DNA end resection is needed for the repair of complex lesions in G1-phase human cells

Abstract

Repair of DNA double strand breaks (DSBs) is influenced by the chemical complexity of the lesion. Clustered lesions (complex DSBs) are generally considered more difficult to repair and responsible for early and late cellular effects after exposure to genotoxic agents. Resection is commonly used by the cells as part of the homologous recombination (HR) pathway in S- and G2-phase. In contrast, DNA resection in G1-phase may lead to an error-prone microhomology-mediated end joining. We induced DNA lesions with a wide range of complexity by irradiation of mammalian cells with X-rays or accelerated ions of different velocity and mass. We found replication protein A (RPA) foci indicating DSB resection both in S/G2- and G1-cells, and the fraction of resection-positive cells correlates with the severity of lesion complexity throughout the cell cycle. Besides RPA, Ataxia telangiectasia and Rad3-related (ATR) was recruited to complex DSBs both in S/G2- and G1-cells. Resection of complex DSBs is driven by meiotic recombination 11 homolog A (MRE11), CTBP-interacting protein (CtIP), and exonuclease 1 (EXO1) but seems not controlled by the Ku heterodimer or by phosphorylation of H2AX. Reduced resection capacity by CtIP depletion increased cell killing and the fraction of unrepaired DSBs after exposure to densely ionizing heavy ions, but not to X-rays. We conclude that in mammalian cells resection is essential for repair of complex DSBs in all phases of the cell-cycle and targeting this process sensitizes mammalian cells to cytotoxic agents inducing clustered breaks, such as in heavy-ion cancer therapy.

Keywords: ATM, Ataxia telangiectasia mutated; ATR, Ataxia telangiectasia and Rad3-related; BLM, Bloom syndrome protein; BRCA1, breast cancer 1, early onset; CENP-F, centromere protein F; CtIP; CtIP, CTBP-interacting protein; DAPI, 4',6-diamidino-2-phenylindole; DSB, double strand break; EXO1; EXO1, exonuclease 1; FCS, fetal calf serum; HR, homologous recombination; IR, ionizing radiation; LET, linear energy transfer; MEF, mouse embryonic fibroblasts; MMEJ, microhomology-mediated end joining; MRE11; MRE11, meiotic recombination 11 homolog A; NHEJ, none homologous end joining; PARP, poly (ADP-ribose) polymerase; RAD51, DNA repair protein RAD51 homolog 1; RPA, replication protein A; WRN, Werner syndrome; complex DNA damage; double-strand break repair; kd, knockdown; resection in G1-phase; siRNA, small interfering RNA; ssDNA, single stranded DNA; wt, wild-type.

Figure 1.

DSB resection occurs in G1- and S/G2-cells upon high-LET irradiation. (A) Human osteosarcoma cells, U2-OS, and normal human fibroblasts, AG1522D, were irradiated with gold ions and fixed 1 h after irradiation. CENP-F immunostaining (red) was used to distinguish between G1- (CENP-F negative) and S/G2-cells (CENP-F positive). RPA immunostaining (green) served as a resection marker. DNA was counter stained with DAPI (blue). Scale bar: 10 μm. (B) Resection of DSBs caused by ionizing radiation is LET dependent. U2-OS, NFFhTERT, and AG1522D cells were irradiated with high-LET (LET ≥ 90 keV/μm; heavy ions) and low-LET irradiation (LET = 2 keV/ μm; X-rays). Cells were fixed 1 h post irradiation and immunostained for RPA (green) and CENP-F (red). DNA was counter stained with DAPI (blue). RPA positive irradiated cells were counted for CENP-F positive (S/G2) cells and CENP-F negative (G1) cells. Each data point represents one experiment, in which at least 50 G1- and S/G2-cells were analyzed. Error bar: binomial error. (C) Confluent normal human fibroblasts AG1522D were irradiated with uranium ions and fixed 1 h after irradiation. Immunostaining was performed against γH2AX (red) and phospho-RPA (green). DNA was counter stained with DAPI (blue). Scale bar: 10 μm. An analogous experiment was performed with lead ions and yielded comparable results.

Figure 2.

MRE11, CtIP, and EXO1 are important for resection of complex DSBs. (A) CtIP is recruited to DSBs in G1. U2-OS cells were irradiated with uranium ions and fixed 1 h after irradiation. Immunostaining was performed against CENP-F (green; cell cycle marker) and CtIP (red). DNA was counter stained with DAPI (blue). (B) The expression of CtIP, MRE11, and EXO1 was decreased by RNAi. DSB resection positive cells (RPA) were counted 1 h after low angle gold, lead, tin, or uranium-ion irradiation in G1 (CENP-F negative) and S/G2 (CENP-F positive) cells. Each bar represents the average of at least four independent experiments ± standard error of the mean (SEM). All knockdown treated samples have significantly less resection positive cells than mock knockdown samples (Student's t-test, p < 0.05). All single or double knockdown samples but CtIP/MRE11 knockdown in G1 show significantly more resection than the triple knockdown (Student's t-test, p < 0.01). (C) ATR is recruited to complex lesions in G1 in an EXO1 dependent manner. U2-OS ATR-GFP cells were depleted for EXO1 by RNAi, irradiated with gold ions, and fixed 1 h after irradiation. γH2AX served as a DSB marker (white) and CENP-F (red) as cell cycle marker. DNA was counter stained with DAPI (blue). Scale bar: 10 μm. Cells where ATR-GFP is recruited to DSBs were counted in G1- (CENP-F negative) and S/G2-cells (CENP-F positive). At least 60 G1- and S/G2-cells were analyzed. Error bar: binomial error of one experiment.

Figure 3.

Resection of complex DSBs is important for their repair and cell survival. (A) NFFhTERT cells depleted for CtIP or mock depleted were irradiated with 1.28 Gy X-rays or carbon ions (LET 170 keV/μm) and treated with Aphidicolin right after irradiation to prevent G1-cells from moving on to G2. Aphidicolin treatment was checked not to affect DSB repair (SI Figure S3).⁵⁰ S phase cells, recognized by their pan-nuclear γH2AX signal, were excluded from the analysis.⁵⁰ The DSB repair kinetics of G2- (CENP-F positive) and G1-cells (CENP-F negative) was monitored by counting γH2AX foci at several time points after irradiation. The numbers of γH2AX foci were normalized to the γH2AX foci number 15 min after irradiation, which served as the number of DSBs induced. Shown is the mean ± SEM foci number/nucleus of one experiment. At least 50 nuclei per data point were analyzed. (B) Survival upon induction of complex DSBs is disabled in resection impaired cells. Clonogenic survival assay of NFFhTERT cells depleted for CtIP or mock depleted by siRNA and irradiated with 0.5 Gy, 1.0 Gy, 2.0 Gy, or 4.0 Gy of photons or 0.64 Gy, 1.28 Gy, 2.56 Gy, or 3.84 Gy of carbon ions of increasing ionizing density. Shown is the mean ± SEM survival of three replicates.