title: Protein kinase mutants of human ATR increase sensitivity to UV and ionizing radiation and abrogate cell cycle checkpoint control.
abstract: In fission yeast, the rad3 gene product plays a critical role in sensing DNA structure defects and activating damage response pathways. A structural homologue of rad3 in humans (ATR) has been identified based on sequence similarity in the protein kinase domain. General information regarding ATR expression, protein kinase activity, and cellular localization is known, but its function in human cells remains undetermined. In the current study, the ATR protein was examined by gel filtration of protein extracts and was found to exist predominantly as part of a large protein complex. A kinase-inactivated form of the ATR gene was prepared by site-directed mutagenesis and was used in transfection experiments to probe the function of this complex. Introduction of this kinase-dead ATR into a normal fibroblast cell line, an ATM-deficient fibroblast line derived from a patient with ataxia-telangiectasia, or a p53 mutant cell line all resulted in significant losses in cell viability. Clones expressing the kinase-dead ATR displayed increased sensitivity to x-rays and UV and a loss of checkpoint control. We conclude that ATR functions as a critical part of a protein complex that mediates responses to ionizing and UV radiation in human cells. These responses include effects on cell viability and cell cycle checkpoint control.
authors.affiliation: Virginia Mason Research Center, 1000 Seneca Street, Seattle, WA 98101 and Department of Immunology, University of Washington School of Medicine, Seattle, WA 98195, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Wright
authors.forename: K S
authors.initials: KS
authors.lastname: Keegan
authors.forename: D R
authors.initials: DR
authors.lastname: Herendeen
authors.forename: N J
authors.initials: NJ
authors.lastname: Bentley
authors.forename: A M
authors.initials: AM
authors.lastname: Carr
authors.forename: M F
authors.initials: MF
authors.lastname: Hoekstra
authors.forename: P
authors.initials: P
authors.lastname: Concannon
mesh_terms: D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle / Q000235:genetics* / Q000528:radiation effects; D018797:Cell Cycle Proteins / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011494:Protein Kinases / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases; D011836:Radiation Tolerance / Q000235:genetics*; D012568:Schizosaccharomyces; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000502:physiology; D014466:Ultraviolet Rays
chemical_list: D018797:Cell Cycle Proteins; D016159:Tumor Suppressor Protein p53; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases