Control of DNA replication and genome stability by cyclin-dependent kinases: substrates and functions – CDKDNA

Regulation of cell proliferation is perhaps the most fundamental control in biology, and when it goes wrong, the consequences are pathological. If we could restore correct proliferation control, many diseases, including cancers, could be eradicated. In all eukaryotes, this control is mediated by Cyclin dependent kinases (CDK), a discovery arising from studies of genetics and biochemistry of the cell cycle, from yeast and amphibian egg systems over 20 years ago. The importance of this discovery was recognised by the award of the Nobel prize for medecine in 2001. But in 2008, the functions of Cdks are still surprisingly poorly understood at a molecular level, partly because of ignorance of their physiological substrates, and the effects of their phosphorylation. For example, in all eukaryotic cells, to initiate DNA replication, activation of Cdks is required and rate-limiting, but the roles of different Cdks and the substrates involved in this regulation have not yet been identified. Another limitation is the functional redundancy of different Cdk complexes. Metazoans encode several highly related Cdks, of several families, whose respective roles are still not well understood. In the current project we combine the use of the biochemical model of Xenopus egg extracts with the vertebrate genetic system of DT40 cells. By using two different systems, we increase our chances of uncovering conserved and imprtant functions. Applying the recent technologies of chemical genetics and phosphoproteomics to these systems, we aim to uncover new physiologically important Cdk substrates, and better understand respective roles of different Cdks. Firstly, we propose that Cdk phosphorylations essential for DNA replication are likely to occur on chromatin. We will attempt to verify this hypothesis by chromatin transfer experiments in Xenopus egg extracts. We predict that a limited subset of Cdk-dependent phosphorylations will be sufficient to allow loading of replication initiation complexes onto chromatin. If this is the case, by manipulating Cdk substrates in the Xenopus system, or by using DT40 genetics, we might be able to completely bypass Cdk requirements for replication intiation. This would be a result of outstanding importance. Nevertheless, it is also possible that Cdks will be required for chromatin remodelling in order to make DNA replication competent. Again, using sequential chromatin transfer experiments, the Xenopus system should allow us to demonstrate the existence of separate Cdk-dependent steps. We will then employ a combination of novel proteomics strategies to identify physiological substrates whose phosphorylation is required for initiation of DNA replication, and biochemical and genetic methods for studying functions of these substrates. For functional study of Cdks, and especially the respective contributions of Cdk1 and Cdk2 to genomic stability, we will use a novel approach based on genetic bypass of chemical inhibition.In this chemical-genetic approach, we are combining chemical inhibition of Cdks with restoration of activity of a single Cdk, using inhibitor-resistant (ir-)Cdk alleles designed using molecular modeling and bioinformatics. Use of ir-Cdks will eliminate the usual caveats in interpreting chemical-inhibition phenotypes. The resulting tools will allow us not only to study Cdk function, but also to investigate specificity of the Cdk inhibitor and provide important information about how resistance to it can arise by mutation of the target kinase. We have already undertaken a proof-of-principle study, and, as a second aspect of this project, we now aim to further this approach. We will use Cdk1 and Cdk2 inhibitors, combined with inhibitor-resistant alleles of the two kinases, to investigate the relative implication of these two Cdks in genomic stability via control of replication origin firing. We have previously shown that reducing Cdk activity to low levels results in a proportional loss of replication initiation events; beyond a certain point, this is likely to result in incomplete DNA replication due to the inability to compensate for stalled replication forks by forks from adjacent origins. This should induce DNA damage and genomic instability. Furthermore, Cdk2 also plays an important role in duplication of centrosomes and in onset of mitosis, although the respective contributions of Cdk1 and Cdk2 to mitosis onset are not understood, nor how Cdk1 compensates in the absence of Cdk2 in somatic cells. Nevertheless, Cdk inhibition is likely to disturb progression through mitosis, again potentially provoking genomic instability.We will therefore also use ir-Cdks to investigate the contributions of Cdk1 and Cdk2 to maintenance of genomic stability by their control of progression through mitosis. These experiments will be undertaken in both Xenopus egg extracts and DT40 cells.