Rif2 interaction with Rad50 counteracts Tel1 functions in checkpoint signalling and DNA tethering by releasing Tel1 from MRX binding.

The yeast Rif2 protein is known to inhibit Mre11 nuclease and the activation of Tel1 kinase through a short motif termed MIN, which binds the Rad50 subunit and simulates its ATPase activity in vitro. The mechanism by which Rif2 restrains Tel1 activation and the consequences of this inhibition at DNA double-strand breaks (DSBs) are poorly understood. In this study, we employed AlphaFold-Multimer modelling to pinpoint and validate the interaction surface between Rif2 MIN and Rad50. We also engineered the rif2-S6E mutation that enhances the inhibitory effect of Rif2 by increasing Rif2-Rad50 interaction. Unlike rif2Δ, the rif2-S6E mutation impairs hairpin cleavage. Furthermore, it diminishes Tel1 activation by inhibiting Tel1 binding to DSBs while leaving MRX association unchanged, indicating that Rif2 can directly inhibit Tel1 recruitment to DSBs. Additionally, Rif2S6E reduces Tel1-MRX interaction and increases stimulation of ATPase by Rad50, indicating that Rif2 binding to Rad50 induces an ADP-bound MRX conformation that is not suitable for Tel1 binding. The decreased Tel1 recruitment to DSBs in rif2-S6E cells impairs DSB end-tethering and this bridging defect is suppressed by expressing a Tel1 mutant variant that increases Tel1 persistence at DSBs, suggesting a direct role for Tel1 in the bridging of DSB ends.

The multistep path to replicative senescence onset: zooming on triggering and inhibitory events at telomeric DNA.

Replicative senescence is an essential cellular process playing important physiological functions, but it is better known for its implications in aging, cancer, and other pathologies. One of the main triggers of replicative senescence is telomere shortening and/or its dysfunction and, therefore, a deep understanding of the molecular determinants is crucial. However, replicative senescence is a heterogeneous and hard to study process, especially in mammalian cells, and some important questions still need an answer. These questions concern i) the exact molecular causes triggering replicative senescence, ii) the role of DNA repair mechanisms and iii) the importance of R-loops at telomeres in regulating senescence onset, and iv) the mechanisms underlying the bypass of replicative senescence. In this review, we will report and discuss recent findings about these mechanisms both in mammalian cells and in the model organism Saccharomyces cerevisiae.

The DNA damage checkpoint: A tale from budding yeast.

Studies performed in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have led the way in defining the DNA damage checkpoint and in identifying most of the proteins involved in this regulatory network, which turned out to have structural and functional equivalents in humans. Subsequent experiments revealed that the checkpoint is an elaborate signal transduction pathway that has the ability to sense and signal the presence of damaged DNA and transduce this information to influence a multifaceted cellular response that is essential for cancer avoidance. This review focuses on the work that was done in Saccharomyces cerevisiae to articulate the checkpoint concept, to identify its players and the mechanisms of activation and deactivation.

To Fix or Not to Fix: Maintenance of Chromosome Ends Versus Repair of DNA Double-Strand Breaks.

Early work by Muller and McClintock discovered that the physical ends of linear chromosomes, named telomeres, possess an inherent ability to escape unwarranted fusions. Since then, extensive research has shown that this special feature relies on specialized proteins and structural properties that confer identity to the chromosome ends, thus allowing cells to distinguish them from intrachromosomal DNA double-strand breaks. Due to the inability of conventional DNA replication to fully replicate the chromosome ends and the downregulation of telomerase in most somatic human tissues, telomeres shorten as cells divide and lose this protective capacity. Telomere attrition causes the activation of the DNA damage checkpoint that leads to a cell-cycle arrest and the entering of cells into a nondividing state, called replicative senescence, that acts as a barrier against tumorigenesis. However, downregulation of the checkpoint overcomes this barrier and leads to further genomic instability that, if coupled with re-stabilization of telomeres, can drive tumorigenesis. This review focuses on the key experiments that have been performed in the model organism Saccharomyces cerevisiae to uncover the mechanisms that protect the chromosome ends from eliciting a DNA damage response, the conservation of these pathways in mammals, as well as the consequences of their loss in human cancer.