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First published online 14 November 2007
doi: 10.1242/jcs.004580

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NBS1 mediates ATR-dependent RPA hyperphosphorylation following replication-fork stall and collapse

Karoline C. Manthey¹, Stephen Opiyo², Jason G. Glanzer¹, Diana Dimitrova¹, James Elliott^3,* and Gregory G. Oakley^1,

¹ Department of Oral Biology, University of Nebraska Medical Center College of Dentistry and Nebraska Center for Cellular Signaling, Lincoln, NE 68583, USA
² Department of Agronomy and Horticulture, University of Nebraska-Lincoln, Lincoln, NE, USA
³ Department of Environmental Health, University of Cincinnati, Cincinnati, OH, USA

Author for correspondence (e-mail: goakley{at}unmc.edu)

Accepted 25 September 2007

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.

Key words: Phosphorylation, DNA-damage response, DNA repair, Replication-fork stall, Replication stress

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