Gaps in DNA synthesized by ultraviolet light-irradiated WI38 human cells

DNA replication in ultraviolet-irradiated human cells was examined by treatment of the extracted DNA with a single-strand specific endonuclease from Neurospora crassa. WI38 cells were uniformly labeled with 32Pi for two generations before irradiation and then labeled with [3H]thymidine after irradiation. The isolated DNA was sedimented in neutral sucrose gradients after incubation with the endonuclease. The endonuclease treatment had no effect on the sedimentation profiles of either [32P]DNA or [3H]DNA from unirradiated control cultures. The endonuclease treatment also did not significantly alter the profile of [32P]DNA from irradiated cultures but did introduce breaks in the 3H pulse-labeled DNA synthesized after irradiation. These results indicate that DNA synthesis after ultraviolet irradiation proceeds in such fashion that gaps are formed along the newly made strand, leaving regions of single strandness in template DNA. As replication proceeds these gaps disappear and 2 h after irradiation (100-250 ergs/mm2) they are barely detectable by the endonuclease assay.