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The creation of new telomeres was studied by generating a site-specific double-strand break in diploid strains of Saccharomyces cerevisiae that are unable to carry out homologous recombination. New telomere formation occurred approximately 1% of the time but only when (T2G4)13 was present proximal to the break site. About half of the healing events occurred at a number of 1- to 9-bp G or G, T sequences located as far as 128 bp distal to the T2G4 repeats. Surprisingly, in 16 events at sites ending in GTGG, the first TG1-3 nucleotides added always included either an 11- or a 13-bp sequence (GTGTGGGTGTG or GTGTGTGGGTGTG), after which each new telomere diverged into a less ordered TG1-3 pattern. Moreover, at 75% of the remaining addition sites, these same 11- or 13-bp sequences were found overlapping the junction between the chromosomal primer and the newly added sequences. We propose that short G, T sequences near an organizing sequence such as (T2G4)13 can act as primers to pair with the template RNA of a telomerase and add new sequences that are complementary to that RNA.

New telomeres in yeast are initiated with a highly selected subset of TG1-3 repeats.