DNA-damage-induced degradation of EXO1 exonuclease limits DNA end resection to ensure accurate DNA repair

Abstract

End resection of DNA double-strand breaks (DSBs) to generate 3'-single-stranded DNA facilitates DSB repair via error-free homologous recombination (HR) while stymieing repair by the error-prone non-homologous end joining (NHEJ) pathway. Activation of DNA end resection involves phosphorylation of the 5' to 3' exonuclease EXO1 by the phosphoinositide 3-kinase-like kinases ATM (ataxia telangiectasia-mutated) and ATR (ATM and Rad3-related) and by the cyclin-dependent kinases 1 and 2. After activation, EXO1 must also be restrained to prevent over-resection that is known to hamper optimal HR and trigger global genomic instability. However, mechanisms by which EXO1 is restrained are still unclear. Here, we report that EXO1 is rapidly degraded by the ubiquitin-proteasome system soon after DSB induction in human cells. ATR inhibition attenuated DNA-damage-induced EXO1 degradation, indicating that ATR-mediated phosphorylation of EXO1 targets it for degradation. In accord with these results, EXO1 became resistant to degradation when its SQ motifs required for ATR-mediated phosphorylation were mutated. We show that upon the induction of DNA damage, EXO1 is ubiquitinated by a member of the Skp1-Cullin1-F-box (SCF) family of ubiquitin ligases in a phosphorylation-dependent manner. Importantly, expression of degradation-resistant EXO1 resulted in hyper-resection, which attenuated both NHEJ and HR and severely compromised DSB repair resulting in chromosomal instability. These findings indicate that the coupling of EXO1 activation with its eventual degradation is a timing mechanism that limits the extent of DNA end resection for accurate DNA repair.

Keywords: ATR; DNA double-strand break; DNA end resection; DNA repair; DNA-damage response; EXO1; Genomic stability; chromosomes; homologous recombination.

Figure 1.

EXO1 was rapidly degraded after DNA damage. a, HEK-293 cells were treated with CPT for the indicated times, and EXO1 protein levels were assessed by Western blotting. Phosphorylation of KAP-1 and p53 were assayed by Western blotting with phospho-specific antibodies to confirm DNA-damage induction. Actin served as a loading control. b, HEK-293 cells were pretreated with the proteasome inhibitor MG-132 or with DMSO as control for 4 h, then treated with CPT for the indicated times. EXO1 levels were assessed by Western blotting. c, HEK-293 cells were treated with DMSO, ionizing radiation (IR), CPT, okadaic acid, or calyculin A as indicated immediately after the addition of the protein synthesis inhibitor CHX. EXO1 levels were assessed by Western blotting at the indicated times after CHX treatment. The plot shows relative EXO1 protein levels after CHX treatment (y axis) versus time (x axis) as determined by scanning and quantifying EXO1 bands and normalizing to actin bands using ImageJ software. All experiments were replicated three times. Error bars depict S.E.

Figure 2.

Phosphorylation of EXO1 by ATR triggered its degradation. a, HEK-293 cells were treated with the ATM/ATR inhibitor caffeine or with DMSO as the control for 1 h before treatment with CPT for the indicated times. EXO1 protein levels were assessed by Western blotting. Phosphorylation of KAP-1 and p53 were assayed by Western blotting with phospho-specific antibodies to confirm DNA-damage induction. Phosphorylation of CHK1 was assessed to confirm ATR inactivation. b, HEK-293 cells were treated with the ATR inhibitor VE822 or with DMSO as the control for 1 h before treatment with CPT. EXO1 protein levels were assessed by Western blotting. c, EXO1 levels were assessed after CPT treatment of HEK-293 cells with siRNA-mediated knockdown of ATR. d, HEK-293 cells were depleted of endogenous EXO1 using siRNA and complemented with V5-tagged, siRNA-resistant wild-type EXO1 (WT) or EXO1 mutated at serine 714 (S714A). Cells were treated with CPT, and EXO1 levels were assessed by Western blotting with anti-V5 antibody. e, HEK-293 cells were depleted of endogenous EXO1 using siRNA and complemented with siRNA-resistant EXO1 mutated at all six SQ sites (Ser-432, Ser-652, Ser-674, Ser-676, Ser-694, and Ser-714) in its C-terminal domain (6A). EXO1 levels after CPT treatment were assessed by Western blotting. All experiments were replicated three times.

Figure 3.

EXO1 was ubiquitinated in a phosphorylation-dependent manner. a, HeLa cells expressing V5-tagged EXO1 (V5-EXO1) and His₆-tagged wild-type (WT) or conjugation-defective (ΔGG) ubiquitin were treated with CPT for the indicated periods of time. His₆-ubiquitin-conjugated proteins were captured under denaturing conditions using Ni²⁺-agarose beads, and ubiquitinated forms of EXO1 were detected by Western blotting with anti-V5 antibody. b, HeLa cells expressing V5-EXO1 and His-Ub were treated with caffeine or DMSO as control before the addition of CPT for 4 h. EXO1 ubiquitination was assessed by Ni²⁺-capture followed by Western blotting. c, HeLa cells expressing wild-type (WT) or phosphorylation site-mutant (6A) V5-EXO1 and His-Ub were treated with CPT for 4 h. EXO1 ubiquitination was assessed by Ni²⁺-capture followed by Western blotting. d, HeLa cells expressing V5-EXO1 were treated with the cullin-RING ubiquitin ligase inhibitor MLN4924 or DMSO for 4 h before the addition of CPT for 4 h. EXO1 ubiquitination was assessed by Ni²⁺-capture followed by Western blotting. e, HEK-293 cells were treated with DMSO or MLN4924 for 4 h before treatment with CPT for the indicated times. EXO1 protein levels were assessed by Western blotting. f, HeLa cells expressing V5-EXO1 and His-Ub in the presence or absence of dominant negative Cullin1 (DN-Cullin1) were treated with CPT for 4 h. EXO1 ubiquitination was assessed by Ni²⁺-capture followed by Western blotting. Cells were transfected with a V5-β-gal vector as control (Mock). g, HEK-293 cells expressing DN-Cullin1 were treated with CPT for the indicated times. EXO1 levels were assessed by Western blotting. All experiments were replicated three times.

Figure 4.

EXO1 degradation prevented hyper-resection and genomic instability. a, U2OS cells were depleted of endogenous EXO1 using siRNA and complemented with V5-tagged, siRNA-resistant wild-type (WT), or phosphorylation site mutant (6A)-EXO1. Cells were treated with CPT or DMSO as control for 4 h and immunostained with anti-RPA antibody (red). Nuclei were stained with DAPI (blue). Representative images are shown. The plot shows average numbers of RPA foci per cell after subtracting background (average numbers of foci in DMSO-treated cells). V5-EXO1 expression was verified by Western blotting with anti-V5 antibody. b, U2OS cells expressing WT- or 6A-EXO1 were treated with CPT or DMSO for 4 h and immunostained for BrdU/ssDNA foci (red). Representative images are shown. The plot shows average numbers of BrdU foci per cell after subtracting background. c, U2OS cells expressing WT- or 6A-EXO1 along with GFP-RPA were laser-micro-irradiated to induce focal DSBs. Time-lapse images of accumulation of GFP-RPA at DSBs (arrows) are shown. The plot shows recruitment of GFP-RPA after DNA damage. d, NHEJ efficiency was measured by quantifying RFP expression (by flow cytometry) in HEK293 cells harboring a GFP/RFP reporter after transfection with an I-SceI plasmid. Cells were depleted of endogenous EXO1 using siRNA and complemented with WT- or 6A-EXO1. The plot shows NHEJ-directed repair relative to WT-EXO1-expressing cells. e, HR efficiency was measured by quantifying GFP expression in HEK-293 cells harboring a DR-GFP reporter after transfection with an I-SceI plasmid. Cells were depleted of endogenous EXO1 using siRNA and complemented with WT- or 6A-EXO1. The plot shows HR-directed repair relative to WT-EXO1 expressing cells. f, U2OS cells depleted of endogenous EXO1 and complemented with WT- or 6A-EXO1 were treated with CPT and immunostained for 53BP1 foci (green) at the indicated times post treatment. Representative images are shown. Rates of DSB repair were determined by scoring 53BP1 foci and plotting average number of 53BP1 foci per cell (y axis) versus time (x axis). g, percentages of metaphase spreads with shattered chromosomes are plotted for CPT-treated U2OS cells with knockdown of endogenous EXO1 and ectopic expression of WT- or 6A-EXO1. Representative metaphases are shown. Scale bar, 5 μm. h, the plot shows clonogenic survival of U2OS cells expressing WT- or 6A-EXO1. Cells were treated with the indicated doses of CPT for 4 h before plating for colony formation. All experiments were replicated three times. Error bars depict S.E. **, p < 0.005; ****, p < 0.0001.

Figure 5.

Proposed model of EXO1 regulation. As cells enter S phase, EXO1 is primed to function in resection by CDK-mediated phosphorylation (circles). The transduction of DNA damage signals by ATR is followed by EXO1 recruitment to DNA breaks and DNA end resection, which promotes DSB repair by HR (23) and cell-cycle checkpoint implementation via ATR (16). Once resection is under way, EXO1 is rapidly ubiquitinated (triangles) and targeted for degradation to restrict its activity and prevent over-resection. Potential phosphorylation sites are numbered.