ATR KINASE ACTIVITY REGULATES THE INTRANUCLEAR TRANSLOCATION OF ATR AND RPA FOLLOWING IONIZING RADIATION

Sharon M. Barr, Cindy G. Leung, Elbert E. Chang and Karlene A. Cimprich*
Department of Molecular Pharmacology
Stanford University, Stanford, CA  94305-5174, USA
*cimprich@stanford.edu

INTRODUCTION 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 [1-4].  Examples of such proteins include the checkpoint kinase ATR (ATM and Rad3-related), which coordinates cell cycle transitions with DNA repair processes, as well as replication protein A (RPA), a multi-functional protein that is required in DNA replication and repair [1, 2]. 

METHOD We used a microscopy-based approach to investigate the mechanism of this translocation. We employed immunofluorescence to examine the localization of endogenous ATR and RPA in HeLa cells and in cells stably transfected to inducibly express Flag- tagged ATR or a kinase inactive mutant of ATR. 

RESULTS 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, potential sites of protein complex assembly and modification [5].  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, suggesting that the damage-induced translocation of both ATR and RPA is an active process that is regulated by ATR’s kinase activity.  We also find that the damage-induced translocation of RPA and the effects of ATR on this process are cell cycle-dependent. Specifically, RPA IRIF formation is enhanced in S and G2 phase in an ATR-dependent manner. These results and new, related findings will be presented. 

DISCUSSION Our data indicate that the intranuclear translocation of both RPA and ATR following irradiation are active processes regulated by the kinase activity of ATR. We propose that the regulation of RPA localization by ATR is indirect, rather than through direct phosphorylation, and suggest a model in which PML bodies could sequester certain proteins until checkpoint activation.

ACKNOWLEDGEMENTS This investigation was supported by grants from the ACS (RPG 99-241-01-CCG) and NIH (GM62193 and CA95357) to KAC, and by grants from the NCI, DHHS (PHS grant number CA09302) and the ACS PF-02-086-01-CCG to SB.

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