A cartoon of a section of chromatin in the nucleus with replication origins in three different states.

John Diffley : Areas of interest

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We aim to understand how the 46 chromosomes in our cells are precisely duplicated in each cell cycle, how this process responds to DNA damage and how it is misregulated in cancer. To do this, we use a variety of approaches including genetics, cell biology and biochemistry. We have reconstituted chromatin replication with purified proteins, which is providing unprecedented insights into chromosome biology.

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 the S phase in a cell cycle in a reasonably precise pattern. The six subunit Origin Recognition Complex (ORC) together with Cdc6 and Cdt1, load the MCM replicative helicase as a double hexamer around double stranded DNA at origins. This occurs in vivo during G1 phase and remains until origin firing during S phase.

During origin firing, the MCM helicase is activated, which involves a remodeling of the MCM ring to encircle single-stranded DNA, accompanied by assembly of a stable CMG (Cdc45-MCM-GINS) complex. CMG then nucleates assembly of a multi-component replisome which must copy not just the entire genome, but also its associated bound proteins.

We have reconstituted the process of chromatin replication with purified proteins. We are using this to understand the mechanism of MCM helicase loading and activation, how the replisome is assembled and regulated by protein kinases and how the DNA replication machinery interfaces with other aspects of chromosome biology.

We use a variety of human cell models to understand how the deregulation of normal replication control mechanisms leads to genomic instability and whether this plays a role in cancer biology.

Selected publications