Fibre autoradiography of repair and replication in DNA from single cells: the effect of DNA synthesis inhibitors.
DNA fibre autoradiography, after incorporation of high specific activity 3H-thymidine and 3H-deoxycytidine, has been used to investigate repair in DNA fibres from single cells following UV, or methyl-methane sulphonate (MMS) treatment. Asynchronously growing human fibroblasts, leucocytes, and HeLa cells at different phases of the cell cycle have been investigated. Isotope incorporation in repair could be differentiated from that involved in replication by the distribution and density of silver grains along the DNA fibres. Grain distribution due to repair was continuous over long stretches of the fibres and was at a low density, occasionally interspersed with short slightly denser segments. Replication labelling on the other hand, was dense and usually in short tandem segments. Repair labelling was of a similar overall density in fibres from a single cell, but differed in intensity from cell to cell. In mutagen treated Go (leucocytes) or G1 (HeLa cells), repair labelling was not increased by the presence of the DNA inhibitors, hydroxyurea (HU) or 5-fluorodeoxyuridine (FUdR). Repair was not detectable in S cells however, without the use of these inhibitors to reduce endogenous nucleoside production. FUdR enhanced the repair labelling in S cells only slightly, while HU increased it beyond that observed in UV irradiated, HU treated, G1 cells. The intensity of repair labelling in fibres from mutagen treated S cells appears to be proportional to the degree of reduction of DNA chain elongation in replicons.
D001345:Autoradiography / Q000379:methods*; D002453:Cell Cycle / Q000187:drug effects; D002478:Cells, Cultured; D004260:DNA Repair / Q000187:drug effects / Q000528:radiation effects; D004261:DNA Replication / Q000187:drug effects / Q000528:radiation effects; D005347:Fibroblasts; D005467:Floxuridine / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D007962:Leukocytes; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D014466:Ultraviolet Rays
D005467:Floxuridine; D008741:Methyl Methanesulfonate; D006918:Hydroxyurea