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

    Research output: Contribution to journalArticle

    6 Citations (Scopus)

    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.

    Original languageEnglish
    Pages (from-to)147-156
    Number of pages10
    JournalJournal of Biochemistry
    Volume141
    Issue number2
    DOIs
    Publication statusPublished - 2007 Feb

    Fingerprint

    Fluorescence microscopy
    Fluorescence Microscopy
    Visualization
    DNA
    Fluorescent Antibody Technique
    Fluorescence
    Monoclonal Antibodies
    Statistical Distributions
    Rec A Recombinases
    Staining and Labeling
    Microscopes
    Antinuclear Antibodies
    Cysteine
    Hydrolysis
    Escherichia coli
    Derivatives

    Keywords

    • DNA-protein interaction
    • Fluorescence label of protein
    • Homologous recombination
    • Microscopic observations
    • RecA protein

    ASJC Scopus subject areas

    • Biochemistry

    Cite this

    Nishinaka, T., Doi, Y., Hashimoto, M., Hara, R., Shibata, T., Harada, Y., ... Yashima, E. (2007). Visualization of RecA filaments and DNA by fluorescence microscopy. Journal of Biochemistry, 141(2), 147-156. https://doi.org/10.1093/jb/mvm033

    Visualization of RecA filaments and DNA by fluorescence microscopy. / Nishinaka, Taro; Doi, Yuko; Hashimoto, Makiko; Hara, Reiko; Shibata, Takehiko; Harada, Yoshie; Kinosita, Kazuhiko; Noji, Hiroyuki; Yashima, Eiji.

    In: Journal of Biochemistry, Vol. 141, No. 2, 02.2007, p. 147-156.

    Research output: Contribution to journalArticle

    Nishinaka, T, Doi, Y, Hashimoto, M, Hara, R, Shibata, T, Harada, Y, Kinosita, K, Noji, H & Yashima, E 2007, 'Visualization of RecA filaments and DNA by fluorescence microscopy', Journal of Biochemistry, vol. 141, no. 2, pp. 147-156. https://doi.org/10.1093/jb/mvm033
    Nishinaka T, Doi Y, Hashimoto M, Hara R, Shibata T, Harada Y et al. Visualization of RecA filaments and DNA by fluorescence microscopy. Journal of Biochemistry. 2007 Feb;141(2):147-156. https://doi.org/10.1093/jb/mvm033
    Nishinaka, Taro ; Doi, Yuko ; Hashimoto, Makiko ; Hara, Reiko ; Shibata, Takehiko ; Harada, Yoshie ; Kinosita, Kazuhiko ; Noji, Hiroyuki ; Yashima, Eiji. / Visualization of RecA filaments and DNA by fluorescence microscopy. In: Journal of Biochemistry. 2007 ; Vol. 141, No. 2. pp. 147-156.
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    AU - Harada, Yoshie

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