Sequential interactions with Mre11-Rad50-Nbs1 activate ATM/Tel1 at DNA double-strand breaks and telomeres

Abstract

AbstractThe Mre11-Rad50-Nbs1 (MRN) protein complex, CtIP/Ctp1/Sae2 and ATM/Tel1 kinase protect genome integrity through their functions in DNA double-strand break (DSB) repair, checkpoint signaling, and telomere maintenance. Nbs1 has a conserved C-terminal motif that binds ATM, but the full extent of ATM interactions with MRN are unknown. Here, we show that Tel1 overexpression in Schizosaccharomyces pombe restores Tel1 activity at DSBs and telomeres in the absence of Nbs1. This activity requires Mre11, indicating that Tel1 overexpression drives low affinity binding to the Mre11-Rad50 subcomplex. Mre11-Rad50 binds DSBs in nbs1Δ cells, and fusing the Tel1-binding motif of Nbs1 to Mre11 fully restores Tel1 signaling in these cells. Tel1 overexpression does not restore Tel1 signaling in cells carrying the rad50-I1192W mutation, which impairs the ability of Mre11-Rad50 to form the ATP-bound closed conformation. From these findings, we propose that Tel1 activation at DNA ends proceeds by a sequential mechanism initiated by high affinity binding to Nbs1 which recruits Tel1, followed by a low affinity interaction with Mre11-Rad50 in the closed conformation to activate Tel1.