Visualization of RecA filaments and DNA by fluorescence microscopy

Taro Nishinaka, Yuko Doi, Makiko Hashimoto, Reiko Hara, Takehiko Shibata, Yoshie Harada, Kazuhiko Kinosita, Hiroyuki Noji, Eiji Yashima

    研究成果: Article

    7 被引用数 (Scopus)

    抄録

    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.

    本文言語English
    ページ(範囲)147-156
    ページ数10
    ジャーナルJournal of Biochemistry
    141
    2
    DOI
    出版ステータスPublished - 2007 2

    ASJC Scopus subject areas

    • Biochemistry

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