John Diffley

Chromosome Replication Laboratory

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) binds to an essential sequence element within yeast origins and remains bound at origins throughout the cell cycle. ORC, together with Cdc6 and Cdt1, loads the MCM replicative helicase as a double hexamer around double stranded DNA at yeast origins. This occurs in vivo during G1 phase and remains until origin firing during S phase.

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. DNA replication is also regulated in response to DNA damage by the genome integrity checkpoint which is required both to stabilise DNA replication forks and to inhibit inappropriate origin activation. We use biochemistry, genetics and cell biology to study these processes in budding yeast and mammalian cells.

Selected publications

Deegan, TD; Yeeles, JTP and Diffley, JFX (2016) Phosphopeptide binding by Sld3 links Dbf4-dependent kinase to MCM replicative helicase activation. EMBO Journal [Epub ahead of print] PubMed abstract

Yeeles, JTP; Deegan, TD; Janska, A; Early, A and Diffley, JFX (2015) Regulated eukaryotic DNA replication origin firing with purified proteins. Nature 519 , 431-435 PubMed abstract

Frigola J, Remus D, Mehanna A, Diffley JF. ATPase-dependent quality control of DNA replication origin licensingNature, 2013;495, 339-343 (Abstract)

Boos D, Sanchez-Pulido L, Rappas M, Pearl LH, Oliver AW, Ponting CP, Diffley JF. Regulation of DNA Replication through Sld3-Dpb11. Interaction Is Conserved from Yeast to HumansCurr Biol. 2011;21(13):1152-7 (Abstract)

Pfander, B., and Diffley, J.F.X. (2011). Dpb11 Coordinates Mec1 Kinase Activation with Cell Cycle-Regulated Rad9 RecruitmentEMBO J 30, 4897-4907. 

Zegerman P, Diffley JF. Checkpoint-dependent inhibition of DNA replication initiation by Sld3 and Dbf4 phosphorylationNature. 2010;467(7314):474-8 

Baxter J, Diffley JFX. Topoisomerase II Inactivation Prevents the Completion of DNA Replication in Budding YeastMol Cell. 2008;30:790-802 

Segurado M, Diffley JF. Separate Roles for the DNA Damage Checkpoint Protein Kinases in Stabilizing DNA Replication ForksGenes Dev. 2008;22:1816-1827 

Zegerman P, Diffley JFX. Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast.
Nature. 2007;445:281-285 

John Diffley

john.diffley@crick.ac.uk
+44 (0)379 61833

  • Qualifications and history
  • 1985 PhD, New York University, USA
  • 1984 Postdoctoral Fellow, Cold Spring Harbor Laboratory, USA
  • 1990 Established lab at the Imperial Cancer Research Fund, UK (in 2002 the Imperial Cancer Research Fund became Cancer Research UK)
  • 2015 Associate Research Director, the Francis Crick Institute, London, UK