Functional and biochemical dissection of the structure-specific nuclease ARTEMIS.
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.
Pannicke
Ulrich
U
Institute for Clinical Transfusion Medicine and Immunogenetics, and Department of Transfusion Medicine, University of Ulm, Ulm, Germany.
Ma
Yunmei
Y
Hopfner
Karl-Peter
KP
Niewolik
Doris
D
Lieber
Michael R
MR
Schwarz
Klaus
K
D000595:Amino Acid Sequence; D002460:Cell Line; D017931:DNA Primers; D004260:DNA Repair / Q000235:genetics*; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism*; D004720:Endonucleases; D005434:Flow Cytometry; D015870:Gene Expression; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic / Q000502:physiology*; D016415:Sequence Alignment
D017931:DNA Primers; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; C499311:RAG2 protein, human; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; C426344:DCLRE1C protein, human; D003851:Deoxyribonucleases; D004720:Endonucleases