title: Visualization of RecA filaments and DNA by fluorescence microscopy.
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.
authors.affiliation: Yashima Super-structured Helix Project, ERATO, Japan Science and Technology Agency, 101 Creation Core Nagoya, 2266-22 Anagahora, Nagoya 463-0003, Japan. nishinak@yp-jst.jp
authors.forename: Taro
authors.initials: T
authors.lastname: Nishinaka
authors.forename: Yuko
authors.initials: Y
authors.lastname: Doi
authors.forename: Makiko
authors.initials: M
authors.lastname: Hashimoto
authors.forename: Reiko
authors.initials: R
authors.lastname: Hara
authors.forename: Takehiko
authors.initials: T
authors.lastname: Shibata
authors.forename: Yoshie
authors.initials: Y
authors.lastname: Harada
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Kinosita
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Noji
authors.forename: Eiji
authors.initials: E
authors.lastname: Yashima
mesh_terms: D004247:DNA / Q000032:analysis*; D004277:DNA, Single-Stranded / Q000032:analysis; D003838:Deoxyadenine Nucleotides / Q000032:analysis; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000032:analysis*; D005455:Fluorescent Antibody Technique; D008856:Microscopy, Fluorescence; D011938:Rec A Recombinases / Q000032:analysis*
chemical_list: D004277:DNA, Single-Stranded; D003838:Deoxyadenine Nucleotides; D029968:Escherichia coli Proteins; D004247:DNA; D011938:Rec A Recombinases