DNA-binding protein from HeLa cells that binds preferentially to supercoiled DNA damaged by ultraviolet light or N-acetoxy-N-acetyl-2-aminofluorene.
A DNA-binding protein was partially purified from extracts of HeLa cells by high-speed centrifugation and chromatography on DEAE-cellulose, phosphocellulose and ultraviolet light-irradiated DNA-cellulose columns. It eluted from the phosphocellulose column with 0.375 M potassium phosphate and from the ultraviolet light-irradiated DNA-cellulose column between 0.5 M and 1 M NaCl. The protein binds preferentially to supercoiled PM2 DNA treated with ultraviolet light or N-acetoxy-N-acetyl-2-aminofluorene, as compared to native supercoiled PM2 DNA. The binding is non-cooperative. Nicked or linear forms of PM2 DNA (damaged or untreated) are not efficient substrates, indicating a requirement of DNA supercoiling for DNA binding. The sedimentation coefficient of the protein estimated by glycerol gradient centrifugation is 2.0-2.5 S, corresponding to a molecular weight of about 20000-25000 if the protein is spherical. The binding to DNA irradiated with ultraviolet light or treated with acetoxyacetylaminofluorene is optimal at around 100-200 mM NaCl and is relatively independent of temperature and pH. MgCl2 and MnCl2 at concentrations between 1 and 5 mM do not markedly affect the binding, but it is inhibited by sucrose, ATP and caffeine. The biological significance of the DNA-binding protein remains to be determined. It does not possess significant glycosylase, endonuclease or exonuclease activities. The dissociation equilibrium constant for the binding reaction of the protein to the ultraviolet light or acetoxyacetylaminofluorene-induced binding sites on DNA is estimated to be 4.10(-11) M. There are at least 1.10(5) DNA-binding protein molecules/HeLa cell.
D015073:2-Acetylaminofluorene / Q000031:analogs & derivatives*; D000099:Acetoxyacetylaminofluorene / Q000494:pharmacology*; D002352:Carrier Proteins / Q000378:metabolism*; D004278:DNA, Superhelical / Q000528:radiation effects; D004268:DNA-Binding Proteins; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D013329:Structure-Activity Relationship; D014466:Ultraviolet Rays
D002352:Carrier Proteins; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D000099:Acetoxyacetylaminofluorene; D015073:2-Acetylaminofluorene