Telomerase-dependent generation of 70-nt-long telomeric single-stranded 3′ overhangs in yeast

Telomeres, the chromatin structures at the ends of eukaryotic chromosomes, are essential for chromosome stability. The telomere terminates with a TG-rich 3′ overhang, which is bound by sequence-specific proteins that both protect the end and regulate the telomerase elongation process. Here, we demonstrate the presence of 3′ overhangs as long as 200 nt in asynchronously growing cells of the budding yeast Saccharomyces castellii. The 3′ overhangs show a wide distribution of 14–200 nt in length, thus resembling the distribution found in human cells. A substantially large fraction of the 3′ overhangs resides in the 70–200 nt range. Remarkably, we found an accumulation of a distinct class of 70-nt-long 3′ overhangs in the S phase of the cell cycle. Cells without a functional telomerase showed the same wide distribution of 3′ overhangs, but significantly, lacked the specific fraction of 70-nt 3′ overhangs. Hence, our data show that the highly defined 70-nt 3′ overhangs are generated by a telomerase-dependent mechanism, which is uncoupled to the mechanisms producing the bulk of the 3′ overhangs. These data provide new insights that will be helpful for deciphering the complex interplay between the specialized telomere replication machinery and the conventional DNA replication.