Final Report Summary - D-END (Telomeres: from the DNA end replication problem to the control of cell proliferation) In eukaryotes, telomeres ensure the integrity of the genome by protecting chromosome ends. Telomeres also shorten with cell division and trigger cell-cycle arrest when reaching a critically short length, a process called replicative senescence. Hence, telomeres provide a molecular alarm clock for the enumeration of generation numbers and the control of cell proliferation. The homeostasis of many organs in humans depends on the proper operation of telomere shortening. This balance is broken in all cancer cells, which lengthen telomeres in a pathological manner. Conversely, abnormally short telomeres are the cause of a spectrum of lethal degeneration syndromes such as pulmonary fibrosis and failures of bone marrow. These telomeropathies of variable penetrance are currently incurable. The main focus of our project was the study of telomere shortening mechanisms, and their consequences for cell proliferation ability, namely, the control of replicative senescence. 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.
