Understanding telomerase insufficiency at the single-cell level

Abstract

Telomeres are the ends of linear eukaryotic chromosomes essential for their stable maintenance. The conventional replication machinery is not able to copy linear DNA molecules to the very end. To overcome “the end replication problem” most eukaryotes rely on telomerase to maintain telomeres to extend back the shortened telomeres and maintain the protection of chromosome ends from fusions and degradation. However, in most stem and somatic cells in humans, the levels of telomerase are developmentally downregulated. The residual telomerase activity is insufficient to compensate for telomere attrition during replication. As a result, telomeres can become critically short, which would trigger activation of the DNA damage response and cell cycle arrest, followed by either senescence or apoptosis. This mechanism is considered a cell proliferation barrier evolved to suppress tumour growth.

In S.cerevisiae, telomerase is constitutively expressed, but the telomere shortening due to the loss of telomerase components also leads to a decrease in the proliferation capacity and frequent arrests. Here, I have engineered yeast cells with telomerase insufficiency (TI) to create a yeast model of human cell populations with downregulated telomerase. Using fluorescent microscopy, I followed the dynamics of the nucleus during mitosis and detected frequent nuclear missegregations in cells with TI. The genetic analysis of the missegregations demonstrated that the observed aberrant mitotic events were not caused by NHEJ or RAD51-dependent recombination at critically short telomeres, but they were dependent on the presence of the DNA damage checkpoint sensor Mec1, and its mediator Rad9. Similar nuclear missegregations were also observed in cells with the HO endonuclease-induced double-stranded breaks (DSBs) and in response to the DSB-inducing drug phleomycin. Therefore, activation of the DNA damage response correlated with the increased frequency of aberrant mitoses.

In response to DNA damage, two largely independent parallel pathways of the DNA damage checkpoint mediated by Chk1 and Rad53 are activated to prevent anaphase entry and mitotic exit. This study reports that mutation or deletion of components of these pathways and subsequently manipulating the efficiency of cohesion cleavage or inhibition of the mitotic exit network affected the probability of a cell cycle arrest to result in aberrant mitosis.

To summarise, these results suggest that nuclear divisions after the activation of the DNA damage checkpoint have an increased frequency of nuclear missegregations. These missegregations might be caused by an altered order of the key events during mitosis, where a delay in cohesin cleavage prevents the timely separation of sister chromatids and leads to the majority of DNA being inherited by either a mother or a daughter cell.