Human CtIP promotes DNA end resection

Abstract

In the S and G2 phases of the cell cycle, DNA double-strand breaks (DSBs) are processed into single-stranded DNA, triggering ATR-dependent checkpoint signalling and DSB repair by homologous recombination. Previous work has implicated the MRE11 complex in such DSB-processing events. Here, we show that the human CtIP (RBBP8) protein confers resistance to DSB-inducing agents and is recruited to DSBs exclusively in the S and G2 cell-cycle phases. Moreover, we reveal that CtIP is required for DSB resection, and thereby for recruitment of replication protein A (RPA) and the protein kinase ATR to DSBs, and for the ensuing ATR activation. Furthermore, we establish that CtIP physically and functionally interacts with the MRE11 complex, and that both CtIP and MRE11 are required for efficient homologous recombination. Finally, we reveal that CtIP has sequence homology with Sae2, which is involved in MRE11-dependent DSB processing in yeast. These findings establish evolutionarily conserved roles for CtIP-like proteins in controlling DSB resection, checkpoint signalling and homologous recombination.

FIGURE 1. CtIP depletion causes hypersensitivity to DSB-inducing agents

a-c, CtIP down-regulation causes replication-dependent camptothecin (CPT) and etoposide hypersensitivity, and weak bleocin hypersensitivity. U2OS cells were pre-incubated with aphidicolin (Aph) where indicated, then treated for 1 h with CPT, etoposide or bleocin. CtIP-1 and CtIP-2 are two independent siRNAs. Survival data represent mean +/- standard error of mean (±SEM) from ≥3 independent experiments. d, e, Aphidicolin suppresses CPT- and etoposide-induced CtIP phosphorylation. Extracts from cells down-regulated for Luciferase or CtIP and treated with CPT or etoposide in the presence or absence of aphidicolin were immunoblotted as indicated. f, g, CtIP depletion impairs Chk1 and RPA phosphorylation but not Chk2 phosphorylation after CPT treatment. Asterisks in d-g: hyper-phosphorylated CtIP and RPA2.

FIGURE 2. CtIP associates with sites of DNA damage in S/G2 and promotes ATR recruitment to DSBs

a, b, CtIP recruitment to laser-induced DSBs occurs in S/G2. Cells were stained for endogenous γH2AX, CtIP or Cyclin A. All cells display local γH2AX signals but only S/G2 cells have pan-nuclear Cyclin A staining. c, GFP-CtIP co-localizes with RPA-ssDNA. Insets: higher magnifications. d, CtIP down-regulation impairs ATR recruitment to DNA damage. GFP-ATR expressing cells were treated, micro-irradiated and monitored (Supplementary Fig. 2c). GFP-ATR DSB tracks were manually scored 15 min after micro-irradiation for control and CtIP-depleted cells (296 and 264 cells, respectively). Data represent mean (±SEM) from two experiments. e, CtIP depletion does not affect γH2AX formation. Cells from d were fixed 15 min after micro-irradiation and immunostained.

FIGURE 3. CtIP depletion impairs DSB resection

a, b, CtIP is required for RPA recruitment to laser- and CPT-induced DSBs. Cells treated with control or CtIP siRNA were either micro-irradiated and 30 min later co-immunostained for γH2AX and RPA2, or were treated with CPT for 1 h and immunostained. c, CtIP depletion impairs ssDNA formation. After siRNA treatment, cells were tested for CPT-induced ssDNA formation by a non-denaturing BrdU staining procedure (see Methods and text for details). For each CPT-treated sample >100 cells were counted and the percentage exhibiting γH2AX foci, or both γH2AX and ssDNA foci, was determined (Supplementary Fig. 3a,b). Data represent the mean (±SEM) from two independent experiments.

FIGURE 4. CtIP interacts with MRN and promotes HR

a, MRN co-immunoprecipitates with CtIP. HeLa nuclear extract (HNE) was immunoprecipitated (IP) with pre-immune or anti-CtIP antibodies and analyzed by immunoblotting (IB). b, CtIP-MRN interaction after DNA damage and after BRCA1-depletion. Where indicated, cells were treated with 1 μM CPT for 1 h. Whole cell extracts (WCE) were immunoblotted directly or after immunoprecipitation. c, The CtIP C-terminus binds MRN. Bacterially-expressed fusions (arrows) were tested for binding MRN in HNE by immunoblotting. d, CtIP stimulates MR-dependent nuclease activity. PhiX174 substrate was incubated with MR (40 ng), BSA (200 ng) or CtIP (200 ng) in 5 mM MgCl₂ or MnCl₂, run on a agarose gel and stained with SYBR Gold. ssc: circular ssDNA. e, CtIP or MRE11 down-regulation impairs HR (see Methods and text for details). Data represent the mean (±SEM) from four independent experiments.

FIGURE 5. Function and evolutionary conservation of the CtIP C-terminus

a. Alignment of CtIP and Sae2 arising from BLAST-searches with the CtIP C-terminus (790-897). b-e, Deletion of the CtIP C-terminus impairs CtIP function. Three days after siRNA transfection, U2OS cells stably expressing GFP- tagged siRNA-resistant wild-type CtIP (WT) or a deletion mutant (1-789) were either treated with 1 μM CPT for 1 h and analyzed by immunoblotting or co-immunostaining, or were micro-irradiated and 30 min later co-immunostained as indicated. Asterisks in b and c: hyper-phosphorylated CtIP and RPA2.