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Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase
Published on: April 29, 2010
NBS1 mediates ATR-dependent RPA hyperphosphorylation following replication-fork stall and collapse
Karoline C Manthey1, Stephen Opiyo, Jason G Glanzer
1Department of Oral Biology, University of Nebraska Medical Center College of Dentistry and Nebraska Center for Cellular Signaling, Lincoln, NE 68583, USA.
Journal of Cell Science
|November 16, 2007
Summary
DNA replication requires protein phosphorylation, a process crucial for DNA repair and cell cycle checkpoints. This study reveals that NBS1 protein and its phosphorylation are essential for hydroxyurea-induced RPA phosphorylation, a key step in DNA damage response.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Post-translational protein phosphorylation regulates cellular processes, including responses to replication stress.
- Replication stress triggers phosphorylation of proteins involved in DNA replication, repair, and checkpoints, such as replication protein A (RPA).
Purpose of the Study:
- To investigate the role of NBS1 (NBN) and its phosphorylation in hydroxyurea (HU)-induced RPA phosphorylation.
- To identify the kinase responsible for RPA phosphorylation during DNA damage response.
Main Methods:
- Utilized phosphospecific and nonphosphospecific anti-NBS1 antibodies in HeLa cells.
- Employed stable transfection of NBS cells with empty vectors, phospho-mutant NBS1 (S343A, S278A/S343A), and wild-type NBS1.
- Assessed ATR retention on chromatin following DNA damage.
Main Results:
- HU-induced RPA hyperphosphorylation was blocked by anti-NBS1 antibodies.
- NBS cells expressing phospho-mutant NBS1 failed to hyperphosphorylate RPA in DNA-damage foci.
- ATR kinase dissociation from chromatin in NBS cells and cells with NBS1 phospho-mutants suggests ATR's role in RPA phosphorylation.
- Expression of a phospho-mutant RPA32 (RPA2) suppressed HU-induced apoptosis.
Conclusions:
- NBS1 and its phosphorylation are required for hydroxyurea-induced RPA hyperphosphorylation.
- ATR is likely the kinase responsible for RPA phosphorylation.
- RPA hyperphosphorylation is critical for the cellular response to DNA damage and prevention of apoptosis.
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