PREFERENTIAL ACCUMULATION OF SINGLE-STRANDED REGIONS IN TELOMERES OF HUMAN FIBROBLASTS

We have demonstrated recently that chronic hyperoxic treatment acceler ates the rate of aging of fibroblasts and the rate of telomere shorten ing in parallel. It was hypothesized that accelerated telomere shorten ing is due to preferential accumulation of oxidative damage in telomer es. To test this hypothesis, we measured the accumulation of sites sen sitive to S1 nuclease treatment in telomeres, in minisatellites, and i n the bulk of the genome of fibroblasts under different models of oxid ative stress as well as after treatment with the alkylating agent, N-m ethyl-N'-nitro-N-nitrosoguanidine. A comparison with qualitative data obtained by alkaline electrophoresis reveals that the sites transferre d to double-strand breaks by treatment with low concentrations of S1 n uclease are, in fact, single-stranded regions in the DNA. These region s may resemble single-stranded overhangs, gaps, or conventional single -strand breaks. The frequency of singlestranded regions is significant ly higher in telomeres than in minisatellites or in the bulk of the ge nome under all conditions tested. Those regions induced in minisatelli tes or in the overall genome by a bolus dose of hydrogen peroxide are completely repaired within 24 h. On the other hand, 50 +/- 12% of H2O2 -induced single-stranded regions remain unrepaired for at least 19 day s in telomeres of human fibroblasts, leading to a significant increase of the telomeric steady-state level of these lesions. This preferenti al accumulation might significantly contribute to telomere shortening. (C) 1998 Academic Press.