A Sensitive SV40 Viral Probe Assay for DNA Strand Breaks and Their Biological Repair in Higher Cells: Techniques and Preliminary Results

An alternative experimental approach to the investigation of intracellular repair of DNA strand breaks by mammalian higher cells has been developed using simian virus 40 (SV40), which has no known intrinsic DNA repair capacity and possesses a minichromosome structure, as an intracellular probe. In this approach unirradiated simian or human cells are infected with irradiated virus and incubated for varying periods. Nuclei are isolated, and viral DNA is extracted and assayed for residual damage. This assay involves separation of the viral DNA by agarose gel electrophoresis into three sharply demarcated bands corresponding to DNA molecules containing a double-strand break (DSB), single-strand breaks (SSB), or no breaks. Quantitative data are obtained by a combination of DNA hybridization with ^{32}{\rm P}-\text{labeled}$ SV40 DNA, autoradiography, and densitometry. Various experiments have been carried out to investigate the feasibility of this approach and to establish the optimal experimental conditions for its use. These experiments indicate that there is rapid and efficient cellular uptake of SV40, independent of prior radiation dose to the virus, and that this multistep experimental procedure gives excellent recovery and quantitation of the three DNA forms when compared with more direct methods of measurement. Radiation dose-response experiments with purified extracellular SV40 virus, using this approach, are quite reproducible and give results closely comparable to those obtained with techniques in current use. Initial time-course incubation experiments with SV40 infected CV-1 monkey kidney cells indicate that this approach can demonstrate slow but extensive intracellular repair of SSB; and limited presumptive early repair of DSB, followed by later and more extensive induction of DSB.