van der Spek P J, Eker A, Rademakers S, Visser C, Sugasawa K, Masutani C, Hanaoka F, Bootsma D, Hoeijmakers J H
Department of Cell Biology and Genetics, Medical Genetic Centre, Erasmus University, Rotterdam, The Netherlands.
Nucleic Acids Res. 1996 Jul 1;24(13):2551-9. doi: 10.1093/nar/24.13.2551.
The xeroderma pigmentosum syndrome complementation group C (XP-C) is due to a defect in the global genome repair subpathway of nucleotide excision repair (NER). The XPC protein is complexed with HHR23B, one of the two human homologs of the yeast NER protein, RAD23 (Masutani at al. (1994) EMBO J. 8, 1831-1843). Using heparin chromatography, gel filtration and native gel electrophoresis we demonstrate that the majority of HHR23B is in a free, non-complexed form, and that a minor fraction is tightly associated with XPC. In contrast, we cannot detect any bound HHR23A. Thus the HHR23 proteins may have an additional function independent of XPC. The fractionation behaviour suggests that the non-bound forms of the HHR23 proteins are not necessary for the core of the NER reaction. Although both HHR23 proteins share a high level of overall homology, they migrate very differently on native gels, pointing to a difference in conformation. Gel filtration suggests the XPC-HHR23B heterodimer resides in a high MW complex. However, immunodepletion studies starting from repair-competent Manley extracts fall to reveal a stable association of a significant fraction of the HHR23 proteins or the XPC-HHR23B complex with the basal transcription/repair factor TFIIH, or with the ERCC1 repair complex. Consistent with a function in repair or DNA/chromatin metabolism, immunofluorescence studies show all XPC, HHR23B and (the free) HHR23A to reside in the nucleus.
着色性干皮病综合征互补组C（XP-C）是由于核苷酸切除修复（NER）的全基因组修复子途径存在缺陷所致。XPC蛋白与HHR23B复合，HHR23B是酵母NER蛋白RAD23的两个人类同源物之一（Masutani等人，（1994年）《欧洲分子生物学组织杂志》8卷，1831 - 1843页）。通过肝素层析、凝胶过滤和非变性凝胶电泳，我们证明大多数HHR23B处于游离的、未复合的形式，只有一小部分与XPC紧密结合。相比之下，我们检测不到任何结合的HHR23A。因此，HHR23蛋白可能具有独立于XPC的额外功能。分级分离行为表明，HHR23蛋白的非结合形式对于NER反应的核心并非必需。尽管两种HHR23蛋白具有高度的总体同源性，但它们在非变性凝胶上的迁移方式非常不同，这表明其构象存在差异。凝胶过滤表明XPC - HHR23B异二聚体存在于高分子量复合物中。然而，从具有修复能力的曼利提取物开始进行的免疫去除研究未能揭示大部分HHR23蛋白或XPC - HHR23B复合物与基础转录/修复因子TFIIH或ERCC1修复复合物之间存在稳定的关联。与在修复或DNA/染色质代谢中的功能一致，免疫荧光研究表明所有XPC、HHR23B和（游离的）HHR23A都存在于细胞核中。
