Phosphorylation-regulated binding of Ctp1 to Nbs1 is critical for repair of DNA double-strand breaks

Abstract

Repair of DNA double-strand breaks (DSBs) is critical for cell survival and for maintaining genome stability in eukaryotes. In Schizosaccharomyces pombe, the Mre11-Rad50-Nbs1 (MRN) complex and Ctp1 cooperate to perform the initial steps that process and repair these DNA lesions via homologous recombination (HR). While Ctp1 is recruited to DSBs in an MRN-dependent manner, the specific mechanism of this process remained unclear. We recently found that Ctp1 is phosphorylated on a domain rich in putative Casein kinase 2 (CK2) phosphoacceptor sites that resembles the SDTD repeats of Mdc1. Furthermore, phosphorylation of this motif is required for interaction with the FHA domain of Nbs1 that localizes Ctp1 to DSB sites. Here, we review and discuss these findings, and we present new data that further characterize the cellular consequences of mutating CK2 phosphorylation motifs of Ctp1, including data showing that these sites are critical for meiosis.

Figure 1

Basal phosphorylation of Ctp1 is carried out by Casein kinase 2. (A) Schematic of Ctp1 depicting conserved N-terminal coiled coil domain (CC), C-terminal core homology region (CXXC/RHR), and a CK2 phosphoacceptor motif (SXT). Motif enlargement shows residue substitutions in the ctp1-5A, ctp1-3A, ctp1-CK, and ctp1-3D strains. (B) ctp1-CK and ctp1-3D cells are sensitive to exogenous DNA damaging agents, similar to ctp1-3A cells. (C) Basal phosphorylation is equally altered in Ctp1-3A and Ctp1-CK relative to wild-type.

Figure 2

The ctp1-5A mutant phenocopies cellular defects observed in ctp1Δ. (A) ctp1-5A cells accumulate increased levels of spontaneous Rad22-YFP foci. (B) Asci from a ctp1-5A × ctp1 5A mating are abnormal. (C) Spore viability in ctp1-5A × ctp1-5A is very low.

Figure 3

CK2-mediated phosphorylation of Ctp1 facilitates interaction with Nbs1. (A) Ctp1 and Nbs1 associate in a manner dependent on phosphorylation of the Ctp1 SXT motif and the Nbs1 FHA domain. Strains were generated that express TAP-tagged Ctp1 and FLAG-tagged Nbs1 in an mre11-H134S background. IgG Sepharose beads were used to precipitate TAP-tagged Ctp1 and any associated proteins. (B) Yeast-two hybrid assays show that Ctp1 associates with Nbs1. Titrating increasing amounts of 3-AT reveals a specific interation between Ctp1 and Nbs1 that is FHA domain-dependent.