NBS1 Mediates ATR-Dependent RPA Hyperphosphorylation Following Replication-Fork Stall and Collapse

Authors

Karoline C. Manthey, University of Nebraska Medical Center College of Dentistry and Nebraska Center for Cellular Signaling, Lincoln, NE
Stephen Opiyo, University of Nebraska-LincolnFollow
Jason G. Glanzer, University of Nebraska Medical Center College of Dentistry and Nebraska Center for Cellular Signaling, Lincoln, NEFollow
Diana Dimitrova, University of Nebraska Medical Center College of Dentistry and Nebraska Center for Cellular Signaling, Lincoln, NE
James Elliott, University of Cincinnati, Cincinnati, OH, USA
Gregory G. Oakley, University of Nebraska Medical Center College of Dentistry and Nebraska Center for Cellular Signaling, Lincoln, NE

Document Type

Article

Date of this Version

2007

Comments

Published in Journal of Cell Science 120, 4221-4229. Copyright 2007. Used by permission.

Abstract

Post-translational phosphorylation of proteins provides a mechanism for cells to switch on or off many diverse processes, including responses to replication stress. Replication-stress-induced phosphorylation enables the rapid activation of numerous proteins involved in DNA replication, DNA repair and cell cycle checkpoints, including replication protein A (RPA). Here, we report that hydroxyurea (HU)-induced RPA phosphorylation requires both NBS1 (NBN) and NBS1 phosphorylation. Transfection of both phosphospecific and nonphosphospecific anti-NBS1 antibodies blocked hyperphosphorylation of RPA in HeLa cells. Nijmegen breakage syndrome (NBS) cells stably transfected with an empty vector or with S343A-NBS1 or S278A/S343A phospho-mutants were unable to hyperphosphorylate RPA in DNA-damage-associated foci following HU treatment. The stable transfection of fully functional NBS1 in NBS cells restored RPA hyperphosphorylation. Retention of ATR on chromatin in both NBS cells and in NBS cells expressing S278A/S343A NBS1 mutants decreased after DNA damage, suggesting that ATR is the kinase responsible for RPA phosphorylation. The importance of RPA hyperphosphorylation is demonstrated by the ability of cells expressing a phospho-mutant form of RPA32 (RPA2) to suppress and delay HU-induced apoptosis. Our findings suggest that RPA hyperphosphorylation requires NBS1 and is important for the cellular response to DNA damage.