Investigating the role of RNase H enzymes in the regulation of telomeric R-loops during replicative senescence

Telomeres are nucleoprotein structures that protect and maintain the ends of eukaryotic linear chromosomes. Telomeres shorten at each round of DNA replication due to the end replication problem. The enzyme telomerase, by adding telomeric repeats to chromosome ends, can counteract this process. In the absence of telomerase, telomeres progressively shorten until they reach a critical length that activates the DNA damage response, thereby halting the cell cycle in a condition referred to as replicative senescence. Telomeres are transcribed into a long, non-coding RNA dubbed TERRA, which can hybridize with its template strand, thereby forming R-loops at S. cerevisiae and human telomeres. Recent data implicate telomeric R-loops in the promotion of homologous recombination at telomeres, leading to telomere lengthening events which can partially compensate for telomere shortening in the absence of telomerase. Telomeric R-loops are regulated by RNase H1 and H2 enzymes, which can degrade the RNA moiety of RNA-DNA hybrids. While the accumulation of telomeric R-loops in cells lacking both enzymes delays senescence onset by promoting homologous recombination at telomeres, the depletion of telomeric R-loops by overexpressing RNase H1 leads to premature senescence onset. This PhD thesis aims to better understand how telomeric R-loops are regulated especially during replicative senescence in S. cerevisiae. We found that RNase H2 localizes to long telomeres and physically interacts with the telomere-associated protein Rif2, which is required for RNase H2 recruitment to telomeres. Accordingly, in the absence of Rif2 telomeric R-loops accumulate, indicating that Rif2 and RNase H2 play a pivotal role in restricting R-loops at long telomeres. Importantly, the interaction between RNase H2 and Rif2 is strongest in late S phase, which is reflected in the degradation of telomeric R-loops in this time frame. We propose that this cell cycle regulated telomeric R-loop degradation is required to avoid collisions of the replication machinery, which replicates long telomeres in late S phase, with R-loops, an event that could have detrimental effects on telomere stability. It was previously shown that, as telomeres shorten, Rif2 localization to telomeres is diminished. We show that decreased Rif2 association to short telomeres leads to impaired recruitment of RNase H2, which is functionally reflected in the accumulation of R-loops at short telomeres. Moreover, while RNase H1 could not be detected at long telomeres, we observed its localization to short telomeres, thereby indicating a distinct requirement for the RNase H enzymes. By analyzing the effect of single RNase H enzymes deletion on the kinetics of senescence onset in telomerase negative cells, we revealed an opposing effect of the two enzymes, suggesting that, differently from what was proposed, RNase H enzymes do not have redundant functions at telomeres. In conclusion, we propose that, while at long telomeres R-loops are timely regulated by Rif2-RNase H2 to avoid collisions with the replication machinery, at short telomeres R-loops are allowed to accumulate, thereby promoting homologous recombination-mediated telomere extension