Characterization of the DNA double strand break repair defect in scid mice.
The scid mutation in CB-17 mice confers a profound immunodeficiency, resulting from an inability to rearrange immunoglobulin and T-cell receptor genes during lymphocyte development. Moreover, we and others have recently demonstrated in these scid mice a hypersensitivity to the lethal effects of ionizing radiation and a defect in DNA double strand break rejoining. In this report, we further characterize the radiosensitivity and repair defect in cells from scid mice. In order to determine whether scid cells were specifically sensitive to agents that produce double strand breaks, restriction enzymes RsaI and Sau3AI were introduced into scid and parental C.B-17 cells by electroporation. scid cells were 2-fold more sensitive than C.B-17 cells to both the blunt and the staggered end cuts produced by these restriction enzymes. However, the scid cells proficiently ligated both staggered and blunt ends of transfected plasmids. To determine whether the extent of DNA rejoining in scid cells was dependent on the initial dose of gamma-rays, final levels of DNA double strand break rejoining in scid and C.B-17 cells were quantitated by asymmetric field inversion gel electrophoresis. The results indicate an apparent difference in repair levels dependent on the dose of gamma-rays, ranging from 75% rejoining at 10 Gy to 40% rejoining at 50 Gy. In contrast, > 90% rejoining was observed in control C.B-17 cells at all doses. Delineating the links between these aberrant recombinational events, abnormal V(D)J recombination, and double strand break repair defects, will aid in the understanding of the basic mechanisms involved in these processes.
Chang
C
C
Department of Radiation Oncology, Stanford University School of Medicine, California 94305.
Biedermann
K A
KA
Mezzina
M
M
Brown
J M
JM
D000818:Animals; D001260:Ataxia Telangiectasia / Q000235:genetics; D002470:Cell Survival; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004247:DNA / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005720:Gamma Rays; D051379:Mice; D016513:Mice, SCID / Q000235:genetics*; D010957:Plasmids
D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D015252:Deoxyribonucleases, Type II Site-Specific; C024405:GATC-specific type II deoxyribonucleases; C040158:GTAC-specific type II deoxyribonucleases