Final Report Summary - D-END (Telomeres: from the DNA end replication problem to the control of cell proliferation)
To investigate telomere shortening mechanisms, we set up in Saccharomyces cerevisiae an accurate and time resolved analysis of telomeric structure. We found that the passage of the replication fork results in two asymmetric strands: one is generated by the lagging strand synthesis and is the same length as the parental strand; the second is generated by synthesis of the leading strand and is shortened by the length of the 3’ overhang. Only the latter is transiently processed by 5’-to-3’ resection and re-synthezised to generate the G-overhang. We have thus provided experimental evidence for many steps of the longstanding model of the DNA end replication problem. Combining mathematical modeling and formal genetics we have also provided evidence for the first telomere reaching a critical short length being the major determinant of the onset of senescence in budding yeast by the accumulation of ssDNA. In addition, we uncovered a role of replication stress response at telomeres, through Rad5, a factor of the DNA damage tolerance pathway involved in the replication of difficult templates. In order to have more insights into the cellular responses to such a critical short telomere, we then focused on the dynamics of replicative senescence at single cell level. We set up a microfluidics device and tracked down the kinetics of consecutive cell divisions from telomerase inactivation to cell death. We found that most lineages are characterized by a sharp on/off transition, consistent with a single event controlling senescence onset, as revealed by mathematical modeling. To our surprise, we also observed the existence of a cryptic noncanonical route leading to replicative senescence characterized by early, transient and stochastic cell cycle delays. These cells may represent the archetype cancer cell precursor—persisting in populations with increasing genomic instability. Taken altogether, our results contributed to the understanding of the mechanisms of telomere replication and shortening as a molecular clock to control cell fate.