RECONSTITUTION OF TFIIH AND REQUIREMENT OF ITS DNA HELICASE SUBUNITS,RAD3 AND RAD25, IN THE INCISION STEP OF NUCLEOTIDE EXCISION-REPAIR

Yeast TFIIH is composed of six subunits: Rad3, Rad25, TFB1, SSL1, p55, and p38. In addition to TFIIH, we have purified a subassembly of the factor that lacks Rad3 and Rad25 and which we refer to as TFIIHi. In t he in vitro nucleotide excision repair (NER) system that consists enti rely of purified proteins, we show that neither TFIIHi nor a mixture o f purified Rad3 and Rad25 proteins is active in NER but that the combi nation of TFIIHi with Rad3 and Rad25 promotes the incision of UV-damag ed DNA. These results provide the first evidence for a direct requirem ent of Rad3, Rad25, and of one or more of the TFIIHi subunits in the i ncision step of NER. The NER efficacy of TFIIH is greatly diminished o r abolished upon substitution of Rad3 with the rad3 Arg-48 mutant prot ein or Rad25 with the rad25 Arg-392 mutant protein, respectively, thus indicating a role of the Rad3 and Rad25 DNA helicase functions in the incision of damaged DNA. Our results further indicate that the carbox yl-terminal domain kinase (CTD) TFIIK is dispensable for the incision of damaged DNA in vitro. These studies reveal the differential require ment of Rad3 DNA helicase and CTD kinase activities in damage specific incision versus RNA polymerase II transcription.