Sugasawa K, Masutani C, Uchida A, Maekawa T, van der Spek P J, Bootsma D, Hoeijmakers J H, Hanaoka F
The Institute of Physical and Chemical Research (RIKEN), Saitama, Japan.
Mol Cell Biol. 1996 Sep;16(9):4852-61. doi: 10.1128/MCB.16.9.4852.
A protein complex which specifically complements defects of XP-C cell extracts in vitro was previously purified to near homogeneity from HeLa cells. The complex consists of two tightly associated proteins: the XPC gene product and HHR23B, one of two human homologs of the Saccharomyces cerevisiae repair gene product Rad23 (Masutani et al., EMBO J. 13:1831-1843, 1994). To elucidate the roles of these proteins in "genome-overall" repair, we expressed the XPC protein in a baculovirus system and purified it to near homogeneity. The recombinant human XPC (rhXPC) protein exhibited a high level of affinity for single-stranded DNA and corrected the repair defect in XP-C whole-cell extracts without extra addition of recombinant HHR23B (rHHR23B) protein. However, Western blot (immunoblot) experiments revealed that XP-C cell extracts contained excess endogenous HHR23B protein, which might be able to form a complex upon addition of the rhXPC protein. To investigate the role of HHR23B, we fractionated the XP-C cell extracts and constructed a reconstituted system in which neither endogenous XPC nor HHR23B proteins were present. In this assay system, rhXPC alone weakly corrected the repair defect, while significant enhancement of the correcting activity was observed upon coaddition of rHHR23B protein. Stimulation of XPC by HHR23B was found with simian virus 40 minichromosomes as well as with naked plasmid DNA and with UV- as well as N-acetoxy-2- acetylfluorene-induced DNA lesions, indicating a general role of HHR23B in XPC functioning in the genome-overall nucleotide excision repair subpathway.
一种能在体外特异性弥补XP - C细胞提取物缺陷的蛋白质复合物，先前已从HeLa细胞中纯化至近乎均一。该复合物由两种紧密结合的蛋白质组成：XPC基因产物和HHR23B，后者是酿酒酵母修复基因产物Rad23的两个人类同源物之一（Masutani等人，《欧洲分子生物学组织杂志》13:1831 - 1843，1994）。为阐明这些蛋白质在“全基因组”修复中的作用，我们在杆状病毒系统中表达了XPC蛋白并将其纯化至近乎均一。重组人XPC（rhXPC）蛋白对单链DNA表现出高度亲和力，并且在不额外添加重组HHR23B（rHHR23B）蛋白的情况下校正了XP - C全细胞提取物中的修复缺陷。然而，蛋白质印迹（免疫印迹）实验表明，XP - C细胞提取物含有过量的内源性HHR23B蛋白，添加rhXPC蛋白后它可能能够形成复合物。为研究HHR23B的作用，我们对XP - C细胞提取物进行了分级分离，并构建了一个内源性XPC和HHR23B蛋白均不存在的重组系统。在该检测系统中，单独的rhXPC只能微弱地校正修复缺陷，而在同时添加rHHR23B蛋白后，校正活性显著增强。在猿猴病毒40微小染色体以及裸露质粒DNA上，以及在紫外线和N - 乙酰氧基 - 2 - 乙酰芴诱导的DNA损伤中，均发现HHR23B对XPC有刺激作用，这表明HHR23B在全基因组核苷酸切除修复子途径中XPC发挥功能方面具有普遍作用。
