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Journal of Biochemistry Advance Access originally published online on January 3, 2007
Journal of Biochemistry 2007 141(2):147-156; doi:10.1093/jb/mvm033
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© 2007 The Japanese Biochemical Society.

Visualization of RecA Filaments and DNA by Fluorescence Microscopy

Taro Nishinaka1,*, Yuko Doi1, Makiko Hashimoto1, Reiko Hara1, Takehiko Shibata2, Yoshie Harada3, Kazuhiko Kinosita, Jr.4, Hiroyuki Noji5 and Eiji Yashima1,6

1Yashima Super-structured Helix Project, ERATO, Japan Science and Technology Agency, 101 Creation Core Nagoya, 2266-22 Anagahora, Shimoshidami, Moriyama-ku, Nagoya 463-0003; 2Laboratory of Cellular and Molecular Biology, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198; 3Department of Molecular Physiology, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613; 4Department of Physics, Faculty of Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555; 5SANKEN, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047; and 6Institute for Advanced Research, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan

*To whom correspondence should be addressed. Tel: +81-52-739-2081, Fax: +81-52-739-2084, E-mail: nishinak{at}yp-jst.jp

Received September 5, 2006; Accepted November 20, 2006


  Abstract

We have developed two experimental methods for observing Escherichia coli RecA-DNA filament under a fluorescence microscope. First, RecA-DNA filaments were visualized by immunofluorescence staining with anti-RecA monoclonal antibody. Although the detailed filament structures below submicron scale were unable to be measured accurately due to optical resolution limit, this method has an advantage to analyse a large number of RecA-DNA filaments in a single experiment. Thus, it provides a reliable statistical distribution of the filament morphology. Moreover, not only RecA filament, but also naked DNA region was visualized separately in combination with immunofluorescence staining using anti-DNA monoclonal antibody. Second, by using cysteine derivative RecA protein, RecA-DNA filament was directly labelled by fluorescent reagent, and was able to observe directly under a fluorescence microscope with its enzymatic activity maintained. We showed that the RecA-DNA filament disassembled in the direction from 5' to 3' of ssDNA as dATP hydrolysis proceeded.

Key Words: DNA–protein interaction, fluorescence label of protein, homologous recombination, microscopic observations, RecA protein

Abbreviations: ATP{gamma}S, adenosine 5'-O-(thiotriphosphate)


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