Cell cycle-and proteasome-dependent formation of etoposide-induced replication protein A (RPA) or Mre11/Rad50/Nbs1 (MRN) complex repair foci.
In response to DNA damage, cells activate a complex protein network designed to sustain genomic integrity. Many of the proteins involved in the network form discrete repair foci, the composition of which is determined by the specific type of damage. Replication protein A (RPA) and the Mre11/Rad50/Nbs1 (MRN) complex both participate in foci and co-localize at certain types of lesions. Following etoposide (ETOP) treatment, cells form foci containing either RPA or the MRN complex, but not both. To investigate this preferential foci formation, we used cell cycle synchronization experimentation. We demonstrate that cells in S phase contain RPA foci but lack phospho-Nbs1 foci. This is consistent with RPA's role in homologous recombination repair of DNA double-strand breaks (DSBs), the predominant form of repair during S phase. Cells synchronized at G0/G1 phase contain phospho-Nbs1 foci, consistent with the MRN complex involvement in non-homologous end joining, the predominant form of repair in G1 phase. Treatment of cells with the proteasome inhibitor MG132 dramatically reduced the percentage of cells forming phospho-Nbs1 foci but did not alter the percentage of cells containing RPA or phospho-RPA foci. ETOP induced similar amounts of damage in all phases of the cell cycle as measured by the comet assay. These data suggest that in response to DNA DSBs, cell cycle-preferred repair pathways differentially engage RPA and the MRN complex in repair foci.
Robison
Jacob G
JG
Division of Nephrology and Hypertension, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039, USA.
Dixon
Kathleen
K
Bissler
John J
JJ
D016590:Aphidicolin / Q000494:pharmacology; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005047:Etoposide / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D051716:Replication Protein A / Q000378:metabolism*
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D007976:Leupeptins; D061988:Proteasome Inhibitors; C497167:RPA1 protein, human; D051716:Replication Protein A; D016590:Aphidicolin; D005047:Etoposide; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde