Abstract
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.
Highlights
A new largescale purification procedure for a humabninding protein which shows an unusual specificity for damdamage-specific DNA bindingprotein is described. aged DNA [14, 15]
We describe a new purification procedure which has allowed us to scale up the purification from one singlet-state-derived thyminelesions as well as triplet- placenta to 100 human placentas
Since a number of proteins with anionic substrates have been shown to contain arginine residues at their active sites [26],the DNA bindingprotein was examined foirts sensitivity to the arginine-specific reagents, 2,3-butanedione and phenylglyoxalH. owever, under the conditions employed, neither of these reagents gave any protein inactivation, suggesting that arginine does not play a critical role in this protein. In previous workfrom this laboratory [14], we have described the partialpurification and characterization of a damage-specific DNA bindingprotein from human cells
Summary
A new largescale purification procedure for a humabninding protein which shows an unusual specificity for damdamage-specific DNA bindingprotein is described. aged DNA [14, 15]. The use of 254 nm irradiation and of313 n m irradiation in the presence of a triplet state sensitizer with alternating copolymers indicated that the proteiwnas recognizing methyl-N-nitrosourea, singlet oxygen, and superoxide. It does not recognize strand breaks, apurinic or apyrimidinic sites, single strand regions, pyrimidine dimers or psoralen-induced cross-links. This pattern of specificity suggests that thebinding protein recognizesmonofunctional base damage which interferes with base pairing [16] and makes it of interest to determine how this protein interacts with DNA. Inhibition of protein have obtained sufficient substantially pure protein to begin bindingbypyridoxalB’-phosphateand
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