The role of protein kinases in DNA replication in Saccharomyces cerevisiae.
Doctoral thesis, UCL (University College London).
The initiation of DNA replication at the onset of S phase in eukaryotic cells is a critically important and tightly regulated process. Multiple origins of replication in the genome must be co-ordinately regulated such that duplication of the chromosomes is complete before cell division, whilst also ensuring that no sections of the DNA are over-replicated. In G1 phase of the cell cycle, a large ‘pre-replicative complex’ (pre-RC) forms at origins consisting of a hexameric Origin Recognition Complex (ORC) as well as Cdc6, Cdt1 and another hexameric complex known as the Minichromosome Maintenance (MCM) complex. At the onset of S phase, two cell cycle regulated protein kinases, the Cyclin Dependent Kinase (CDK) and Cdc7, are activated. Phosphorylation of various proteins by these two enzymes triggers formation of large ‘replisome’ complexes, initiation of DNA replication from each origin, and disassembly of the pre-RCs. Pre-RC re-assembly is subsequently inhibited until kinase activity falls again after cell division. In this study, we have set about identifying substrates of both CDK and Cdc7 involved in DNA replication in the budding yeast Saccharomyces cerevisiae. Two techniques are employed, the in vitro phosphorylation of arrays of peptides and phosphorylation of pre-RCs assembled in cell-free yeast extracts. Peptide arrays provide a high throughput technique for screening large numbers of potential substrates in a single experiment, whilst pre-RC phosphorylation allows consideration of both tertiary and quaternary structures of the in vivo kinase substrate. Several potential novel substrates of both CDK and Cdc7 are revealed. Pre-RC phosphorylation also reveals a previously unreported phosphorylation of Orc1 by a third kinase which has been identified as Casein Kinase II (CKII).
|Title:||The role of protein kinases in DNA replication in Saccharomyces cerevisiae|
|Open access status:||An open access version is available from UCL Discovery|
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