RPA phosphorylation facilitates mitotic exit in response to mitotic DNA damage.
Human replication protein A (RPA) becomes phosphorylated on the RPA2 subunit by cyclin B-Cdc2 during mitosis, although the functional role of this modification is unclear. We find that this modification stimulates RPA2 to become hyperphosphorylated in response to mitotic DNA damage caused by bleomycin treatment. Cells in which endogenous RPA2 was replaced by a mutant subunit lacking both Cdc2 sites had a significant defect in mitotic release into a 2N G(1) phase after exposure to bleomycin. An increased percentage of these mutant cells also was positive initially for cyclin B expression and BubR1 chromatin staining, indicative of an extended spindle assembly checkpoint. The mutant cells that experienced mitotic DNA damage also underwent apoptosis at higher levels than cells expressing the WT subunit. Even so, we did not find the mutation had any dramatic effects on the level of DNA repair in mitosis. Cells lacking ATM (a checkpoint factor and RPA2 kinase) also were severely defective in mitotic exit and were unable to support RPA hyperphosphorylation after mitotic DNA damage. Although checkpoint 1 effector kinase (Chk1) had a more complex role, inhibition of Chk1 activity with UCN-01 also reduced mitotic exit. Chk1 activation and mitotic RPA hyperphosphorylation were found to be independent events. Our results demonstrate that mitotic RPA hyperphosphorylation facilitates release of cells from a damaged mitosis into a 2N G(1) phase, thereby increasing cell viability.
Anantha
Rachel William
RW
Department of Biochemistry and New York University Cancer Institute, New York University School of Medicine, New York, NY 10016, USA.
Sokolova
Elena
E
Borowiec
James A
JA
D017209:Apoptosis; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D016213:Cyclins / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism; D016193:G1 Phase; D006801:Humans; D008938:Mitosis; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051716:Replication Protein A / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism
D018797:Cell Cycle Proteins; D016213:Cyclins; D004268:DNA-Binding Proteins; D051716:Replication Protein A; D025521:Tumor Suppressor Proteins; D011494:Protein Kinases; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D018844:Cyclin-Dependent Kinases