Biophysical characterization of DNA repair complex TFIIH

TFIIH是转录因子II H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500 kDa。到目前为止，它可以从HeLa细胞中提纯，也可以在昆虫细胞中有限过度表达。由于其体积大，材料供应有限，TFIIH的生物物理和生化特性仅限于低电子显微镜。一些遗传性疾病是由于TFIIH(XPB和XPD)的成分突变和失活造成的。有趣的是，XPB和XPD含有解旋酶基序，但TFIIH没有显示DNA解离活性。古细菌来源的XPD的结构已经确定，各种保守的残基被映射到结构上。但这些结构并没有显示XPD是如何与DNA相互作用的，或者它是如何与其他9种成分结合形成TFIIH的。我们的合作者杉泽博士和他的同事们已经成功地从昆虫细胞的过度表达中提纯了少量的TFIIH。我们已经收到神户大学杉泽博士的表达克隆，并计划在昆虫细胞中生产TFIIH的全部10个亚基。我们需要提高产率并提纯足够的TFIIH络合物，以进行初步的生物物理表征，并有望最终进行结晶学研究。

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.