Cancer Research UK

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The London Research Institute research groups are based at Lincoln’s Inn Fields and Clare Hall. Our major research themes are: the biology of tumours and tissues, cellular regulatory mechanisms and genomic integrity and cell cycle.

John Diffley : Chromosome Replication

Goals

Previous and current research

Maintaining the integrity of the genome requires the precise duplication of all of the cell's chromosomes in each cell cycle. Errors in this process can cause the mutations leading a cell down the path to cancer. DNA replication in eukaryotic cells initiates from a large number of chromosomal sites known as origins. These initiation events do not occur synchronously but, rather, occur throughout S phase in a reasonably precise pattern. For much of our research, we have used the budding yeast Saccharomyces cerevisiae. The six subunit Origin Recognition Complex (ORC) binds to an essential sequence element within yeast origins and remains bound at origins throughout the cell cycle. A prereplicative complex (pre-RC) containing, in addition to ORC, the essential replication proteins Cdc6, Cdt1 and Mcm2-7 assembles at yeast origins in vivo at the end of mitosis and remains until origin firing during S phase. A subset of pre-RC components including the Mcm2-7 complex then plays an essential role during the elongation phase of DNA replication. The cyclin dependent kinase, Cdc28, plays a dual role in DNA replication: it is essential for initiating DNA replication and it prevents the assembly of new pre-RCs, thus limiting origin firing to once per cell cycle. We have also been interested in understanding how DNA replication is regulated in response to DNA damage. We have shown that the genome integrity checkpoint is required both to stabilise DNA replication forks and to inhibit inappropriate origin activation in response to DNA damage.

Future projects

Using a cell free system to assemble pre-RCs, we will characterise all of the pre-RC components and understand their regulation in detail. We will elucidate mechanisms by which the genome integrity checkpoint regulates both replication fork stability and late origin firing. Finally, we will begin to extend our findings in yeast to metazoans.