Purification and characterization of a DNA-binding protein activated by ionizing radiation.
Exposure of mammalian cells to a variety of agents leads to the activation of pre-existing proteins and the induction of specific genes. We have recently described the appearance of a specific DNA-binding protein in nuclei from cells exposed to ionizing radiation (Singh, S. P., and Lavin, M. F. (1990) Mol. Cell. Biol. 10, 5279-5285). This protein is present in the cytoplasm of unperturbed cells and is apparently translocated to the nucleus in response to radiation damage. We describe here the purification and characterization of this specific DNA-binding protein. Purification involved the use of affinity chromatography employing a multimeric form of the DNA-binding motif conjugated to cyanogen bromide-activated Sepharose. Three DNA-binding species were recognized by UV-cross-linking and South-Western analysis. The major species or that with the highest affinity was approximately 70 kDa in size. DNase-1 footprint analysis revealed a single binding site in the kappa immunoglobulin gene enhancer and in a putative control sequence upstream from the c-myc gene. At salt concentrations as high as 1 M, up to 40% of the DNA-binding activity was maintained and the Kd was calculated to be 1.205 x 10(-6) M-1. Binding activity was found to be modulated by phosphorylation. Removal of phosphate groups from the protein resulted in a major loss of binding activity. It is not clear at this stage whether the factor(s) described here plays a role in transcription control or a more general DNA-processing role in response to radiation damage.
Teale
B
B
Queensland Institute of Medical Research, Bancroft Centre, Royal Brisbane Hospital, Herston, Australia.
Singh
S
S
Khanna
K K
KK
Findik
D
D
Lavin
M F
MF
D000469:Alkaline Phosphatase / Q000378:metabolism; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D002472:Cell Transformation, Viral; D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism; D016172:DNA Fingerprinting; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000528:radiation effects; D004591:Electrophoresis, Polyacrylamide Gel; D004742:Enhancer Elements, Genetic; D005803:Genes, Immunoglobulin; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D014466:Ultraviolet Rays
D004268:DNA-Binding Proteins; D007145:Immunoglobulin kappa-Chains; D004247:DNA; D000469:Alkaline Phosphatase