Defective Artemis nuclease is characterized by coding joints with microhomology in long palindromic-nucleotide stretches.
T-B-NK+ severe combined immunodeficiency (SCID) is caused by a defect in V(D)J recombination. A subset of these patients has a mutation in one of the non-homologous end joining (NHEJ) genes, most frequently the Artemis gene. Artemis is involved in opening of hairpin-sealed coding ends. The low levels of residual DH-JH junctions that could be amplified from patients' bone marrow precursor B cells showed high numbers of palindromic (P)-nucleotides. In 25% of junctions, microhomology was observed in the P-nucleotide regions, whereas this phenomenon was never observed in junctions amplified from bone marrow precursor B cells from healthy controls. We utilized this difference between Artemis-deficient cells and normal controls to develop a V(D)J recombination assay to determine hairpin-opening activity. Mutational analysis of the Artemis gene confirmed and extended the mapping of an N-terminal nuclease active site, which contains several indispensable aspartate residues. C-terminal deletion mutants did not show such severe defects in the V(D)J recombination assay using transient overexpression of (mutated) Artemis protein. However, a C-terminal deletion mutation causes T-B-NK+ SCID, indicating that the Artemis C terminus is essential for V(D)J recombination at the normal Artemis expression level. The V(D)J recombination assays used in this study contribute to the diagnostic strategy for T-B-NK+ SCID patients.
van der Burg
Mirjam
M
Department of Immunology, Erasmus MC, Rotterdam, The Netherlands.
Verkaik
Nicole S
NS
den Dekker
Alexander T
AT
Barendregt
Barbara H
BH
Pico-Knijnenburg
Ingrid
I
Tezcan
Ilhan
I
vanDongen
Jacques J M
JJ
van Gent
Dik C
DC
D001665:Binding Sites; D002478:Cells, Cultured / Q000378:metabolism; D004268:DNA-Binding Proteins; D004720:Endonucleases; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain / Q000235:genetics*; D005816:Genetic Complementation Test; D006801:Humans; D007133:Immunoglobulin Joining Region / Q000235:genetics*; D007223:Infant; D009687:Nuclear Proteins / Q000737:chemistry* / Q000172:deficiency / Q000502:physiology; D009690:Nucleic Acid Conformation; D054448:Precursor Cells, B-Lymphoid / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011836:Radiation Tolerance / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D016511:Severe Combined Immunodeficiency / Q000175:diagnosis / Q000235:genetics*; D013329:Structure-Activity Relationship; D045567:VDJ Recombinases / Q000032:analysis* / Q000502:physiology
D004268:DNA-Binding Proteins; D007133:Immunoglobulin Joining Region; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D045567:VDJ Recombinases; C426344:DCLRE1C protein, human; D004720:Endonucleases