Radioimmune Assay of Induction and Removal of uv Lesions in Total and Staphylococcal Nuclease-Resistant DNA of Mammalian Chromatin

Abstract

We have used a radioimmune assay (RIA) to measure the induction and removal of uv lesions in total (DNA) and staphylococcal nuclease-resistant DNA (DNA) of nuclei from uv-irradiated mammalian cells. Antibodies prepared against uv-irradiated single-stranded DNA were specific for uv-irradiated DNA. In Chinese hamster ovary (CHO) cells uv lesion induction was shown by RIA to be linear with dose in the range of 20 to 80 J/m/sup 2/. Lesion induction per unit DNA in the DNA/sub r/ of nuclei extracted from CHO and HeLa cells irradiated with 7 and 15 J/m/sup 2/ was 80% of the induction in the DNA/sub t/. This difference is insufficient to account for the two- to fourfold difference of repair synthesis reported for these fractions. There was no detectable removal of the lesions induced by 15 J/m/sub 2/ in CHO cells in 24 hr of repair incubation. In HeLa cells, however, 40 and 50% of the lesions induced by 15 J/m/sup 2/ of uv light were removed in 6 and 24 hr of repair incubation, respectively. Although the production of lesions is lower in DNA, than in DNA/sub t/, at 4 and 6 hr of repair incubation there are approximately equal numbers of lesions inmore » these DNAs of HeLa cells. This can be explained by (1) higher rate of removal of lesions in staphylococcal nuclease-sensitive DNA (DNAs) or (2) nucleosomal rearrangement during repair.« less