Identification and characterization of XPC-binding domain of hHR23B.
hHR23B was originally isolated as a component of a protein complex that specifically complements nucleotide excision repair (NER) defects of xeroderma pigmentosum group C cell extracts in vitro and was identified as one of two human homologs of the Saccharomyces cerevisiae NER gene product Rad23. Recombinant hHR23B has previously been shown to significantly stimulate the NER activity of recombinant human XPC protein (rhXPC). In this study we identify and functionally characterize the XPC-binding domain of hHR23B protein. We prepared various internal as well as terminal deletion products of hHR23B protein in a His-tagged form and examined their binding with rhXPC by using nickel-chelating Sepharose. We demonstrate that a domain covering 56 amino acids of hHR23B is required for binding to rhXPC as well as for stimulation of in vitro NER reactions. Interestingly, a small polypeptide corresponding to the XPC-binding domain is sufficient to exert stimulation of XPC NER activity. Comparison with known crystal structures and analysis with secondary structure programs provided strong indications that the binding domain has a predominantly amphipathic alpha-helical character, consistent with evidence that the affinity with XPC is based on hydrophobic interactions. Our work shows that binding to XPC alone is required and sufficient for the role of hHR23B in in vitro NER but does not rule out the possibility that the protein has additional functions in vivo.
|Keywords||0 (DNA-Binding Proteins), 0 (Oligonucleotides), 0 (Recombinant Proteins), 0 (Ubiquitins), 156533-33-4 (RAD23 protein, human), 156533-34-5 (XPCC protein, human), Amino Acid Sequence, Antibody Specificity, Base Sequence, Binding Sites/genetics, Cell Line, DNA Repair, DNA-Binding Proteins/*chemistry/genetics/*metabolism, Human, Immunochemistry, In Vitro, Molecular Sequence Data, Mutagenesis, Site-Directed, Oligonucleotides/genetics, Recombinant Proteins/chemistry/genetics/metabolism, Sequence Deletion, Sequence Homology, Amino Acid, Support, Non-U.S. Gov't, Ubiquitins/genetics, Xeroderma Pigmentosum/genetics/*metabolism|
Masutani, C., Araki, M., Sugasawa, K., van der Spek, P.J., Yamada, A., Uchida, A., … Hanaoka, F.. (1997). Identification and characterization of XPC-binding domain of hHR23B.. Molecular and Cellular Biology, 17, 6915–6923. Retrieved from http://hdl.handle.net/1765/3125