Fibre autoradiography of repair and replication in DNA from single cells: the effect of DNA synthesis inhibitors
Ockey, C.H.
European Journal of Cell Biology 27(1): 25-33
1982
ISSN/ISBN: 0171-9335 PMID: 6211354 Document Number: 190097
DNA fiber autoradiography, after incorporation of high specific activity 3H-thymidine and 3H-deoxycytidine, was used to investigate repair in DNA fibers from single cells following UV, or methyl methanesulfonate treatment. Asynchronously growing human fibroblasts, leukocytes, and HeLa cells at different phases of the cell cycle were investigated. Isotope incorporation in repair could be differentiated from that involved in replication by the distribution and density of Ag grains along the DNA fibers. Grain distribution due to repair was continuous over long stretches of the fibers and was at a low density, occasionally interspersed with short slightly denser segments. Replication labeling was dense and usually in short tandem segments. Repair labeling was of a similar overall density in fibers from a single cell, but differed in intensity from cell to cell. In mutagen treated Go (leukocytes) or G1 (HeLa cells), repair labeling was not increaed by the presence of the DNA inhibitors, hydroxyurea (HU) or 5-fluorodeoxyuridine (FUdR). Repair was not detectable in S cells without the use of these inhibitors to reduce endogenous nucleoside production. FUdR enhanced the repair labeling in D cells only slightly, while HU increased it beyond that observed in UV irradiated, HU treated, G1 cells. The intensity of repair labeling in fibers from mutagen treated S cells appears to be proportional to the degree of reduction of DNA chain elongation in replicons.