Repair Endonuclease-sensitive Sites in Daughter DNA of Ultraviolet-irradiated Human Cells

Abstract

WE have used cells that were derived from individuals with xeroderma pigmentosum (XP) and are defective in excision repair¹ to demonstrate the appearance of ultraviolet lesions in the DNA made after ultraviolet irradiation. Normal human and XP cells were irradiated and labelled with either ³H-thymidine or ³²PO₄ for 6–8 h, the label was removed, and the cells were further incubated for 18 h to allow for DNA chain elongation and joining of the short DNA segments synthesised after irradiation. The cells were then osmotically shocked, treated with a repair endonuclease contained in Micrococcus luteus extract for 5 min at 37° C and pH 8.0, and sedimented in 5–20% alkaline sucrose gradients. The enzymatic assay² and the sedimentation procedure³ have been described elsewhere. Figure 1 shows that daughter DNA from irradiated and non-irradiated normal human cells contains no repair endonuclease-sensitive sites. This is expected because normal human cells excise and repair most of the pyrimidine dinners formed after ultraviolet irradiation⁴. Figure 2 shows that DNA synthesised by irradiated XP cells does contain repair endonuclease-sensitive sites, although the number appears to be small. Under our experimental conditions there is approximately one sensitive site for every fifteen to thirty-five dimers in the parental DNA⁵.