NBS1 mediates ATR-dependent RPA hyperphosphorylation following replication-fork stall and collapse.
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.
Manthey
Karoline C
KC
Department of Oral Biology, University of Nebraska Medical Center College of Dentistry and Nebraska Center for Cellular Signaling, Lincoln, NE 68583, USA.
Opiyo
Stephen
S
Glanzer
Jason G
JG
Dimitrova
Diana
D
Elliott
James
J
Oakley
Gregory G
GG
D017304:Annexin A5 / Q000378:metabolism; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D004261:DNA Replication; D005347:Fibroblasts / Q000378:metabolism; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051716:Replication Protein A / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics; D013539:Simian virus 40 / Q000502:physiology; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection
D017304:Annexin A5; D018797:Cell Cycle Proteins; C112156:NBN protein, human; D009687:Nuclear Proteins; D051716:Replication Protein A; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases