Biophysical characterization of DNA repair complex TFIIH

TFIIH是转录因子IIH的缩写，是一种基础转录因子。但它对DNA核苷酸切除修复和细胞周期控制也是必不可少的。TFIIH由10个亚基组成，分子量为500kDa。迄今为止，它可以从HeLa细胞中纯化或在昆虫细胞中有限的过表达。由于其体积大，材料供应有限，TFIIH的生物物理生化表征仅限于低电子显微镜。许多遗传疾病是由于TFIIH （XPB和XPD）成分的突变和失活引起的。有趣的是，XPB和XPD含有解旋酶基序，而TFIIH没有DNA解绕活性。确定了古细菌来源的XPD的结构，并将各种保守残基映射到结构上。但这些结构并没有显示出XPD是如何与DNA相互作用的，也没有显示出XPD是如何与其他9种成分结合形成TFIIH的。我们的合作者Kaoru Sugasawa博士和他的同事已经成功地从昆虫细胞中的过表达中纯化了少量的TFIIH。我们已经收到了神户大学Sugasawa博士的表达克隆，并计划在昆虫细胞中产生全部10个TFIIH亚基。我们需要提高产量并纯化足够的TFIIH复合物，以进行初步的生物物理表征，并有望最终进行晶体学研究。

TFIIH, which stands for Transcription Factor II H, is a basal transcription factor. But it is also essential for DNA nucleotide-excision repair as well as cell cycle control. TFIIH is composed of 10 subunits and has a molecular weight of 500kDa. So far it can be purified from HeLa cells or limited over-expression in insect cells. With its large size and limited material supply, biophysical and biochemical characterization of TFIIH is limited to low electron microscopy. A number if genetic diseases are due to mutation and inactivation of the components in TFIIH (XPB and XPD). Interestingly XPB and XPD contain helicase motifs, but TFIIH shown no DNA unwinding activity. Structure of XPD from archaeal sources have been determined and various conserved residues are mapped to the structure. But these structures don't show how XPD interact with DNA or how it is associated with other 9 components to form TFIIH. Our collaborator, Dr. Kaoru Sugasawa, and his colleagues have successfully purify small amount of TFIIH from over-expression in insect cells. We have received the expression clones from Dr. Sugasawa in Kobe University and plan to produce all 10 subunits of TFIIH in insect cells. We need to improve the yield and purify enough TFIIH complexes for initial biophysical characterization and hopefully eventual crystallographic studies.