Differential involvement of phosphatidylinositol 3-kinase-related protein kinases in hyperphosphorylation of replication protein A2 in response to replication-mediated DNA double-strand breaks.
Replication protein A2 (RPA2), a component of the RPA heterotrimer, is hyperphosphorylated and forms nuclear foci in response to camptothecin (CPT) that directly induces replication-mediated DNA double-strand breaks (DSBs). Ataxia-telangiectasia mutated and Rad3-related kinase (ATR) and DNA-dependent protein kinase (DNA-PK) are activated by CPT, and RPA2 is hyperphosphorylated in a DNA-PK-dependent manner. To distinguish the roles of phosphatidylinositol 3-kinase-related protein kinases including DNA-PK, ataxia-telangiectasia mutated (ATM), and ATR, in the response to replication-mediated DSBs, we analyzed RPA2 focus formation and hyperphosphorylation during exposure to CPT. ATR knock-down with siRNA suppressed CPT-induced RPA2 hyperphosphorylation and focus formation. CPT-induced RPA2 focus formation was normally observed in DNA-PK- or ATM-deficient cells. Comparison between CPT and hydroxyurea (HU) indirectly inducing DSBs showed that RPA2 hyperphosphorylation is DNA-PK-dependent in CPT-treated cells and DNA-PK-independent in HU-treated cells. Although RPA2 foci rapidly formed in response to HU and CPT, the RPA2 hyperphosphorylation in HU-treated cells occurred later than in the CPT-treated cells, indicating that the DNA-PK dependency of RPA2 hyperphosphorylation is likely to be related to the mode of DSB induction. These results suggest that DNA-PK is responsible for the RPA2 hyperphosphorylation following ATR-dependent RPA2 focus formation in response to replication-mediated DSBs directly induced by CPT.
Department of Pathological Biochemistry, Medical Research Institute, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101-0062, Japan.
Ryo
R
Sakasai
Keitaro
K
Shinohe
Yosuke
Y
Ichijima
Naoyuki
N
Okita
Atsushi
A
Shibata
Kinji
K
Asahina
Hirobumi
H
Teraoka
D064007:Ataxia Telangiectasia Mutated Proteins; D002166:Camptothecin / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D004261:DNA Replication; D051747:DNA-Activated Protein Kinase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D007668:Kidney / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D051716:Replication Protein A / Q000235:genetics / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism
D064007:Ataxia Telangiectasia Mutated Proteins; D002166:Camptothecin; D018797:Cell Cycle Proteins; D004247:DNA; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D006918:Hydroxyurea; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D034741:RNA, Small Interfering; D051716:Replication Protein A; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; C576597:ATR protein, human