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Differential involvement of phosphatidylinositol 3‐kinase‐related protein kinases in hyperphosphorylation of replication protein A2 in response to replication‐mediated DNA double‐strand breaks
Author(s) -
Sakasai Ryo,
Shinohe Keitaro,
Ichijima Yosuke,
Okita Naoyuki,
Shibata Atsushi,
Asahina Kinji,
Teraoka Hirobumi
Publication year - 2006
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1111/j.1365-2443.2006.00942.x
Subject(s) - hyperphosphorylation , replication protein a , biology , dna replication , kinase , dna damage , microbiology and biotechnology , origin recognition complex , dna pkcs , eukaryotic dna replication , dna , protein kinase a , biochemistry , dna binding protein , gene , transcription factor
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.