Winkler G S, Vermeulen W, Coin F, Egly J M, Hoeijmakers J H, Weeda G
Department of Cell Biology and Genetics, Medical Genetics Center, Erasmus University, P. O. Box 1738, 3000 DR Rotterdam, The Netherlands.
J Biol Chem. 1998 Jan 9;273(2):1092-8. doi: 10.1074/jbc.273.2.1092.
TFIIH is a high molecular weight complex with a remarkable dual function in nucleotide excision repair and initiation of RNA polymerase II transcription. Mutations in the largest subunits, the XPB and XPD helicases, are associated with three inherited disorders: xeroderma pigmentosum, Cockayne's syndrome, and trichothiodystrophy. To facilitate the purification and biochemical characterization of this intricate complex, we generated a cell line stably expressing tagged XPB, allowing the immunopurification of the XPB protein and associated factors. Addition of two tags, a N-terminal hexameric histidine stretch and a C-terminal hemagglutinin epitope, to this highly conserved protein did not interfere with its functioning in repair and transcription. The hemagglutinin epitope allowed efficient TFIIH immunopurification to homogeneity from a fractionated whole cell extract in essentially one step. We conclude that the predominant active form of TFIIH is composed of nine subunits and that there is one molecule of XPB per TFIIH complex. The affinity-purified complex exhibits all expected TFIIH activities: DNA-dependent ATPase, helicase, C-terminal domain kinase, and participation in in vitro and in vivo nucleotide excision repair and in vitro transcription. The affinity purification procedure described here is fast and simple, does not require extensive chromatographic procedures, and yields highly purified, active TFIIH.
TFIIH是一种高分子量复合物，在核苷酸切除修复和RNA聚合酶II转录起始过程中具有显著的双重功能。最大的亚基XPB和XPD解旋酶发生突变与三种遗传性疾病相关：着色性干皮病、科凯恩综合征和毛发硫营养不良症。为了便于对这种复杂的复合物进行纯化和生化特性分析，我们构建了一个稳定表达带标签XPB的细胞系，从而能够免疫纯化XPB蛋白及相关因子。给这种高度保守的蛋白添加两个标签，即N端的六聚组氨酸延伸序列和C端的血凝素表位，并不影响其在修复和转录过程中的功能。血凝素表位使得从分级分离的全细胞提取物中基本上一步就能高效免疫纯化TFIIH至均一状态。我们得出结论，TFIIH的主要活性形式由九个亚基组成，且每个TFIIH复合物中有一个XPB分子。亲和纯化的复合物表现出所有预期的TFIIH活性：依赖DNA的ATP酶、解旋酶、C端结构域激酶，以及参与体外和体内的核苷酸切除修复及体外转录。本文所述的亲和纯化方法快速简便，无需大量的色谱操作步骤，并且能够得到高度纯化的活性TFIIH。
