title: ATR kinase activity regulates the intranuclear translocation of ATR and RPA following ionizing radiation.
abstract: Upon damage of DNA in eukaryotic cells, several repair and checkpoint proteins undergo a dramatic intranuclear relocalization, translocating to nuclear foci thought to represent sites of DNA damage and repair. Examples of such proteins include the checkpoint kinase ATR (ATM and Rad3-related) as well as replication protein A (RPA), a single-stranded DNA binding protein required in DNA replication and repair. Here, we used a microscopy-based approach to investigate whether the damage-induced translocation of RPA is an active process regulated by ATR. Our data show that in undamaged cells, ATR and RPA are uniformly distributed in the nucleus or localized to promyelocytic leukemia protein (PML) nuclear bodies. In cells treated with ionizing radiation, both ATR and RPA translocate to punctate, abundant nuclear foci where they continue to colocalize. Surprisingly, an ATR mutant that lacks kinase activity fails to relocalize in response to DNA damage. Furthermore, this kinase-inactive mutant blocks the translocation of RPA in a cell cycle-dependent manner. These observations demonstrate that the kinase activity of ATR is essential for the irradiation-induced release of ATR and RPA from PML bodies and translocation of ATR and RPA to potential sites of DNA damage.
authors.affiliation: Department of Molecular Pharmacology, Stanford University, California 94305-5174, USA.
authors.forename: Sharon M
authors.initials: SM
authors.lastname: Barr
authors.forename: Cindy G
authors.initials: CG
authors.lastname: Leung
authors.forename: Elbert E
authors.initials: EE
authors.lastname: Chang
authors.forename: Karlene A
authors.initials: KA
authors.lastname: Cimprich
mesh_terms: D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism / Q000528:radiation effects*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D004249:DNA Damage / Q000502:physiology* / Q000528:radiation effects; D004260:DNA Repair / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000528:radiation effects*; D006801:Humans; D007150:Immunohistochemistry; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011839:Radiation, Ionizing; D051716:Replication Protein A
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases