In vitro function of ARTEMIS mutants. (A) DNA-PKcs assay of ARTEMIS and ARTEMIS mutants. Wild-type ARTEMIS and mutant ARTEMIS ARM27 were subjected to a DNA-PKcs phosphorylation assay in the absence and presence of exogenous 35 bp DNA. The positions of phosphorylated DNA-PKcs (p-DNA-PKcs) and phosphorylated ARTEMIS (p-ARTEMIS) are indicated. The ARTEMIS mutant ARM27 is indistinguishable from wild-type ARTEMIS in the ability of being phosphorylated by DNA-PKcs. (B) In vitro nuclease assay of ARTEMIS and ARTEMIS mutants. To analyze 5′ overhang processing and hairpin opening activities of ART-WT, ARM27, ARM12, and ARM14, a 20-bp hairpin with a 6 nt 5′ overhang was used as the substrate. The asterisks indicate the positions of the radioactive label. Wild-type ARTEMIS and mutant ARTEMIS proteins were incubated with the substrate indicated in the absence and presence of DNA-PKcs. Autoradiographs of sequencing gels are shown. M1 marks the position of the hairpin opening product if the hairpin was opened at the tip. Positions and sizes of the major hairpin opening and overhang processing products are indicated by arrows and numbers (in nt). The exonucleolytic product by ARTEMIS (1 nt) is indicated by “exo” adjacent to the size of the product. Diagrams adjacent to the arrows are depicted to show the cleavage positions in the substrate that result in the corresponding products. (C) A 21-bp DNA with a 15 nt 3′ overhang was used as the substrate. The asterisks indicate the positions of the radioactive label. Diagrams adjacent to the arrows are depicted to show the cleavage positions in the substrate that result in the corresponding products. M2 and M3 were generated by incubating the labeled strand of the 3′ overhang substrate with Klenow enzyme at room temperature for 30 and 60 minutes, respectively.