Overproduction, purification, and characterization of the XPC subunit of the human DNA repair excision nuclease.
Xeroderma pigmentosum complementation group C gene (XPC) encodes a protein of 125 kDa which is present in a tight complex with a 58-kDa protein encoded by the human homolog of the yeast RAD23 gene, HHR23B (Masutani, C., Sugasawa, K., Yanagisawa, J., Sonoyama, T., Ui, M., Enomoto, T., Takio, K., Tanaka, K., van der Spek, P. J., Bootsma, D., Hoeijmakers, 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 biochemical properties. We find that both XPC and the heterodimer bind DNA with high affinity and UV-damaged DNA with slightly higher preference. Surprisingly, we find that the XPC subunit alone is sufficient for reconstitution of the human excision nuclease and that the HHR23B subunit has no detectable effect on the excision activity of the reconstituted system.
Reardon
J T
JT
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA.
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D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002848:Chromatography, DEAE-Cellulose; D003001:Cloning, Molecular; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; C086984:XPC protein, human