A damage-specific DNA binding protein. Large scale purification from human placenta and characterization.
A new large scale purification procedure for a human damage-specific DNA binding protein is described. Physical characterization suggests that the protein can dissociate into active subunits. A maximum molecular weight of 400,000 was obtained using gel exclusion chromatography, while electrophoresis through a pH 8 polyacrylamide gradient gel suggested a minimum molecular weight for the active protein of 120,000. Binding to UV-irradiated DNA revealed a broad pH optimum, no temperature dependence between 0 and 37 degrees C, and inhibition by intercalating agents. The use of 254 nm irradiation and of 313 nm irradiation in the presence of a triplet state sensitizer with alternating copolymers indicated that the protein was recognizing singlet-state-derived thymine lesions as well as triplet-state-derived adenine lesions. Inhibition of protein binding by pyridoxal 5'-phosphate and NaBH4 suggested a role for lysine at the DNA binding site, while sulfhydryl group involvement was indicated by the sensitivity of the protein to p-hydroxymercuribenzoate inhibition.
D002352:Carrier Proteins / Q000302:isolation & purification*; D004261:DNA Replication; D004279:DNA, Viral / Q000528:radiation effects; D004268:DNA-Binding Proteins; D005260:Female; D006801:Humans; D007364:Intercalating Agents / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010920:Placenta / Q000032:analysis*; D011089:Polydeoxyribonucleotides; D011247:Pregnancy; D011485:Protein Binding; D014466:Ultraviolet Rays
D002352:Carrier Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D007364:Intercalating Agents; D046911:Macromolecular Substances; D011089:Polydeoxyribonucleotides