Repair of Ionizing Radiation Damage in Primate αDNA Transfected into Rat Cells

Abstract

The time-course of repair of irradiated primate αDNA was studied after transfection and recovery from rat NRK cells. Rat cells were chosen for transfection because they have no αDNA. Plasmid pBUC4α10, containing 10 tandem 172 bp αDNA subunits in its 5 kbp DNA, was irradiated and introduced into the rat cells by electroporation. The transfected αDNA was then recovered from NRK nuclei free of extraneous rat DNA, permitting study of the fate of the transfected αDNA in time-course experiments. αDNA continuously entered nuclei for processing in the first 2·5 h after transfection. The pool of damaged bases in αDNA in NRK nuclei was detectable 2·5 h after transfection.

Radiation-induced αDNA fragments of electrophoretic mobility intermediate between those of unit nucleotide length were prominent in sequencing gel analyses of αDNA for 5–150 min after transfection. These intermediate mobility fragments initially disappeared with T_½ of 6–25 min; DNA fragments with 3'-phosphoryl termini disappeared with T_½ of 10–20 min. The αDNAs 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 β-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).