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 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.
Auclair
Yannick
Y
Maisonneuve-Rosemont Hospital Research Center, Faculty of Medicine, University of Montreal, Montréal, Québec, Canada.
Rouget
Raphael
R
Affar
El Bachir
el B
Drobetsky
Elliot A
EA
D000015:Abnormalities, Multiple / Q000473:pathology; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D004247:DNA / Q000378:metabolism / Q000528:radiation effects; D004260:DNA Repair / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology* / Q000201:enzymology* / Q000528:radiation effects; D005434:Flow Cytometry; D015894:Genome, Human / Q000235:genetics*; D006801:Humans; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency / Q000378:metabolism*; D011740:Pyrimidine Dimers / Q000378:metabolism; D016196:S Phase / Q000528:radiation effects; D015398:Signal Transduction / Q000528:radiation effects; D012867:Skin / Q000473:pathology; D013577:Syndrome; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D014466:Ultraviolet Rays
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D011740:Pyrimidine Dimers; D025521:Tumor Suppressor Proteins; D004247:DNA; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases