22. Analysis of Plasmid DNA Structure after -irradiation by Atomic Force Microscope

Masahiro Murakami, Akira Furukawa, Kiyomi Eguchi-Kasai, Reiko Kanda, Hideo Hirokawa^* and Isamu Hayata
(^*Max-Planck Institute for Molecular Genetics, Germany)

Keywords: DNA damage, atomic force microscope, plasmid DNA

DNA damage (DNA single-strand break, DNA double-strand break, etc.) induced with ionizing radiation is considered one of the main causes of cell inactivation. Several methods including agarose gel electrophoresis, pulsed-field gel electrophoresis, neutral filter elution method, neutral sedimentation and electron microscopy have been applied to analyze this type DNA damage.

A new method employing an atomic force microscope (AFM) for nanometer-level structure analysis of DNA damage induced with -irradiation was established. Formal changes of plasmid DNA (about 3 kbp; length about 1m) after irradiation with ⁶⁰Co -rays at doses of 1.9k, 5.6k, and 8.3kGy were visually analyzed by AFM. The tortile feature of the plasmid DNA was depicted better with AFM than with a transmission electron microscope (TEM). Three forms of plasmid DNA, closed circular (intact DNA), open circular (DNA with a single-strand break) and linear form (DNA with a double-strand break) were observed in the sample irradiated with -rays at the dose of 1.9kGy. On the other hand, in the samples irradiated with -rays at doses of 5.6 kGy and 8.3 kGy, open circular and linear forms were observed, but no closed circular form was observed. A shortening of the length of the linear form of DNA irradiated with 5.6kGy and 8.3kGy -rays was observed by AFM. The average lengths of the linear form of DNA irradiated with 1.9kGy, 5.6kGy and 8.3kGy of -rays were 0.96±0.06 m, 0.69±0.04 m and 0.69±0.04 m, respectively. Shortening of the length of the linear form of DNA under experimental conditions of 5.6kGy and 8.3kGy -ray irradiation were significant when T-test was applied (p<0.05). The shortening of the linear form is possible as a result of the destruction of DNA secondary structure caused by complicated DNA damage, such as the combination of DNA single-strand break(s), DNA double-strand break or DNA base damage(s) on the same DNA molecule, induced with low LET-ionizing radiation. Shortening of the DNA strand has also been observed as a result of cisplatin-DNA interaction by TEM [Macquet JP, Butour JL. : Biochimie 60, 901-914, 1978] and AFM [Onoa GB, Cervantes G, Moreno V, Prieto MJ. : Nucleic Acids Research 26, 1473-1480, 1998]. The present observations support our interpretation, because DNA damage caused by cisplatin-DNA interaction other than multiple DNA double-strand breaks produces shortening of the DNA length. Our findings indicate the possibility that AFM imaging may be able to visualize a novel type of structural change to DNA following ionizing radiation, which is not observed by gel electrophoretic analysis.