Biophysical characterization of DNA repair complex TFIIH

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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.