Radiation-activated DNA-binding protein constitutively present in ataxia telangiectasia nuclei.
We have recently described the appearance of a specific DNA-binding protein in nuclei from human cells exposed to ionizing radiation which was not detected in nuclear extracts from unperturbed cells (Singh, S. P., and Lavin, M. F. (1990) Mol. Cell. Biol. 10, 5279-5285). We report here a similar activity which is constitutively present in nuclei of both unirradiated and irradiated cells from patients with the human genetic disorder ataxia telangiectasia (A-T). Activity was present in unirradiated nuclear extracts from 3 A-T cell lines of different complementation groups, but was not detected or was present only at a low level in 4 controls. Active protein was detected in the cytoplasm of both cell types. Exposure to ionizing radiation did not change the amount of DNA binding activity in A-T nuclei but led to an increase in nuclei from 4 control cell lines. Purification of the binding activities from A-T nuclei and control cytoplasm was carried out by affinity chromatography, as described previously for control extracts (Teale, B., Singh, S. P., Khanna, K. K., Findik, D., and Lavin, M. F. (1992) J. Biol. Chem. 267, 10295-10301). Southwestern analysis and UV cross-linking confirmed the presence of a major DNA-binding species at 70 kDa in both cases with a minor binding activity at 47 kDa also evident. It was not possible to distinguish between the binding activities from A-T and control cells under different conditions, and phosphorylation was required for binding activity in both cases. Footprint analysis revealed that the same sequence was being recognized by the control and A-T proteins. The constitutive presence of a specific radiation-responsive DNA-binding protein in A-T cells may be indicative of a continuous state of stress in these cells.
Teale
B
B
Queensland Cancer Fund Research Unit, Queensland Institute of Medical Research, Bancroft Centre, Royal Brisbane Hospital, Herston, Australia.
Khanna
K K
KK
Singh
S P
SP
Lavin
M F
MF
D001260:Ataxia Telangiectasia / Q000378:metabolism*; D001483:Base Sequence; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000378:metabolism* / Q000528:radiation effects; D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000528:radiation effects*; D005720:Gamma Rays; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D013601:T-Lymphocytes
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D004247:DNA