Sedimentation of DNA from Human Fibroblasts Irradiated with Ultraviolet Light: Possible Detection of Excision Breaks in Normal and Repair-Deficient Xeroderma Pigmentosum Cells

Usual alkaline sucrose techniques, which produce single-stranded DNA from unirradiated mammalian cells of <tex-math>$2-5\times 10^{8}$</tex-math> daltons (120-165 S) prove inadequate for resolution of strand breaks transiently present during excision repair of ultraviolet (uv) damage. These breaks seem to be relatively few in number and sedimentation changes are at the limit of resolution. Using a simple modification employing brief lysis in alkali, DNA with sedimentation coefficients up to 350 S have been obtained from human fibroblasts. 350-S DNA from unirradiated fibroblasts consists of complementary strands cosedimenting with choline-labeled material originating in the cytoplasm, in a conformation that allows renaturation to double-stranded DNA on neutralization after gradient fractionation. Large changes in sedimentation coefficient occur immediately after irradiation of normal fibroblasts with doses as low as <tex-math>$5-10\ {\rm ergs}/{\rm mm}^{2}$</tex-math>, but not xeroderma pigmentosum (XP) fibroblasts. The kinetics of the uv-induced changes in sedimentation of DNA from normal fibroblasts are consistent with the hypothesis that they are caused by strand breaks associated with excision repair that act as sites for strand separation in alkali. At various times after irradiation, strand breaks accumulate in DNA from XP (deSanctis-Cacchione) fibroblasts, suggesting either that these cells may possess uvspecific endonucleolytic activity but lack a later stage in repair or that breaks accumulate during cell death by action of nucleases which may be different from repair enzymes.