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

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 G₀/G₁ or G₂/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.