A yeast four-hybrid system identifies Cdk-activating kinase as a regulatorof the XPD helicase, a subunit of transcription factor IIH

To understand the role of the various components of TFIIH, a DNA repair/tra nscription factor, a yeast four-hybrid system was designed. When the ternar y Cdk-activating kinase (CAK) complex composed of Cdk7, cyclin H, and MAT1 was used as bait, the xeroderma pigmentosum (XP) D helicase of transcriptio n factor IIH (TFIIH), among other proteins, was identified as an interactin g partner. Deletion mutant analyses demonstrated that the coiled-coil and t he hydrophobic domains of MAT1 interlink the CAK complex directly with the N-terminal domain of XPD. Using immunoprecipitates from cells coinfected wi th baculoviruses, we further validated the bridging function of XPD, which anchors CAK to the core TFIIH. In addition we show that upon interaction wi th MAT1, CAK inhibits the helicase activity of XPD. This inhibition is over come upon binding to p44, a subunit of the core TFIIH. It is not surprising that under these conditions some XPD mutations affect interactions not onl y with p44, but also with MAT1, thus preventing either the CAK inhibitory f unction within CAK(.)XPD and/or the role of CAK within TFIIH and, consequen tly, explaining the variety of the XP phenotypes.