REPAIR OF IONIZING-RADIATION DAMAGE IN PRIMATE ALPHA-DNA TRANSFECTED INTO RAT-CELLS

The time-course of repair of irradiated primate alpha-DNA was studied after transfection and recovery from rat NRK cells. Rat cells were chosen for transfection because they have no alpha-DNA. Plasmid pBUC4-alpha-10, containing 10 tandem 172 bp alpha-DNA subunits in its 5 kbp DNA, was irradiated and introduced into the rat cells by electroporation. The transfected alpha-DNA was then recovered from NRK nuclei free of extraneous rat DNA, permitting study of the fate of the transfected alpha-DNA in time-course experiments. Alpha-DNA continuously entered nuclei for processing in the first 2.5 h after transfection. The pool of damaged bases in alpha-DNA in NRK nuclei was detectable 2.5 h after transfection. Radiation-induced alpha-DNA fragments of electrophoretic mobility intermediate between those of unit nucleotide length were prominent in sequencing gel analyses of alpha-DNA for 5-150 min after transfection. These intermediate mobility fragments initially disappeared with T1/2 of 6-25 min; DNA fragments with 3'-phosphoryl termini disappeared with T1/2 of 10-20 min. The alpha-DNAs of intermediate mobility are presumed to be intermediates in DNA repair. Residual DNA base damage which had not been processed in the transfected cells could later be unmasked in vitro by conversion to strand breaks by beta-elimination using heat and piperidine or endonuclease III of E. coli. Irradiation of the recipient NRK cells with 5 Gy 4 hours before transfection prolonged the time during which intermediate mobility species could be found, consistent with the increased frequency of intermediate mobility species observed in DNA of monkey CV-1 cells pretreated with small doses of radiation before 300 Gy (Bases et al. 1990).