SUBSTRATE-SPECIFICITY OF THE CDK-ACTIVATING KINASE (CAK) IS ALTERED UPON ASSOCIATION WITH TFIIH

The transcription/DNA repair factor TFIIH consists of nine subunits, s everal exhibiting known functions: helicase/ATPase, kinase activity an d DNA binding. Three subunits of TFIIH, cdk7, cyclin H and MAT1, form a ternary complex, cdk-activating kinase (CAK), found either on its ow n or as part of TFIIH. In the present work, we demonstrate that purifi ed human CAK complex (free CAK) and recombinant CAK (rCAK) produced in insert cells exhibit a strong preference for the cyclin-dependent kin ase 2 (cdk2) over a ctd oligopeptide substrate (which mimics the carbo xyterminal domain of the RNA polymerase II). In contrast, TFIIH prefer entially phosphorylates the ctd as well as TFIIE alpha, but not cdk2, TFIIH was resolved into four subcomplexes: the kinase complex composed of cdk7, cyclin H and MAT1; the core TFIIH which contains XPB, p62, p 52, p44 and p34; and two other subcomplexes in which XPD is found asso ciated with either the kinase complex or with the core TFIIH. Using th ese fractions, we demonstrate that TFIIH lacking the CAK subcomplex co mpletely recovers its transcriptional activity in the presence of free CAK. Furthermore, studies examining the interactions between TFIIH su bunits provide evidence that CAK is integrated within TFIIH via XPB an d XPD.