Purification of a HeLa cell nuclear protein that binds selectively to DNA irradiated with ultra-violet light.
Ultraviolet (UV) light induces a variety of lesions in DNA of which the pyrimidine dimer represents the major species. Pyrimidine dimers exist as both a cyclobutane type and a 6-4' (pyrimidine-2'-one) photoproduct. We have purified a protein of M(r) approximately 125,000 from HeLa cell nuclei which binds efficiently to double-stranded DNA irradiated with UV light but not to undamaged DNA. This protein was designated UVBP1 (UV damage binding protein 1). UVBP1 did not recognise DNA damaged by cisplatin. Using oligonucleotides with a single dipyrimidine site for induction of UV photoproducts, binding of UVBP1 to a TC-containing substrate was shown to be more efficient than to substrates containing a TT, a CT or a CC pair. This binding specificity implies selective recognition of the 6-4' photoproduct. Further evidence for this was provided by the finding that hot alkali treatment of the substrate (which selectively hydrolyses 6-4' photoproducts) abrogated binding of UVBP1, whereas incubation with DNA photolyase to remove cyclobutane dimers did not. No detectable DNA helicase, ATPase or exonuclease activity was associated with the purified protein. We suggest that UVBP1 may be involved in the lesion recognition step of DNA excision repair and could contribute to the preferential repair of 6-4' photoproducts from the DNA of UV-irradiated mammalian cells.
van Assendelft
G B
GB
Imperial Cancer Research Fund, University of Oxford, John Radcliffe Hospital, UK.
Rigney
E M
EM
Hickson
I D
ID
D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D002945:Cisplatin / Q000494:pharmacology; D004247:DNA / Q000378:metabolism* / Q000528:radiation effects; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000378:metabolism; D006367:HeLa Cells / Q000737:chemistry*; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000302:isolation & purification* / Q000378:metabolism; D011740:Pyrimidine Dimers / Q000378:metabolism; D014466:Ultraviolet Rays
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011740:Pyrimidine Dimers; C082518:UVBP1 protein, human; D004247:DNA; D004255:Deoxyribodipyrimidine Photo-Lyase; D002945:Cisplatin