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Genes Cells. 2006 Mar;11(3):237-46.

Differential involvement of phosphatidylinositol 3-kinase-related protein kinases in hyperphosphorylation of replication protein A2 in response to replication-mediated DNA double-strand breaks.

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Department of Pathological Biochemistry, Medical Research Institute, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101-0062, Japan.


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.

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