OVERPRODUCTION, PURIFICATION, AND CHARACTERIZATION OF THE XPC SUBUNITOF THE HUMAN DNA-REPAIR EXCISION NUCLEASE

Xeroderma pigmentosum complementation group C gene (XPC) encodes a pro tein of 125 kDa which is present in a tight complex with a 58-kDa prot ein encoded by the human homolog of the yeast RAD23 gene, HHR23B (Masu tani, C., Sugasawa, K., Yanagisawa, J., Sonoyama, T., Ui, M., Enomoto, T., Takio, K., Tanaka, K., van der Spek, P. J., Bootsma, D., Hoeijmak ers, J. H. J., and Hanaoka, F. (1994) EMBO J. 13, 1831-1843). The XPC- HHR23B complex is required for excision of thymine dimers from DNA in a human excision nuclease system reconstituted from purified proteins. In order to understand the role of the XPC-HHR23B complex in excision repair, we have overexpressed each subunit alone and the heterodimer in heterologous systems, purified them, and characterized their bioche mical properties. We find that both XPC and the heterodimer bind DNA w ith high affinity and UV-damaged DNA with slightly higher preference. Surprisingly, we find that the XPC subunit alone is sufficient for rec onstitution of the human excision nuclease and that the HHR23B subunit has no detectable effect on the excision activity of the reconstitute d system.