Functional and biochemical dissection of the structure-specific nuclease ARTEMIS

Pannicke U, Ma Y, Hopfner KP, Niewolik D, Lieber MR, Schwarz K
Source: EMBO J
Publication Date: (2004)
Issue: 23(9): 1987-1997
Research Area:
Immunotherapy / Hematology
Primary human dermal ARTEMIS-negative and ARTEMIS-positive fibroblasts were co-nucleofected with a RAG1 and RAG2 expression plasmid, a V(D)J recombination substrate plasmid, and expression plasmids for either wild-type or nine different mutant ARTEMIS proteins.
During V(D)J recombination, the RAG1 and RAG2 proteins form a complex and initiate the process of rearrangement by cleaving between the coding and signal segments and generating hairpins at the coding ends. Prior to ligation of the coding ends by DNA ligase IV/XRCC4, these hairpins are opened by the ARTEMIS/DNA-PKcs complex. ARTEMIS, a member of the metallo-beta-lactamase superfamily, shares several features with other family members that act on nucleic acids. ARTEMIS exhibits exonuclease and, in concert with DNA-PKcs, endonuclease activities. To characterize amino acids essential for its catalytic activities, we mutated nine evolutionary conserved histidine and aspartic acid residues within ARTEMIS. Biochemical analyses and a novel in vivo V(D)J recombination assay allowed the identification of eight mutants that were defective in both overhang endonucleolytic and hairpin-opening activities; the 5' to 3' exonuclease activity of ARTEMIS, however, was not impaired by these mutations. These results indicate that the hairpin-opening activity of ARTEMIS and/or its overhang endonucleolytic activity are necessary but its exonuclease activity is not sufficient for the process of V(D)J recombination.