Periodic Reporting for period 1 - RTEL1inHHS (Characterization of RTEL1 mutations in Hoyeraal-Hreidarsson Syndrome)
Reporting period: 2017-01-01 to 2018-12-31
Terc is the RNA component of telomerase, an enzyme that solves the end replication problem by extending telomere repeats, being therefore essential for stem cell renewal and tissue homeostasis. However, telomerase is a “double-edged sword” as its re-expression in ∼90% of all human cancers is sufficient to drive transformation and provide unlimited proliferative capacity. We made the unexpected discovery that telomerase is also the driver of telomere catastrophe in Rtel1-deficient cells. We establish that this pathological effect of telomerase results from its aberrant binding and stabilization of reversed replication forks within the telomere, which inhibits telomere replication. Once bound by telomerase, the only option to resolve the stalled replication fork is to recruit SLX1/4 to excise the offending DNA secondary structure, which results in dramatic consequences for the telomere. Our conclusion that telomerase binds inappropriately to reversed replication forks that form within telomeres in Rtel1-null cells is supported by PLA and RNAscope experiments, which revealed that telomerase binds aberrantly to telomeres in these cells. PLA for TERT-RAD51 also placed telomerase in close proximity to reversed replication forks in the absence of RTEL1. Furthermore, blocking fork reversal abolished aberrant telomerase binding to telomeres in Rtel1-null cells. In turn, this prevented the accumulation of SLX1/4 at telomeres, which catalyzes t-loop excision, overcame the toxic effect of telomerase in Rtel1−/− cells, and suppressed telomere dysfunction. We propose that blocking fork reversal prevents the fork from slowing and allows the replisome to replicate unimpeded through the telomere displacing the t-loop in its wake. The importance of fork reversal for this phenomenon is also supported by our analysis of fork restart activities in the context of Rtel1 and telomerase deficiency. Inhibition of fork restart mimics the pathological effect of telomerase and is sufficient to induce telomere dysfunction in Rtel1−/−Tert−/−cells. We hypothesized that binding of telomerase to the reversed fork would prevent efficient replication restart, possibly by outcompeting fork restart activities. In agreement with this possibility, analysis of replication dynamics at telomeres by SMARD demonstrated that telomerase inhibits active telomere replication in cells lacking RTEL1, whereas removing telomerase mitigates this effect. These results establish that telomerase binding to reversed replication forks is inhibitory for replication fork restart within telomeres. This work was published in the prestigious Cell journal (Margalef et al., 2018).
We believe that our research opens a full field for further exploration and offers the possibility that the same or similar mechanisms can be operating in other telomeric diseases or in other genomic unstable conditions.