Telomeres protect the ends of linear chromosomes from abnormal recombination events and buffer them against terminal DNA loss. Models of telomere replication predict that two daughter molecules have one end that is blunt, the product of leading-strand synthesis, and one end with a short G-rich 3′-overhang. However, experimental data from proliferating cells are not completely consistent with this model. For example, telomeres of human chromosomes have long G-rich 3′-overhangs, and the persistence of blunt ends is uncertain. Here we show that the product of leading-strand synthesis is not always blunt but can contain a long C-rich 5′-tail, the incompletely replicated template of the leading strand. We examined the presence of Grich and C-rich single-strand DNA in fibroblasts and HeLa cells. Although there were no significant changes in the length distribution of the 3′-overhang, the 5′-overhangs were mostly present in S phase. Similar results were obtained using telomerase-negative fibroblasts. The amount and the length distribution of the 5′ C-rich tails strongly correlate with the proliferative rate of the cell cultures. Our results suggest that, contrary to what has commonly been supposed, completion of leading-strand synthesis is inefficient and could well drive telomere shortening.
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