John Diffley is known for his elegant use of genetics, biochemistry, molecular and cellular biology to elaborate the events that occur at origins of eukaryotic DNA replication throughout the cell cycle. His most significant contribution was the discovery and characterisation of the pre-replicative complex (pre-RC), which contains the origin recognition complex (ORC), Cdc6 and the Mcm2-7 complex. His laboratory showed that cyclin-dependent kinases prevent pre-RC assembly in budding yeast by inhibiting ORC, and by regulating Cdc6 proteolysis and Mcm2-7 nuclear localisation.

Additionally, cyclin-dependent kinase promotes initiation by phosphorylating Sld2 and Sld3. Recently, his laboratory has reconstituted regulated initiation with purified proteins. His laboratory also contributed to understanding how replication origins are regulated by checkpoint kinases in response to DNA damage. Together, John’s work has led to a deeper understanding of how chromosomal DNA replication is controlled.