53BP1 is associated with replication protein A and is required for RPA2 hyperphosphorylation following DNA damage.
p53-binding protein 1 (53BP1) acts as an 'adaptor/mediator' for transducing DNA damage signals, especially following detection of DNA double-strand breaks. In an effort to broaden our understanding of the protein network surrounding 53BP1, we isolated possible 53BP1 binding partners by co-immunoprecipitation, and identified them via tandem mass spectrometric analysis. The 53BP1-associated proteins included RPA1 and RPA2, two components of the replication protein A (RPA) complex. The presence of RPA components in the immunoprecipitates was confirmed by immunoblotting, and we found that the association between 53BP1 and RPA2 was disrupted following DNA damage induced by treatment with camptothecin, a topoisomerase I inhibitor. To investigate the functional meaning of the 53BP1 and RPA interaction, we established U2OS osteosarcoma cell lines stably expressing dominant-negative fragments of 53BP1. We found that camptothecin-induced RPA2 phosphorylation was inhibited in these cells, and also following 53BP1 knockdown by siRNA transfection. On the cellular level, camptothecin-induced apoptosis was augmented in the dominant-negative cell lines, resulting in increased chemosensitivity to this drug. Taken together, these results suggest that 53BP1 is involved in DNA damage-induced RPA2 hyperphosphorylation, and inhibition of 53BP1 function may sensitize cancer cells to camptothecin treatment.
Yoo
Eunjae
E
Research Institute, National Cancer Center, 809 Madu-dong, Ilsan-gu, Goyang, Gyeonggi 411-769, Korea.
Kim
Byung U
BU
Lee
Seung Youn
SY
Cho
Chae Hyun
CH
Chung
Jay H
JH
Lee
Chang-Hun
CH
D000972:Antineoplastic Agents, Phytogenic / Q000633:toxicity; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D002166:Camptothecin / Q000633:toxicity; D045744:Cell Line, Tumor; D004249:DNA Damage / Q000187:drug effects / Q000502:physiology*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013058:Mass Spectrometry; D012516:Osteosarcoma / Q000378:metabolism*; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation; D034741:RNA, Small Interfering; D051716:Replication Protein A; D014162:Transfection; D000071857:Tumor Suppressor p53-Binding Protein 1
D000972:Antineoplastic Agents, Phytogenic; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D034741:RNA, Small Interfering; C497167:RPA1 protein, human; D051716:Replication Protein A; C498301:TP53BP1 protein, human; D000071857:Tumor Suppressor p53-Binding Protein 1; D002166:Camptothecin