Base Sequence Damage in DNA from X-irradiated Monkey CV-1 Cells

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Abstract
Two kinds of 3′-ends were detected in DNA scission fragments of highly repetitive primate component αDNA which were isolated from irradiated monkey CV-1 cells. The fragments' 3′-ends were characterized by 5′–32P-end labelling the DNA, followed by examination in high-resolution polyacrylamide gels under denaturing conditions. Hydrolysis of the labelled fragments' termini with exonuclease III of E. coli or by the 3′-phosphatase activity of T4 polynucleotide kinase generated a third, slowest migrating species in each mobility size class. Reference to mobility size class standards makes it highly probable that the fragment ends generated by X-rays in cells are 3′-phosphoryl and 3′-phosphoglycolate, and that they are converted to slower migrating fragments with 3′-OH ends, similar to results obtained with DNA irradiated in water (Henner et al. 1982, 1983 a, b). Densitometer measurements of gel autoradiograms showed that X-ray induction of DNA fragments with 3′-phosphoryl and 3′-phosphoglycolate ends was dose-dependent over a range 100–900 Gy. In CV-1 cells the frequency of single-strand breaks in αDNA was 8·6 × 10−7 breaks/nt/Gy. The two kinds of ends disappeared in post-radiation incubation with a half-time of 1·6 h. These results provide a new means to study X-ray damage and repair of specific sequences in animal cells.