Sites in human nuclei where DNA damaged by ultraviolet light is repaired: visualization and localization relative to the nucleoskeleton

Abstract

The repair of damage induced in DNA by ultraviolet light involves excision of the damage and then repair synthesis to fill the gap. We investigated the sites of repair synthesis using MRC-5 fibroblasts and HeLa cells in G1 phase. Cells were encapsulated in agarose microbeads to protect them during manipulation, irradiated, incubated to allow repair to initiate, and permeabilized with streptolysin O to allow entry of labelled triphosphates; [32P]dTTP was incorporated into acid-insoluble material in a dose-dependent manner. Incubation with biotin-16-dUTP allowed sites of incorporation to be indirectly immunolabeled using a FITC-conjugated antibody; sites were not diffusely spread throughout nuclei but concentrated in discrete foci. This is similar to sites of S phase activity that are attached to an underlying nucleoskeleton. After treatment with an endonuclease, most repaired DNA electroeluted from beads with chromatin fragments; this was unlike nascent DNA made during S phase and suggests that repaired DNA is not as closely associated with the skeleton. However, the procedure destroyed repair activity, so repaired DNA might be attached in vivo through a polymerase that was removed electrophoretically. Therefore this approach cannot be used to determine decisively whether repair sites are associated with a skeleton in vivo.