Functional and biochemical dissection of the structure-specific nuclease ARTEMIS

EMBO J. 2004 May 5;23(9):1987-97. doi: 10.1038/sj.emboj.7600206. Epub 2004 Apr 8.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Cell Line
  • DNA Primers
  • DNA Repair / genetics*
  • DNA-Activated Protein Kinase
  • DNA-Binding Proteins / metabolism
  • Deoxyribonucleases / metabolism*
  • Endonucleases
  • Flow Cytometry
  • Gene Expression
  • Homeodomain Proteins / metabolism
  • Humans
  • Immunoblotting
  • Models, Molecular*
  • Mutagenesis, Site-Directed
  • Nuclear Proteins / chemistry
  • Nuclear Proteins / genetics*
  • Nuclear Proteins / metabolism*
  • Phosphorylation
  • Plasmids / genetics
  • Protein Structure, Tertiary
  • Protein-Serine-Threonine Kinases / metabolism
  • Recombination, Genetic / physiology*
  • Sequence Alignment

Substances

  • DNA Primers
  • DNA-Binding Proteins
  • Homeodomain Proteins
  • Nuclear Proteins
  • RAG2 protein, human
  • V(D)J recombination activating protein 2
  • RAG-1 protein
  • DNA-Activated Protein Kinase
  • PRKDC protein, human
  • Protein-Serine-Threonine Kinases
  • DCLRE1C protein, human
  • Deoxyribonucleases
  • Endonucleases