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Biophysical Journal 85:402-408 (2003)
© 2003 The Biophysical Society

Interarm Interaction of DNA Cruciform Forming at a Short Inverted Repeat Sequence

Mikio Kato * {ddagger}, Shingo Hokabe *, Shuji Itakura *, Shinsei Minoshima {dagger}, Yuri L. Lyubchenko {ddagger}, Theodor D. Gurkov §, Hiroshi Okawara §, Kuniaki Nagayama § and Nobuyoshi Shimizu {dagger}

* Department of Life Sciences, Osaka Prefecture University College of Integrated Arts and Sciences, Sakai 599-8531, Japan; {dagger} Department of Molecular Biology, Keio University School of Medicine, Tokyo 160-8582, Japan; {ddagger} Department of Microbiology, Arizona State University, Tempe, Arizona 85287 USA; and § Center for Integrative Bioscience, Okazaki National Research Institutes, Okazaki 444-8585, Japan

Correspondence: Address reprint requests to Mikio Kato, Dept. of Life Sciences, Osaka Prefecture University College of Integrated Arts and Sciences, 1-1 Gakuencho, Sakai 599-8531, Japan. Tel. and Fax: +81-72-254-9746; E-mail: mkato{at}el.cias.osakafu-u.ac.jp, mikio_kato{at}mac.com.

A novel interarm interaction of DNA cruciform forming at inverted repeat sequence was characterized using an S1 nuclease digestion, permanganate oxidation, and microscopic imaging. An inverted repeat consisting of 17 bp complementary sequences was isolated from the bluegill sunfish Lepomis macrochirus (Perciformes) and subcloned into the pUC19 plasmid, after which the supercoiled recombinant plasmid was subjected to enzymatic and chemical modification. In high salt conditions (200 mM NaCl, or 100–200 mM KCl), S1 nuclease cut supercoiled DNA at the center of palindromic symmetry, suggesting the formation of DNA cruciform. On the other hand, S1 nuclease in the presence of 150 mM NaCl or less cleaved mainly the 3'-half of the repeat, thereby forming an unusual structure in which the 3'-half of the inverted repeat, but not the 5'-half, was retained as an unpaired strand. Permanganate oxidation profiles also supported the presence of single-stranded part in the 3'-half of the inverted repeat in addition to the center of the symmetry. Both electron microscopy and atomic force microscopy have detected a thick protrusion on the supercoiled DNA harboring the inverted repeat. We hypothesize that the cruciform hairpins at conditions favoring triplex formation adopt a parallel side-by-side orientation of the arms allowing the interaction between them supposedly stabilized by hydrogen bonding of base triads.




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