Mechanism of MRX inhibition by Rif2 at telomeres

Abstract

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 (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.

Rif2 protects Rap1-depleted telomeres from MRX-mediated degradation in Saccharomyces cerevisiae

“…Surprisingly, the recruitment of neither Rif2 nor Sir3 is affected. Rif2 can interact with the Xrs2 and Rad50 subunits of the MRX complex ( Hirano et al, 2009 ; Hailemariam et al, 2019 ; Roisné-Hamelin et al, 2021 ), which binds to DNA ends and telomeres ( Oh and Symington, 2018 ); Rif2 can also interact directly with double-strand DNA ( Cassani et al, 2016 ; Hailemariam et al, 2019 ). Sir3 possesses multiple domains that can interact with histones ( Gartenberg and Smith, 2016 ).…”

“…Rif2 also inhibits MRX-mediated resection of telomeric DNA ends ( Bonetti et al, 2010a ; Bonetti et al, 2010b ). Rif2 inhibition of the MRX complex involves a direct interaction between the BAT/MIN motif of Rif2 with the ATPase domain of Rad50 ( Roisné-Hamelin et al, 2021 ; Khayat et al, 2021 ). Therefore, we hypothesized that Rif2 could be inhibiting MRX-mediated degradation of tlc1-tm telomeres.…”

Sir3 Heterochromatin Protein Promotes NHEJ by Direct Inhibition of Sae2

“…This dichotomy is conserved in mammalian cells in which NHEJ is prevented at telomeres, but not near them (van Steensel et al 1998;Muraki et al 2015). At yeast telomeres, a key NHEJ repressor is Sir4, which acts, at least in part, in a Sir3 independent manner (Marcand et al 2008;Roisné-Hamelin et al 2021;Khayat et al 2021). Sir4 thus seems to have two opposite functions in NHEJ regulation depending on chromosomic context: a strong repressive function at telomeres, and a stimulating function at subtelomeres.…”

Rif2 protects Rap1-depleted telomeres from MRX-mediated degradation inSaccharomyces cerevisiae

“…Surprisingly, the recruitment of neither Rif2 nor Sir3 is affected. Rif2 can interact with the Xrs2 and Rad50 subunits of the MRX complex (Hirano et al, 2009;Hailemariam et al, 2019;Roisné-Hamelin et al, 2021), which binds to DNA ends and telomeres (Oh & Symington, 2018); Rif2 can also interact directly with double-strand DNA (Cassani et al, 2016;Hailemariam et al, 2019). Sir3 possesses multiple domains that can interact with histones (Gartenberg & Smith, 2016).…”

“…7C), indicating that Rap1-independent recruitment of Rif2 to tlc1-tm sequence requires a DNA end. Since the MRX complex binds to DNA ends and telomeres (Oh & Symington, 2018), and Rif2 is known to interact with both Rad50 and Xrs2 (Hirano et al, 2009;Hailemariam et al, 2019;Roisné-Hamelin et al, 2021), Rif2 recruitment to tlc1-tm telomeres might require the MRX complex. Consistent with this hypothesis, we find that Rif2 association to the tlc1tm telomeres is lost in rad50∆ strains (Fig.…”