Research Fellow, The Wellcome Trust/CR UK Gurdon Institute
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The Regulation of Replication Initiation in Eukaryotes
How is the entire genome replicated accurately before cell division? Eukaryotes replicate their genomes from multiple origins. This means that replication initiation must be strictly controlled so that no origin fires more than once in a given cell cycle and that enough origins fire to allow complete replication of every chromosome. In our lab we are interested in how this strict regulation of DNA replication is achieved during the cell cycle and when replication forks stall. We work primarily with the budding yeast Saccharomyces cerevisiae but a key research focus is to extend this analysis into the nematode C.elegans and to mammalian cells.
If replication forks encounter a block to progression, such as DNA damage, a cascade of checkpoint kinases become activated. In budding yeast, these kinases are called Mec1 and Rad53 and in vertebrates they are called ATR and Chk2. These kinases exert at least two effects on DNA replication in all eukaryotes tested so far. Firstly they stabilise replication forks that have stalled and secondly they prevent further origin firing until the block to DNA synthesis is removed. In order to understand how these kinases regulate the replication machinery directly we have done saturating proteomic screens for substrates of these kinases in vitro. Biochemical and genetic approaches are being used to determine which of these substrates are important in vivo.
We have previously shown that two replication initiation factors, Sld3 and Sld2, are the minimal substrates of CDK required for the initiation of DNA replication. CDK phosphorylation of Sld3 and Sld2 allows binding to a third essential protein, Dpb11 to form a pre-initiation complex (pre-IC). How this complex contributes to the initiation of DNA replication is not known, but it is likely that the pre-IC is an essential intermediate in the establishment of bi-directional replication forks. We intend to elucidate the essential functions of the pre-IC in budding yeast by combining biochemical reconstitution of the pre-IC with the powerful genetics approaches available in this organism.
- Mantiero D, Mackenzie A, Donaldson A, Zegerman P (2011) Limiting replication initiation factors execute the temporal programme of origin firing in budding yeast. EMBO J 30(23): 4805-4814.
- Zegerman P, Diffley JF (2010) Checkpoint-dependent inhibition of DNA replication initiation by Sld3 and Dbf4 phosphorylation. Nature 467(7314): 474-478.
- Zegerman P, Diffley JF (2009) DNA replication as a target of the DNA damage checkpoint. DNA Repair (Amst) 8(9): 1077-1088.
- Zegerman P, Diffley JF (2007) Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast. Nature 445(7125): 281-285.