SITES IN HUMAN NUCLEI WHERE DNA DAMAGED BY ULTRAVIOLET-LIGHT IS REPAIRED - VISUALIZATION AND LOCALIZATION RELATIVE TO THE NUCLEOSKELETON

The repair of damage induced in DNA by ultraviolet light involves exci sion of the damage and then repair synthesis to fill the gap. We inves tigated the sites of repair synthesis using MRC-5 fibroblasts and HeLa cells in G(1) phase. Cells were encapsulated in agarose microbeads to protect them during manipulation, irradiated, incubated to allow repa ir to initiate, and permeabilized with streptolysin O to allow entry o f labelled triphosphates; [P-32]dTTP was incorporated into acid-insolu ble material in a dose-dependent manner. Incubation with biotin-16-dUT P allowed sites of incorporation to be indirectly immunolabelled using a FITC-conjugated antibody; sites were not diffusely spread throughou t nuclei but concentrated in discrete foci. This is similar to sites o f 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 associ ated with the skeleton. However, the procedure destroyed repair activi ty, so repaired DNA might be attached in vivo through a polymerase tha t was removed electrophoretically. Therefore this approach cannot be u sed to determine decisively whether repair sites are associated with a skeleton in vivo.