Study Of The Interaction Between SIK2and DNA-PKcs

Objective: The function of SIK2in irradiation damage repair was deduced by comparing the DNA repaircapacity post irradiation damage between HeLa-shNC cell and HeLa-shSIK2. The interaction betweenSIK2and DNA-PKcs was assayed in vivo and in intro to confirm their interaction relationship, map theinteraction region and identify the correlation between interaction and cell cycle.Methods: HeLa-shNC cells and HeLa-shSIK2cells were exposed to4Gy60Co γ ray, then SCGE (singlecell gel electrophoresis) was performed to detect their tail moment to speculate their DNA repair capacitypost irradiation damage. Initially the interaction between SIK2and DNA-PKcs was confirmed by cell coimmunoprecipitation (Co-IP). HeLa cells was synchronized by TdR double-blocking, then Co-IP wasperformed to screen their interaction intensity at2h,9h and12h after TdR release. The primers forconstructing SIK2and its truncated mutants were designed, and the gene fragment of SIK2, SIK2-Δ1（280~926）, SIK2-Δ2（400~926）, SIK2-Δ3（1~400） and SIK2-Δ4（700~926）were amplified bypolymerase chain reaction (PCR) and inserted into pGEX-4T-2vector to construct recombinant SIK2truncated plasmids fused with GST. The plasmids were transformed into E. coil BL21respectively andinduced with IPTG to overexpress GST fusioned protein. The recombinant proteins were proved byCoomassie Blue Staining and Western blot and GST pull-down assay was carried out to map the interactionregion of SIK2and DNA-PKcs.Results:1. SCGE shows that the tail moment of HeLa-shSIK2is longer than HeLa-shNC at1h and2h postirradiation, indicating that the DNA repair capacity of HeLa-shNC is stronger than that of HeLa-shSIK2post irradiation damage, which imples that SIK2participates in DNA repair post irradiation damage.2.Using TdR double-blocking to synchronize the cells in G1/S phase and release, co-immunoprecipitationwas performed to detected their interaction at S phase, G2/M phase and G1phase. The results shows thatthere is more immune complexes in G2/M phase and G1phase than that in S phase, which suggests thattheir interaction may be mainly occurs in G2/M and G1phase.3. SIK2and its truncated mutant plasmidswere constructed and overexpressed in E. coil BL21. Coomassie Blue Staining and Western blot shows thatGST-SIK1~926, GST-SIK280~926, GST-SIK400~926, GST-SIK1~400, GST-SIK700~926and GST are130KD,110KD,84KD,72KD,54KD and26KD respectively, consistent with their expected molecularsizes.4. When performing GST pull-down to map their interaction region with GST-fused SIK2truncatantsand endogenetic DNA-PKcs, we found that GST-SIK1~926, GST-SIK280~926and GST-SIK400~926caninteract with DNA-PKcs, but GST-SIK700~926cannot. Therefore, we speculate that the domain which canbind DNA-PKcs may be located in the PKA target domain of SIK2.Conclusions:1. SIK2participates in DNA repair post irradiation damage.2. SIK2can interact withDNA-PKcs, and the interaction mainly occurs in G2/M phase and G1phase.3. Recombinant plasmid ofSIK2and its truncated mutants were constructed and the GST fused recombinant proteins wereoverexpressed in E. coil BL21successfully.4. SIK2can be combined with DNA-PKcs, and the binding site is located in the PKA target domain of SIK2.