Ctp1-dependent clipping and resection of DNA double-strand breaks by Mre11 endonuclease complex are not genetically separable

Abstract

Homologous recombination (HR) repair of programmed meiotic double-strand breaks (DSBs) requires endonucleolytic clipping of Rec12Spo11-oligonucleotides from 5' DNA ends followed by resection to generate invasive 3' single-stranded DNA tails. The Mre11-Rad50-Nbs1 (MRN) endonuclease and Ctp1 (CtIP and Sae2 ortholog) are required for both activities in fission yeast but whether they are genetically separable is controversial. Here, we investigate the mitotic DSB repair properties of Ctp1 C-terminal domain (ctp1-CD) mutants that were reported to be specifically clipping deficient. These mutants are sensitive to many clastogens, including those that create DSBs devoid of covalently bound proteins. These sensitivities are suppressed by genetically eliminating Ku nonhomologous end-joining (NHEJ) protein, indicating that Ctp1-dependent clipping by MRN is required for Ku removal from DNA ends. However, this rescue requires Exo1 resection activity, implying that Ctp1-dependent resection by MRN is defective in ctp1-CD mutants. The ctp1-CD mutants tolerate one but not multiple broken replication forks, and they are highly reliant on the Chk1-mediated cell cycle checkpoint arrest, indicating that HR repair is inefficient. We conclude that the C-terminal domain of Ctp1 is required for both efficient clipping and resection of DSBs by MRN and these activities are mechanistically similar.