Accumulation of single-strand breaks is the major cause of telomere shortening in human fibroblasts

Telomere shortening triggers replicative senescence in human fibroblasts. T he inability of DNA polymerases to replicate a linear DNA molecule complete ly (the end replication problem) is one cause of telomere shortening. Other possible causes are the formation of single-stranded overhangs at the end of telomeres and the preferential vulnerability of telomeres to oxidative s tress. To elucidate the relative importance of these possibilities, amount and distribution of telomeric single-strand breaks, length of the G-rich ov erhang, and telomere shortening rate in human MRC-5 fibroblasts were measur ed. Treatment of nonproliferating cells with hydrogen peroxide increases th e sensitivity to S1 nuclease in telomeres preferentially and accelerates th eir shortening by a corresponding amount as soon as the cells proliferate. A reduction of the activity of intracellular peroxides using the spin trap alpha-phenyl-t-butyl-nitrone reduces the telomere shortening rate and incre ases the replicative life span. The length of the telomeric single-stranded overhang is independent of DNA damaging stresses, but single-strand breaks accumulate randomly all along the telomere after alkylation. The telomere shortening rate and the rate of replicative aging can be either accelerated or decelerated by a modification of the amount of oxidative stress. Quanti tatively, stress-mediated telomere damage contributes most to telomere shor tening under standard conditions. (C) 2000 Elsevier Science Inc.