br Materials and methods br

Materials and methods
Results
Discussion DNA-PKcs and the Ku heterodimer (Ku70 and Ku80) constitute the DNA-PK complex, which is a serine/threonine kinase. DNA-PK phosphorylates H2AX and other substrates, such as main NHEJ factors (Artemis, XRCC4, and DNA ligase IV) [4]. Therefore, DNA-PK deficiency causes a decrease in NHEJ efficiency [5], [6]. In this study, however, we showed that aberrant DNA-PK causes a decrease in HR efficiency in Medaka embryonic cell lines (Fig. 3C and E). The core protein components of NHEJ include the Ku heterodimer, DNA-PKcs, XRCC4 and DNA ligase IV [4], [20], [21]. The DNA-PKcs, Ku heterodimer are recruited immediately to DSB sites and DNA-PK phosphorylates many substrates, including DNA-PKcs itself [22]. DNA-PKcs is trans-autophosphorylated at the ABCDE cluster, which contains six close serine or threonine residues, including Thr2609, Ser2612, Thr2620, Ser2624, Thr2638, and Thr2647 [18], [22], [23]. In addition, it has been reported that the autophosphorylation of Thr2609 in the ABCDE cluster induces the dissociation of DNA-PK from DSB sites and other repair factors, including both NHEJ and HR repair factors, act on DSB sites [22], [23], [24]. XRCC4 and DNA ligase IV bind DNA-PK complex at the DSB sites, which results in the activation of the process for repair by NHEJ. Previous studies reported that Ku heterodimer, XRCC4 and Lig IV can form the complex at the DSB sites independently of DNA-PK activity [20], [21], and the complex inhibits end resection of the DSBs which is required for the process for repair by HR [25], [26]. These reports proposed a hypothesis that DNA-PK inhibition causes the retention of Ku heterodimer, DNA-PKcs, XRCC4 and DNA ligase IV complex at DSB sites, which prevents HR process by the inhibition of the end resection of DSBs. Therefore, the DNA-PK inhibition that prevents the dissociation of DNA-PK from DSB sites causes defects in both NHEJ and HR process [6], [27]. However, the hypothesis has not been tested in vivo. RIC1 ABT263 showed low level of DNA-PK activity and decrease in the number of phosphorylated DNA-PKcs (Thr2609) foci after γ-irradiation compared with CAB cells (Fig. 2, Fig. 4B). Furthermore, we previously reported that RIC1 showed delayed DSB repair in pre-early gastrula cells and embryonic cell lines [12], [13]. These results suggest that aberrant DNA-PK prevents DNA-PKcs autophosphorylation at Thr2609, which presumably induces the retention of the DNA-PK complex at DSB sites and inhibits both of the NHEJ and HR processes. 53BP1 is involved in classical NHEJ. Therefore, we predicted that the inhibition of NHEJ process suppresses 53BP1 foci formation. However RIC1 cells showed a slight increase in the number of 53BP1 foci after DSB compared with CAB cells (Fig. 4C). In addition, RIC1 cells showed a slight increase in NHEJ efficiency (Fig. 3D). Several studies reported that 53BP1 is involved with alternative NHEJ under the condition of DNA-PK dysfunction [18], [19]. Therefore, we suggest that aberrant DNA-PK not inhibit alternative NHEJ which is a complementary mechanism to classical NHEJ and HR. It is known that DNA-PK binds and activates many DSB repair factors. Therefore, the regulation of DSB repair mechanism by DNA-PK and direct (or indirect) involvement of DNA-PK in HR repair pathway is unclear yet. In this study, we showed that a low level of DNA-PK activity causes decreased H2AX phosphorylation and HR efficiency in fish cells. These results are comparable to previous reports in mammals. Furthermore, our findings suggested that DNA-PK independent NHEJ repairs DSBs under the condition of decreased DNA-PK activity, which causes reduction of HR efficiency. Therefore, further analysis of the dynamics of DSB repair factors using Medaka mutants may reveal an in vivo molecular mechanism for the regulation of classical NHEJ, alternative NHEJ and HR by DNA-PK.
Acknowledgments We thank Dr Masayuki Hidaka for technical assistance. We also thank Dr Maria Jasin for providing the pDRGFP plasmid and Dr Jochen Dahm-Daphi for providing the pEJ plasmid. This publication was supported by a Grant-in-Aid for Scientific Research (s) (21221003) from the Ministry of Education, Sports, Science and Technology (MEXT) of Japan and by the FY2006 Ground-based Research Program for Space Utilization Research from the Japan Space Forum (to HM).