screening of target kinases of AtATR

AtATRの基質キナーゼのにおけるスクリーニング

坂本 綾子; 根本 圭一郎*; 関 原明*; 篠崎 一雄*; 澤崎 達也*

Sakamoto, Ayako; Nemoto, Keiichiro*; Seki, Motoaki*; Shinozaki, Kazuo*; Sawasaki, Tatsuya*

ATR protein is a conserved member of phosphatidylinositol 3-kinase-related kinases involved in cell-cycle checkpoint responses. ATR is activated through DNA damage or stalled replication fork and subsequently phosphorylates downstream cell-cycle components to inhibit the progression of cell cycle. In Arabidopsis, ATR-disrupted mutant shows hypersensitive to DNA damaging treatments or replication inhibitors. However, either upstream or downstream signaling cascade of ATR largely remains unknown in plants. To identify downstream components involved in the ATR-mediated checkpoint responses, we screened Riken Arabidopsis Full-Length (RAFL) cDNA clones by using AlphaScreen luminescence system. Here, the AtATR protein with GST-FLAG tag was synthesized in wheat cell-free extract using synthesized mRNA transcribed , which is bound to protein A-conjugated acceptor beads through anti-FLAG antibody. Similarly, biotin-tagged putative kinases derived from RAFL cDNA clones were synthesized and bound to streptavidin-coated donor beads. When interaction between AtATR and a putative kinase happens, 520-620 nm emission light from acceptor bead induced by a singlet oxygen from donor bead becomes measurable. After screening of 759 clones, some candidate kinases showed higher luminescence signal than background level, indicating that they have protein-protein interactions with ATR.