Asymmetrical end structures of leading and lagging telomeres in Saccharomyces cerevisiae dictate the nature of the end replication problem

The end replication problem refers to incomplete replication of parental DNA at telomeres, a process whose molecular depiction is hampered by the complex nature of telomere ends. Here, we recapitulate this process using a synthetic de novo telomere in Saccharomyces cerevisiae and delineate distinct molecular fates of telomere ends in vivo. We show that the lagging-strand telomeres carry a ∼10 nt 3' overhang, while the leading-strand telomeres have a Yku-protected blunt end, a feature that is common to native telomeres. Additionally, RNase H2 is primarily responsible for removing the terminal RNA primer. Consistently, the absence of RNase H2 activity results in the retention of the RNA primer on the lagging-strand telomere, which attenuates telomere erosion and delays senescence in telomerase-null cells. These findings highlight incongruent end structures on yeast telomeres and clarify that the primary culprit behind the end replication problem is the incompletely replicated lagging-strand telomere.