Discovery and Evolution of New Domains in Yeast Heterochromatin Factor Sir4 and Its Partner Esc1

International audienceSir4 is a core component of heterochromatin found in yeasts of the Saccharomycetaceae family, whose general hallmark is to harbor a three-loci mating-type system with two silent loci. However, a large part of the Sir4 amino acid sequences has remained unexplored, belonging to the dark proteome. Here, we analyzed the phylogenetic profile of yet undescribed foldable regions present in Sir4 as well as in Esc1, an Sir4-interacting perinuclear anchoring protein. Within Sir4, we identified a new conserved motif (TOC) adjacent to the N-terminal KU-binding motif. We also found that the Esc1-interacting region of Sir4 is a Dbf4-related H-BRCT domain, only present in species possessing the HO endonuclease and in Kluveryomyces lactis. In addition, we found new motifs within Esc1 including a motif (Esc1-F) that is unique to species where Sir4 possesses an H-BRCT domain. Mutagenesis of conserved amino acids of the Sir4 H-BRCT domain, known to play a critical role in the Dbf4 function, shows that the function of this domain is separable from the essential role of Sir4 in transcriptional silencing and the protection from HO-induced cutting in Saccharomyces cerevisiae. In the more distant methylotrophic clade of yeasts, which often harbor a two-loci mating-type system with one silent locus, we also found a yet undescribed H-BRCT domain in a distinct protein, the ISWI2 chromatin-remodeling factor subunit Itc1. This study provides new insights on yeast heterochromatin evolution and emphasizes the interest of using sensitive methods of sequence analysis for identifying hitherto ignored functional regions within the dark proteome

Mechanism of MRX inhibition by Rif2 at telomeres

International audienceSpecific proteins present at telomeres ensure chromosome end stability, in large part through unknown mechanisms. In this work, we address how the Saccharomyces cerevisiae ORC-related Rif2 protein protects telomere. We show that the small N-terminal Rif2 BAT motif (Blocks Addition of Telomeres) previously known to limit telomere elongation and Tel1 activity is also sufficient to block NHEJ and 5' end resection. The BAT motif inhibits the ability of the Mre11-Rad50-Xrs2 complex (MRX) to capture DNA ends. It acts through a direct contact with Rad50 ATP-binding Head domains. Through genetic approaches guided by structural predictions, we identify residues at the surface of Rad50 that are essential for the interaction with Rif2 and its inhibition. Finally, a docking model predicts how BAT binding could specifically destabilise the DNA-bound state of the MRX complex. From these results, we propose that when an MRX complex approaches a telomere, the Rif2 BAT motif binds MRX Head in its ATP-bound resting state. This antagonises MRX transition to its DNA-bound state, and favours a rapid return to the ATP-bound state. Unable to stably capture the telomere end, the MRX complex cannot proceed with the subsequent steps of NHEJ, Tel1-activation and 5' resection

Avelumab Versus Docetaxel in Patients With Platinum-Treated Advanced NSCLC: 2-Year Follow-Up From the JAVELIN Lung 200 Phase 3 Trial

International audienceAbstract Specific proteins present at telomeres ensure chromosome end stability, in large part through unknown mechanisms. In this work, we address how the Saccharomyces cerevisiae ORC-related Rif2 protein protects telomere. We show that the small N-terminal Rif2 BAT motif ( B locks A ddition of T elomeres) previously known to limit telomere elongation and Tel1 activity is also sufficient to block NHEJ and 5’ end resection. The BAT motif inhibits the ability of the Mre11-Rad50-Xrs2 complex (MRX) to capture DNA ends. It acts through a direct contact with Rad50 ATP-binding Head domains. Through genetic approaches guided by structural predictions, we identify residues at the surface of Rad50 that are essential for the interaction with Rif2 and its inhibition. Finally, a docking model predicts how BAT binding could specifically destabilise the DNA-bound state of the MRX complex. From these results, we propose that when an MRX complex approaches a telomere, the Rif2 BAT motif binds MRX Head in its ATP-bound resting state. This antagonises MRX transition to its DNA-bound state, and favours a rapid return to the ATP-bound state. Unable to stably capture the telomere end, the MRX complex cannot proceed with the subsequent steps of NHEJ, Tel1-activation and 5’ resection