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

    7 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

    Keywords

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

    ASJC Scopus subject areas

    • Biochemistry

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  • Cite this

    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, 141(2), 147-156. https://doi.org/10.1093/jb/mvm033