Protein Expression and Purification of NHEJ Factors

Ku70 and Ku80 were both cloned into pFastBac Dual vector (Thermo Fisher) and co-expressed in insect Sf9 cells. Ku70 contains an N-terminal hexahistidine tag followed by a TEV cleavage site. XLF and XRCC4 were individually cloned into pHAT5 vector [40 (link)] and transformed into Rosetta™2 (DE3) cells (Invitrogen). Both XLF and XRCC4 contain a C-terminal hexahistidine tag. Cell cultures were grown in LB medium until OD₆₀₀ was approximately 0.6. IPTG was added to a final concentration of 1 mM. Proteins were expressed at 16°C overnight for XLF and at 37°C for 4 hours for XRCC4. The PAXX and full-length LigaseIV-XRCC4 co-expression constructs are as described previously [11 (link), 41 ]. XLF_1-233 (C-terminal truncation mutant, which can not bind to Ku70/80) was cloned into pETG-41A (Gateway™ Destination vector, EMBL) and expressed as descripted before [42 (link)]. The catalytically-dead LigaseIV(K273A)-XRCC4 protein complex was generated from the LigaseIV-XRCC4 co-expression plasmid (A gift from Prof. Ming-Daw Tsai) by the QuikChange method (Agilent Technologies). Full-length Ligase IV fused with a hexahistidine tag at the C-terminus and ccXRCC4 (residue 138-213) were amplified from the Ligase IV-XRCC4 co-expression plasmid and an XRCC4 plasmid, of which cysteines were mutated to alanines, respectively and cloned into pRSFDuet1 vector (Novagen).