RESUMO
Artemis is a mammalian protein, the absence of which results in SCID in Athabascan-speaking Native Americans (SCIDA). This novel protein has been implicated in DNA double-strand break repair and V(D)J recombination. We have cloned the Artemis murine counterpart, mArt, and generated a mouse with a targeted disruption of mArt. Artemis-deficient mice show a similar T-B- NK+ immunodeficiency phenotype, and carry a profound impairment in coding joint rearrangement, while retaining intact signal ends and close to normal signal joint formation. mArt-/- embryonic fibroblasts show increased sensitivity to ionizing radiation. Hemopoietic stem cell (HSC) transplantation using 500-5000 enriched congenic, but not allogeneic mismatched HSC corrected the T cell and partially corrected the B cell defect. Large numbers (40,000) of allogeneic mismatched HSC or pretreatment with 300 cGy of radiation overcame graft resistance, resulting in limited B cell engraftment. Our results suggest that the V(D)J and DNA repair defects seen in this mArt-/- mouse model are comparable to those in humans with Artemis deficiency, and that the recovery of immunity following HSC transplantation favors T rather than B cell reconstitution, consistent with what is seen in children with this form of SCID.
Assuntos
Transplante de Medula Óssea/imunologia , Rearranjo Gênico do Linfócito B/genética , Rearranjo Gênico do Linfócito T/genética , Marcação de Genes , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/imunologia , Animais , Diversidade de Anticorpos/genética , Diversidade de Anticorpos/efeitos da radiação , Transplante de Medula Óssea/patologia , Morte Celular/genética , Morte Celular/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem Celular , Linhagem Celular Transformada , Proteínas de Ligação a DNA , Endonucleases , Feminino , Rearranjo Gênico do Linfócito B/efeitos da radiação , Rearranjo Gênico do Linfócito T/efeitos da radiação , Marcação de Genes/métodos , Humanos , Regiões Constantes de Imunoglobulina/genética , Região de Junção de Imunoglobulinas/genética , Região de Junção de Imunoglobulinas/efeitos da radiação , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/biossíntese , Proteínas Nucleares/fisiologia , Tolerância a Radiação/genética , Tolerância a Radiação/imunologia , Receptores de Antígenos de Linfócitos T/genética , Imunodeficiência Combinada Severa/patologia , Imunodeficiência Combinada Severa/terapia , Transdução GenéticaRESUMO
The somatic hypermutation mechanism produces high-rate mutagenesis specifically targeted to rearranged immunoglobulin (Ig) variable (V) gene segments during the germinal center (GC) stage of B lymphocyte differentiation. The mechanism of this process remains uncertain, partly due to the lack of a direct assay for hypermutation activity. In this study, a gene-specific DNA repair assay was used to compare the rate and quality of DNA repair in the mantle zone (MZ) and GC B cells at rearranged and unrearranged Ig V genes. GC B cells were distinguished from MZ B cells by a retarded repair rate specific for rearranged Ig V genes. In addition, a unique feature of GC cells after DNA repair was the appearance of predominant mutations in rearranged Ig VH5 gene PCR products. These predominant mutations also occurred in natural mutants of VH5 genes. However, repair-associated mutations reflected, at least in part, "template-jumping" during amplification of the residually damaged genomic template. Overall, these findings reflect a repair abnormality associated with the hypermutation process by the criteria of sequence- and B cell stage-specificity. We conclude that locus-specific retardation of DNA repair is a component of the hypermutation mechanism. RFLP or SSCP analysis provides a simple assay to monitor this repair abnormality as a surrogate biochemical marker for hypermutation during B cell differentiation.