ATR kinase is required for global genomic nucleotide excision repair exclusively during S phase in human cells

Abstract

Global-genomic nucleotide excision repair (GG-NER) is the only pathway available to humans for removal, from the genome overall, of highly genotoxic helix-distorting DNA adducts generated by many environmental mutagens and certain chemotherapeutic agents, e.g., UV-induced 6-4 photoproducts (6-4PPs) and cyclobutane pyrimidine dimers (CPDs). The ataxia telangiectasia and rad-3-related kinase (ATR) is rapidly activated in response to UV-induced replication stress and proceeds to phosphorylate a plethora of downstream effectors that modulate primarily cell cycle checkpoints but also apoptosis and DNA repair. To investigate whether this critical kinase might participate in the regulation of GG-NER, we developed a novel flow cytometry-based DNA repair assay that allows precise evaluation of GG-NER kinetics as a function of cell cycle. Remarkably, inhibition of ATR signaling in primary human lung fibroblasts by treatment with caffeine, or with siRNA specifically targeting ATR, resulted in total inhibition of 6-4PP removal during S phase, whereas cells repaired normally during either G(0)/G(1) or G(2)/M. Similarly striking S-phase-specific defects in GG-NER of both 6-4PPs and CPDs were documented in ATR-deficient Seckel syndrome skin fibroblasts. Finally, among six diverse model human tumor strains investigated, three manifested complete abrogation of 6-4PP repair exclusively in S-phase populations. Our data reveal a highly novel role for ATR in the regulation of GG-NER uniquely during S phase of the cell cycle, and indicate that many human cancers may be characterized by a defect in this regulation.

Fig. 1.

GG-NER of 6–4PPs in individual phases of the cell cycle in HDLFs. (A) Representative histogram overlay illustrating repair of 6–4PPs in each phase of the cell cycle in HDLF-shCTRL. (B) Bivariate distributions of 6–4PP (FITC; log scale) versus DNA content (PI; linear scale) in HDLF-shCTRL. (C) Graphical depictions of 6–4PP removal in HDLFs differing in p53 status and (D), in XPA-deficient HDSFs. Mean ± SEM from three independent experiments is shown. *, P < 0.05; two-tailed paired t test (S phase relative to G₁).

Fig. 2.

Pharmacological abrogation of signaling through ATR but not through ATM completely abolishes GG-NER of 6–4PPs in HDLFs during S but not during G₀/G₁ or G₂/M. (A) Representative results depicting cell cycle-specific 6–4PP repair in HDLFs treated with 10 mmol/l of caffeine; (top panel) Bivariate distributions of 6–4PP (FITC) versus DNA content (PI); (bottom panel) representative histogram overlay illustrating repair of 6–4PP. (B) Graphical depiction of 6–4PP repair in wild-type HDLFs treated or not with 10 mmol/l caffeine followed by irradiation with UV. (C) Graphical depictions of 6–4PP repair in wild-type HDLFs treated with 30 μmol/l wortmannin, and in the ATM-deficient cell line AG04405A, following irradiation with UV. Mean ± SEM from three independent experiments is shown. *, P < 0.05; two-tailed paired t test (S phase relative to G₁).

Fig. 3.

ATR is strictly required for GG-NER of 6–4PPs exclusively during S phase of the cell cycle in HDLFs. (A) Western blot showing expression of ATR (top panel), and immunostaining of γH2AX (bottom panel), 2 hours after irradiation with UV in HDLFs transfected with control siRNAs versus siRNAs targeting ATR. (B) Graphical depictions of 6–4PP repair in HDLFs transfected with control siRNAs or with siRNAs targeting ATR and irradiated with UV. Mean ± SEM from three independent experiments is shown. *, P < 0.05; two-tailed paired t test (S phase relative to G₁).

Fig. 4.

Removal of UV-induced DNA photoproducts is abrogated uniquely during S phase in ATR-deficient Seckel syndrome skin fibroblasts. (A) Immunostaining of ATR protein (top panel) and of γH2AX (bottom panel) 2 hours post-UV in Seckel syndrome F02–98 skin fibroblasts versus the closely related normal counterpart 1BR. (B) Graphical depiction of 6–4PP repair and (C) of CPD repair, in F02–98 versus 1BR. Mean ± SEM from three independent experiments is shown. *, P < 0.05; two-tailed paired t test (S phase relative to G₁).

Fig. 5.

Cell cycle-specific repair of UV DNA photoproducts in human tumor cell lines. Graphical depictions of 6–4PP repair in HDSFs and in various tumor strains irradiated with 25 J/m² of UV. Mean ± SEM of three independent experiments is shown. *, P < 0.05; two-tailed paired t test (S relative to G₁).