Electron Microscopic Visualization of N-acetoxy-N-2-acetylaminofluorene Binding Sites in ColE1 DNA by means of Specific Antibodies

ColE1 DNA has been allowed to react in vitro with N-acetoxy-N-2-[$^{14}$C]acetylaminofluorene in the range of 0-15 N-2-[$^{14}$C]acetylaminofluorene residues bound per molecule of DNA, at the C8 of guanine residues. Purified rabbit antibodies to both N-2-(guanosine-8-yl)-acetylaminofluorene and nati...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1979-12, Vol.76 (12), p.6076-6080
Main Authors: De Murcia, Gilbert, Lang, Marie-Claude E., Freund, Anne-Marie, Robert P. P. Fuchs, Daune, Michel P., Sage, Evelyne, Leng, Marc
Format: Article
Language:English
Subjects:
2-Acetylaminofluorene
2-Acetylaminofluorene - analogs & derivatives
Acetoxyacetylaminofluorene
Acetoxyacetylaminofluorene - immunology
Acetoxyacetylaminofluorene - metabolism
Antibodies
Bacteriocin Plasmids
Base Sequence
Binding Sites
Biochemistry
Biochemistry, Molecular Biology
Carcinogens
Deoxyguanine Nucleotides
DNA
DNA, Bacterial
DNA, Bacterial - metabolism
Electron micrographs
Electrophoresis
Electrophoresis, Agar Gel
Gels
Immunoglobulins
Life Sciences
Molecules
Structural Biology
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Summary:ColE1 DNA has been allowed to react in vitro with N-acetoxy-N-2-[$^{14}$C]acetylaminofluorene in the range of 0-15 N-2-[$^{14}$C]acetylaminofluorene residues bound per molecule of DNA, at the C8 of guanine residues. Purified rabbit antibodies to both N-2-(guanosine-8-yl)-acetylaminofluorene and native DNA that had reacted with N-acetoxy-N-2-acetylaminofluorene were shown by electron microscopy to recognize specifically the acetylaminofluorene-modified ColE1 DNA. The antibodies bound to DNA were visualized either per se or after reaction with goat anti-rabbit immunoglobulins coupled with ferritin. There was a linear relationship between the average number of antibodies bound per DNA molecule and the number of N-2-(deoxyguanosine-8-yl)-acetylaminofluorene residues per DNA molecule. The slope of this straight line was equal to 0.4. Due to the bivalence of the immunoglobulins one would expect a value of 0.5; we actually observed an important fraction of the bound antibodies crosslinking two parts of the same (or of another) DNA molecule.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.76.12.6076