The transient accumulation and activation of complexes of cyclins and cyclin-dependent kinases (Cdks) triggers the commitment to enter the cell cycle and to initiate chromosomal DNA replication in eukaryotes. In vertebrates, the cyclin E/Cdk2 kinase has been studied for its central role in promoting activities in 61. These activities include control of G1- and S-phase specific transcription, regulation of tumor suppressors, and the initiation of chromosomal DNA replication. The amphibian egg from Xenopus laevis provides a unique model for studying the cellular regulation and requirements for DNA replication. Using sperm nuclei and cell-free extracts from Xenopus eggs, the complex reactions of nuclear assembly, chromatin assembly, and DNA replication proceed efficiently in the absence of protein synthesis. Nonetheless, the requirement for cyclin-dependent kinases in DNA replication and the control that limits replication to one round per cell cycle are faithfully recapitulated in these extracts. Dr. Jackson has devised a Xenopus extract system that is dependent on exogenously added cyclins for the initiation of DNA replication. This system will be used to identify targets of the cyclin-dependent kinases and mechanisms of replication. The research described proposes to (1) determine the domains of the cyclin E kinase required for promoting the initiation of DNA replication; (2) identify cyclin E-associated effectors of DNA replication; and (3) to study how the cyclin E kinase controls a component of the initiation machinery: the protein encoded by a recently cloned vertebrate homologue of the S. cerevisiae CDC6 gene. The regulatory proteins that modulate G1 cyclin/Cdk kinases are important for controlling cellular growth and senescence. Defects in these regulators and cyclins themselves are implicated in a wide variety of cancers. At this time, we have only a faint outline of Cdk regulation and the control of timing and fidelity of DNA replication. Insight into the normal control of DNA replication is important to understanding how replication is altered in diseased cells. By focusing on identifying components of the regulatory mechanism and replication machinery, we may find new targets for chemotherapeutic control of neoplastic cells. Finally, understanding the mechanisms that constrain chromosomes to replicate accurately once per cell cycle will have implications for understanding aging, tumor progression, the generation of mutations that cause birth defects and metabolic disease, and the mechanism of amplification of drug resistance genes following chemotherapy.
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