A yeast four-hybrid system identifies Cdk-activating kinase as a regulator of the XPD helicase, a subunit of transcription factor IIH.
To understand the role of the various components of TFIIH, a DNA repair/transcription factor, a yeast four-hybrid system was designed. When the ternary Cdk-activating kinase (CAK) complex composed of Cdk7, cyclin H, and MAT1 was used as bait, the xeroderma pigmentosum (XP) D helicase of transcription factor IIH (TFIIH), among other proteins, was identified as an interacting partner. Deletion mutant analyses demonstrated that the coiled-coil and the hydrophobic domains of MAT1 interlink the CAK complex directly with the N-terminal domain of XPD. Using immunoprecipitates from cells coinfected with baculoviruses, we further validated the bridging function of XPD, which anchors CAK to the core TFIIH. In addition we show that upon interaction with MAT1, CAK inhibits the helicase activity of XPD. This inhibition is overcome upon binding to p44, a subunit of the core TFIIH. It is not surprising that under these conditions some XPD mutations affect interactions not only with p44, but also with MAT1, thus preventing either the CAK inhibitory function within CAK.XPD and/or the role of CAK within TFIIH and, consequently, explaining the variety of the XP phenotypes.
Sandrock
B
B
Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/Université Louis Pasteur, B. P. 163, 67404 Illkirch Cedex, France.
Egly
J M
JM
D001483:Base Sequence; D056748:Cyclin H; D018844:Cyclin-Dependent Kinases; D016213:Cyclins / Q000378:metabolism; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004268:DNA-Binding Proteins; D015336:Molecular Probe Techniques; D009154:Mutation; D010641:Phenotype; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D019473:Transcription Factors, TFII; D051759:Xeroderma Pigmentosum Group D Protein; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D056748:Cyclin H; D016213:Cyclins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D011506:Proteins; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D051758:Transcription Factor TFIIH; D017346:Protein Serine-Threonine Kinases; D018844:Cyclin-Dependent Kinases; D004265:DNA Helicases; D051759:Xeroderma Pigmentosum Group D Protein; D000095043:Cyclin-Dependent Kinase-Activating Kinase