Visualization of RecA filaments and DNA by fluorescence microscopy.
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.
Nishinaka
Taro
T
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
Doi
Yuko
Y
Hashimoto
Makiko
M
Hara
Reiko
R
Shibata
Takehiko
T
Harada
Yoshie
Y
Kinosita
Kazuhiko
K
Noji
Hiroyuki
H
Yashima
Eiji
E
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*
D004277:DNA, Single-Stranded; D003838:Deoxyadenine Nucleotides; D029968:Escherichia coli Proteins; D004247:DNA; D011938:Rec A Recombinases