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.