IDENTIFICATION AND CHARACTERIZATION OF XPC-BINDING DOMAIN OF HHR23B

hHR23B was originally isolated as a component of a protein complex tha t specifically complements nucleotide excision repair (NER) defects of xeroderma pigmentosum group C cell extracts in vitro and was identifi ed as one of two human homologs of the Saccharomyces cerevisiae NER ge ne product Rad23. Recombinant hHR23B has previously been shown to sign ificantly stimulate the NER activity of recombinant human XPC protein (rhXPC). In this study we identify and functionally characterize the X PC-binding domain of hHR23B protein. We prepared various internal as w ell as terminal deletion products of hHR23B protein in a His-tagged fo rm and examined their binding with rhXPC by using nickel-chelating Sep harose. We demonstrate that a domain covering 56 amino acids of hHR23B is required for binding to rhXPC as well as for stimulation of in vit ro NER reactions. Interestingly, a small polypeptide corresponding to the XPC-binding domain is sufficient to exert stimulation of XPC NER a ctivity. Comparison with known crystal structures and analysis with se condary structure programs provided strong indications that the bindin g domain has a predominantly amphipathic alpha-helical character, cons istent with evidence that the affinity with XPC is based on hydrophobi c interactions. Our work shows that binding to XPC alone is required a nd sufficient for the role of hKR23B in in vitro NER but does not rule out the possibility that the protein has additional functions in vivo .