TEMPORAL REGULATION OF CYCLIN A-P107 AND P33(CDK2) COMPLEXES BINDING TO A HUMAN THYMIDINE KINASE PROMOTER ELEMENT IMPORTANT FOR G1-S PHASE TRANSCRIPTIONAL REGULATION

The cyclins are an extensive family of proteins whose cell cycle-dependent synthesis is postulated to control multiple events during the cell cycle. The synthesis of A-type cyclins begins at the start of S phase. In mammalian cells, association with the cdc-type kinases suggests that cyclin A complexes are important for DNA replication and regulating other DNA-bound substrates required for S phase. We report here that a 25-bp promoter element previously shown to be important for the G1-S activation of the human thymidine kinase (htk) promoter in growth-stimulated cells is a cellular target of cyclin A and the p33cdk2 complexes. Though the p33cdk2 and other nuclear factor complexes exhibit constitutive binding to the htk G1-S regulatory domain, the binding activity of a cyclin A/p107 protein complex is greatly enhanced when the cells enter S phase, correlating with the increase in the tk mRNA levels and the replication of DNA. The binding activity of the cyclin A complex is maintained throughout S phase. Mutation of the DNA sequences on either half of the 25-bp protein binding site results in the loss of its ability to compete efficiently in vitro for the htk complexes, including that of cyclin A-containing complex. The loss of high-affinity binding for the htk complexes also substantially reduces the S-phase regulation of the htk promoter in vivo. Our results support the hypothesis that a cyclin A complex, in association with the p33cdk2 kinase, mediates the S-phase-regulated transcription of the htk promoter in growth-stimulated cells.