A method for detecting pyrimidine dimers in the DNA of bacteria irradiated with low doses of ultraviolet light.
Cells of Escherichia coli treated with lysozyme and Brij-58 become permeable to proteins, but do not release their DNA. I incubated permeable cells with an endonuclease that produces single-strand breaks in DNA-containing pyrimidine dimers. The enzyme entered the permeable cells, and, if they had been irradiated with ultraviolet light, caused breaks in their DNA. The frequency of breaks was estimated from the sedimentation pattern of the DNA in alkaline sucrose gradients. The procedure is sensitive enough to detect the dimers produced by a dose of 10 erg/mm(2) at 254 nm, or about 50 dimers per E. coli genome. This method exemplifies and extends the use of permeabilized cells for examining biological processes at the molecular level.
D002499:Centrifugation, Density Gradient; D003090:Coliphages / Q000201:enzymology; D004269:DNA, Bacterial / Q000032:analysis / Q000528:radiation effects*; D003902:Detergents; D004720:Endonucleases; D004926:Escherichia coli / Q000528:radiation effects; D004987:Ethers; D009113:Muramidase; D011092:Polyethylene Glycols; D011108:Polymers; D011743:Pyrimidines / Q000032:analysis*; D011830:Radiation Effects; D013941:Thymine; D014316:Tritium; D014466:Ultraviolet Rays
D004269:DNA, Bacterial; D003902:Detergents; D004987:Ethers; D011108:Polymers; D011743:Pyrimidines; D014316:Tritium; D011092:Polyethylene Glycols; D004720:Endonucleases; D009113:Muramidase; D013941:Thymine