Purification of an ultraviolet-inducible, damage-specific DNA-binding protein from primate cells.
A UV-inducible, damage-specific DNA-binding (DDB) protein with high affinity for double-stranded UV-irradiated DNA has been identified recently in monkey kidney (CV-1) cells (Hirschfeld, S., Levine, A. S., Ozato, K., and Protić, M. (1990) Mol. Cell. Biol. 10, 2041-2048). We have now purified the DDB protein from extracts of CV-1 cells using hydroxylapatite, phosphocellulose, Mono S, and DNA-affinity column chromatography. The DDB activity, either from mock-treated or UV-induced cells, is heterodisperse in column chromatography, and separation of three forms of the protein was obtained on a phosphocellulose column. Analysis of purified preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that greater than 90% of all three forms is a protein of approximately 126 kDa. The size of the native DDB protein was deduced from gel filtration and native polyacrylamide gel electrophoresis to be approximately 210 kDa, which suggests that the native DDB protein in solution is a homodimer. Preparations of partially purified DDB protein from UV-treated cells have enhanced levels of DDB activity and the protein when compared with similar preparations from mock-treated cells. This damage-recognition protein, alone or in conjunction with other subunits, may be of general importance for the initial recognition of DNA damage in mammals.
Abramić
M
M
Section on Viruses and Cellular Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Levine
A S
AS
Protić
M
M
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002460:Cell Line; D002522:Chlorocebus aethiops; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D006882:Hydroxyapatites; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000528:radiation effects*; D011485:Protein Binding; D013379:Substrate Specificity; D014466:Ultraviolet Rays
D004268:DNA-Binding Proteins; D006882:Hydroxyapatites; D009838:Oligodeoxyribonucleotides; D017886:Durapatite