Pobiega, S. | bioRxiv Science

bioRxiv2024

Restriction on Ku's Inward Translocation Caps Telomere Ends

Safeguarding chromosome ends against fusions via nonhomologous end joining (NHEJ) is essential for preserving genome integrity. Paradoxically, the conserved NHEJ core factor Ku binds telomere ends. How it is prevented from promoting NHEJ remains unclear, as does the mechanism that allows Ku to coexist with telomere-protective DNA binding proteins, e.g., Rap1 in Saccharomyces cerevisiae. Here, we reveal a direct role for Rap1 in the inhibition of Kus NHEJ function at telomeres. A single Rap1 molecule bound near a DNA end inhibits NHEJ in vivo without disrupting Ku presence. Consistent with this, Rap1 and Ku form a complex on short DNA duplexes in vitro. Cryo-EM and molecular modelling analysis of this complex shows that Rap1 obstructs Kus inward translocation on DNA - an essential step for NHEJ at broken ends. Nanopore sequencing of telomere fusions confirms the importance of this pathway in protecting native telomere ends. Collectively, our findings uncover a mechanism of telomere end protection mediated by restricting Kus inward translocation, a functional switch that prevents promiscuous NHEJ repair at telomeres.

molecular biology↗

bioRxiv2021

A New Assay Capturing Chromosome Fusions Shows a Protection Trade-off at Telomeres and NHEJ Vulnerability to Low Density Ionising Radiation

Chromosome fusions threaten genome integrity and promote cancer by engaging catastrophic mutational processes, namely chromosome breakage-fusion-bridge cycles and chromothripsis. Chromosome fusions are frequent in cells incurring telomere dysfunctions or those exposed to DNA breakage. Their occurrence and therefore their contribution to genome instability in unchallenged cells is unknown. To address this issue, we constructed a genetic assay able to capture and quantify rare chromosome fusions in budding yeast. This chromosome fusion capture assay (CFC) relies on the controlled inactivation of one centromere to rescue unstable dicentric chromosome fusions. It is sensitive enough to quantify the basal rate of end-to-end chromosome fusions occurring in wild-type cells. These fusions depend on canonical nonhomologous end-joining (NHEJ). Our results show that chromosome end protection results from a trade-off at telomeres between positive effectors (Rif2, Sir4, telomerase) and a negative effector partially antagonizing them (Rif1). The CFC assay also captures NHEJ-dependent chromosome fusions induced by ionising radiation. It provides evidence for chromosomal rearrangements stemming from a single photon-matter interaction.

molecular biology↗