. In contrast, pulses caused more DSBs than CW sonication for pulse widths shorter than a second. These effect of ultrasound on DNA were interpreted in terms of the time‐dependent decay in the probability of breakage during the duration of a pulse. We propose a simple phenomenological model by considering a characteristic decay in the probability of DSBs during single‐pulse sonication, which reproduces the essence of the experimental trend. In addition, a data analysis revealed a characteristic scaling behavior between the number of pulses and the number of DSBs.

Branched-Chain Polyamine Found in Hyperthermophiles Induces Unique Temperature-Dependent Structural Changes in Genome-Size DNA

Abstract A pentavalent branched‐chain polyamine, N 4‐bis(aminopropyl)spermidine 3(3)(3)4, is a unique polycation found in the hyperthermophilic archaeon Thermococcus kodakarensis, which grows at temperatures between 60 and 100u2009°C. We studied the effects of this branched‐chain polyamine on DNA structure at different temperatures up to 80u2009°C. Atomic force microscopic observation revealed that 3(3)(3)4 induces a mesh‐like structure on a large DNA (166u2005kbp) at 24u2009°C. With an increase in temperature, DNA molecules tend to unwind, and multiple nano‐loops with a diameter of 10–50u2005nm are generated along the DNA strand at 80u2009°C. These results were compared to those obtained with linear‐chain polyamines, homocaldopentamine 3334 and spermidine, the former of which is a structural isomer of 3(3)(3)4. These specific effects are expected to neatly concern with its role on high‐temperature preference in hyperthermophiles.

Action of newly developed tetrazolato-bridged dinuclear platinum(II) complexe as the candidate of effective antitumor drug on the structure of large DNA

We report the action of tetrazolato-bridged dinuclear platinum(II) complex, 5-H-Y (2+), and SPD (3+) on the higher-order and secondary structure of DNA through the observations with fluorescence microscopy and CD measurements. From the measurement on the higher-order structure of a giant DNA in solution with fluorescence microscopy, it was found that 5-H-Y (2+) causes shrinking of DNA above 10 μM. On the contrary, SPD (3+) showed negligible change on the higher-order structure of DNA up to 30 μM. From circular dichroism (CD) measurements, it becomes clear that 5-H-Y (2+) induces the transition of the secondary structure of DNA from B-form to C-form.

Folding transition of long duplex DNA from mammalian cells.

Conformational change in individual giant DNAs from pig liver is studied by use of fluorescence microscopy. With the addition of spermidine (a trivalent amine), each DNA chains undergo abrupt transition from an elongated coiled state into a folded compact state. It is found that the all-or-none characteristics in the folding transition for the mammalian DNA is similar to that in phage DNAs.