Age-dependent excision repair of damaged thymine from γ-irradiated DNA by isolated nuclei from human fibroblasts

Abstract

THE lifespan of diploid human fibroblasts in culture is limited¹, although there are substantial variations in lifespan with different culturing conditions and growth media^2–4. Before death, the cells pass through a period of senescence, characterised by numerous structural changes⁵. According to the error catastrophe hypothesis, a breakdown of the fidelity of the expression of genetic information resulting in the production of faulty proteins^6–11, and the accumulation of mutations in the genome¹², may represent important mechanisms of cellular ageing. It would be predicted that the deterioration of the quality of the genome would be accelerated by deficient or error-prone DNA repair. Several steps in the excision repair of damaged DNA residues have been demonstrated in mammalian cells exposed to exogenous physical or chemical agents. In the case of ionising radiation, the removal from the DNA of thymine damaged by γ rays was observed in Chinese hamster ovary¹³ and human WI-38 cells¹⁴, and the filling of the resulting gaps, indicated by repair replication or unscheduled synthesis, has been demonstrated in numerous cell lines¹⁵. Recently published experiments¹⁶ have demonstrated that the capacity of skin fibroblasts to perform ultraviolet-induced repair synthesis increases as a function of the lifespan of the donor species. We report that isolated nuclei or nuclear sonicates from senescent diploid human lung fibroblasts WI–38 have lost their ability to excise from DNA thymine damaged by γ rays.