A conserved Ctp1/CtIP C-terminal peptide stimulates Mre11 endonuclease activity

The Mre11-Rad50-Nbs1 complex (MRN) is important for repairing DNA double-strand breaks (DSBs) by homologous recombination (HR). The endonuclease activity of MRN is critical for resecting 5′-ended DNA strands at DSB ends, producing 3′-ended single-strand DNA, a prerequisite for HR. This endonuclease activity is stimulated by Ctp1, the Schizosaccharomyces pombe homolog of human CtIP. Here, with purified proteins, we show that Ctp1 phosphorylation stimulates MRN endonuclease activity by inducing the association of Ctp1 with Nbs1. The highly conserved extreme C terminus of Ctp1 is indispensable for MRN activation. Importantly, a polypeptide composed of the conserved 15 amino acids at the C terminus of Ctp1 (CT15) is sufficient to stimulate Mre11 endonuclease activity. Furthermore, the CT15 equivalent from CtIP can stimulate human MRE11 endonuclease activity, arguing for the generality of this stimulatory mechanism. Thus, we propose that Nbs1-mediated recruitment of CT15 plays a pivotal role in the activation of the Mre11 endonuclease by Ctp1/CtIP.

DNA double-strand breaks (DSBs) are potentially lethal lesions that threaten genomic integrity and cell viability. DSBs can occur spontaneously as a result of faulty DNA metabolism or by exposure to genotoxins. In eukaryotes, these DSBs have “dirty ends” that lack ligatable 3ʹ-hydroxyl/5ʹ-phosphate groups and are often firmly attached to proteins such as the Ku70-80 heterodimer and topoisomerases (1, 2). During meiosis, the topoisomerase-like Spo11 protein generates DSBs and remains covalently attached to the 5ʹ DNA ends (3). To enable further processing, these DSB ends must be converted to “clean” ends with 3ʹ-hydroxyl/5ʹ-phosphate groups properly exposed. This step is achieved by endonucleolytic cleavage, or clipping, by Mre11 (4–7).In mammals, the MRE11, RAD50, and NBS1 complex (Mre11-Rad50-Nbs1 MRN]), together with CtIP, is involved in the clipping reaction. MRE11 is the nuclease subunit that has both endonuclease and 3′-to-5′ exonuclease activities, but only the former is essential for clipping (4, 8–12). RAD50, a member of the Structural Maintenance of Chromosomes protein family, binds to MRE11 to form an (MRE11)₂-(RAD50)₂ ring structure (MR complex) (13–15). NBS1 binds to the MR complex via MRE11 to form the MRN complex (16). Homologs of CtIP include Ctp1 in Schizosaccharomyces pombe and Sae2 in Saccharomyces cerevisiae (17–19). Upon phosphorylation, these proteins physically interact with their cognate MRN complex via the N-terminal forkhead-associated domain of NBS1, leading to activation of the MRE11 endonucleolytic clipping activity (20–24). However, the mechanistic details underlying this activation have not yet been determined.Through biochemical reconstitution using fission yeast proteins, we made three key findings regarding how Ctp1 activates MRN. First, MRN activation is mediated by Ctp1 phosphorylation, which promotes the direct association of Ctp1 with the Nbs1 subunit of MRN. Second, the highly conserved extreme C terminus of Ctp1 retains the ability to promote the endonuclease activity of MRN. Strikingly, a synthetic polypeptide comprising the 15 amino acids from the extreme C terminus of Ctp1 was sufficient for the full activation of MRN. Third, we verified the evolutionary significance of these findings by demonstrating that the conserved C-terminal polypeptide of CtIP can also stimulate the endonuclease activity of human MRN. Together, our results strongly suggest that the Ctp1-promoted MRN activation mechanism consists of at least two fundamentally separable elements: phosphorylation-induced Ctp1-MRN association and activation of MRN by the C-terminal peptide of Ctp1. Thus, recruitment of the Ctp1 C terminus to MRN is likely pivotal in this activation mechanism.