JOHN DIFFLEY, American/British dual national, is awarded the 2016 Louis-Jeantet Prize for Medicine for his contributions to understanding how DNA replication, a process essential to life, initiates.
When a cell in an organism divides to yield two identical daughter cells, its DNA is first duplicated, or “replicated”, as two identical copies. John Diffley has become one of the worldwide leaders in the study of the mechanisms governing this process of duplication. His work has allowed us to understand how DNA replication is initiated, and how it is subsequently regulated throughout the cell cycle and in response to DNA damage. Since any mistakes in this process can lead to genetic mutations causing tumours, this research could be significant in the fight against cancer.
John Diffley will use the prize money to conduct further research into the mechanisms involved in the replication of chromosomes in yeast and human cells.
The information below refers to time of the award.
John Diffley was born 1958 in New York (USA) and studied in his home town (New York University) where he received his BA and PhD. Following a period as a post-doctoral fellow at Cold Spring Harbor Laboratory in New-York, he left for the UK in 1990. He continued his research at the Clare Hall Laboratories, where he became the director in 2006. In the same year he was made Deputy Director of the London Research Institute, and in 2015 became Associate Research Director at the Francis Crick Institute.
John Diffley was elected as a member of the European Molecular Biology Organization (EMBO) in 1998. He is also a Fellow of the Royal Society, of the American Association for the Advancement of Science, the Academia Europaea, the Academy of Medical Sciences and the European Academy of Cancer Sciences. In 2003 he won the American Paul Marks prize for cancer research.
Cell duplication, in which a cell becomes two daughter cells, is essential for all life, from bacteria to human beings. The first stage of this process involves the copying, or replication, of the DNA of the mother cell in a precisely regulated manner to make exactly two complete copies, one for each of the daughter cells. This “once per cell cycle” genome duplication is crucial for organisms to maintain a stable genetic composition during their lives and through evolution. In human cells, this means the precise duplication of over a billion base pairs each time a cell divides. To do this, replication initiation from 50-100,000 chromosomal sites, known as “replication origins”, must be tightly coordinated to ensure no origin is used more than once in a cell cycle.
John Diffley enjoys worldwide recognition for his work on the mechanism driving the initiation of DNA replication, using yeast, and human cells for his research. He and his team used chromosomal replication origins to characterise and ultimately reconstitute the protein machinery required to initiate DNA replication once in each cell cycle.
Any error in DNA replication or its initiation can result in genome instability that may contribute to the development of cancers. John Diffley’s research work could therefore have significant implications for cancer biology.
Shorten and reproduced with permission from EMBO Molecular Medicine. Diffley JFX (2016) “On the road to replication”. EMBO Mol Med doi:10.15252/emmm.201505965
John F.X. Diffley
The Francis Crick Institute Clare Hall Laboratory Blanche Lane
South Mimms Hertfordshire EN6 3LD U.K.
Tel: +44 (0)1707-625869
FAX: +44 (0)1707-625801
I had come to Clare Hall straight from a postdoc in Bruce Stillman’s lab at Cold Spring Harbor. When I first arrived in Bruce’s lab, he had just embarked on a major project to dissect cell extracts that supported the replication of SV40 DNA, and during my time at CSH many of the ‘household names’ in DNA replication like PCNA, RPA, RFC, etc. were discovered. I, however, had a different agenda. As a student I had been fascinated by electron micrographs of DNA from early Drosophila embryos showing multiple replication ‘bubbles’ along the chromosome (Kriegstein & Hogness, 1974). Although the idea that metazoan chromosomes were replicated from multiple replication origins had been demonstrated years earlier by fiber autoradiography, actually seeing these structures piqued my curiosity, and I became interested in the idea of trying to understand the events that led to the formation of these bubbles — the initiation of chromosomal DNA replication. For this SV40 was not ideal since it relies on the viral-encoded protein large T antigen (TAg) for origin recognition and replicative helicase activity. Little did I realise at the time that the ‘cellular TAg’ I was chasing actually comprised some 32 gene products, and it would take us more than 25 years to reconstitute the initiation of DNA replication with purified proteins!
My initial strategy was to use yeast replication origins, which had been identified years earlier, as a tool to identify origin binding proteins by biochemical approaches. Hopefully, we would be able to develop extracts that could replicate plasmids containing yeast origins, analogous to the SV40 system. Indeed, I spent many months making and testing extracts from S phase-arrested cells for origin-dependent incorporation of radio-labelled nucleotides into DNA to no avail. Fortunately, by the time my postdoc was drawing to a close, I had been productive enough to convince ICRF to hire me, but it was clear to me that different approaches would be needed to crack this problem. In my new lab, I decided to establish techniques to look at proteins binding to replication origins in vivo. This was before chromatin immunoprecipitation reached the masses, and so we settled on developing genomic footprinting, a technique that uses DNase1 to probe for protection of DNA sequences on chromatin in situ. With this, my first student Julie Cocker and I soon had evidence that the essential ‘A element’ in yeast origins was bound by a protein, and was flanked by a distinctive set of DNase1 hypersensitive sites at 10bp intervals (Diffley & Cocker, 1992). In a biochemical tour de force (Bell & Stillman, 1992), Steve Bell in the Stillman lab had identified and purified a six subunit protein that specifically recognised the A element and generated a pattern of protection and hypersensitivity nearly identical to our in vivo pattern, and so ORC (Origin Recognition Complex) was born! Because our footprints were initially performed on asynchronous cells and the A element was completely protected, we suggested that ORC was likely to be bound at origins all or most of the cell cycle, and thus its binding was not likely to be the trigger for DNA replication. In 1994, using synchronous cell populations, we showed that ORC was indeed bound throughout the cell cycle, but during G1 phase the ORC footprint was extended by an additional ~70bp of protection, a complex we called the ‘prereplicative complex’ or pre-RC (Diffley et al, 1994).
Over the next few years we showed that the pre-RC footprint required not only ORC, but also Cdc6, Cdt1 and the MCM complex (Figure 2). Pre-RCs first assemble at origins right at the end of mitosis, coincident with the inactivation of cyclin dependent kinase (CDK), and in a collaboration with the laboratory of Kim Nasmyth (Dahmann et al, 1995), we showed that premature inactivation of CDK before anaphase was sufficient to promote pre-RC assembly. This led us to propose the idea that CDK has a dual role in replication: on the one hand, it is required to promote DNA replication in S phase – as we would later show, by phosphorylating two key firing factors, Sld2 and Sld3 (Zegerman & Diffley, 2007). But on the other hand it prevents pre-RC assembly outside of G1 phase. Thus pre-RCs can only assemble during G1 phase when CDK activity is low; activation of CDK in S phase then both triggers DNA replication, and prevents re-assembly of pre-RCs at origins that have already fired. This idea neatly explained how multiple replication origins could be regulated to fire just once in each cell cycle (Figure 1).
It also helped explain why my early attempts to get S phase extracts to support replication failed: the hydroxyurea-arrested budding yeast cells I had been using to make extracts have high CDK levels, which would block pre-RC assembly. In addition to CDK, these cells also have high levels of the Rad53 DNA damage checkpoint kinase, which blocks origin activation (Santocanale & Diffley, 1998). So, the years we had spent trying to understand how events at replication origins are regulated in vivo were pointing us in the right direction, and by the close of the 1990s we felt we knew enough to get back to the biochemistry.
The in vivo experiments had taught us that the initiation reaction would need to be reconstituted in two sequential biochemical steps: first pre-RCs would need to be assembled in the absence of CDK activity, then origin firing would require high CDK activity. We knew cells arrested in G1 phase were competent for pre-RC assembly in vivo, but we also knew from Lucy Drury’s work that the critical assembly factor, Cdc6, was highly unstable in G1 phase. So, Takashi Seki used extracts from G1 arrested cells that conditionally overexpressed Cdc6 and showed that he could assemble MCM onto DNA in a Cdc6- and cell cycle- dependent manner (Seki & Diffley, 2000). When Dirk Remus joined the lab, he performed mass spectrometry on pre-RCs assembled in extracts and identified ORC, Cdc6, Cdt1 and MCM, but no additional proteins, suggesting we had the complete list of pre-RC components. Dirk then purified these proteins and reconstituted the reaction. In collaboration with electron microscopists Fabienne Beuron and Ed Morris, Dirk showed that MCM is loaded as a head-to-head double hexamer, and that this double hexamer is bound as topologically closed rings around double stranded DNA (Remus et al, 2009).
The next step was never going to be easy: activation of the MCM helicase involves melting the DNA duplex, re-opening the MCM ring, separation of the double hexamer into two single hexamers, extrusion of the lagging strand template from the interior and re-closing of the ring around the leading strand template. We also knew this step requires a long list of firing factors. But we were developing effective workflows for expression and purification of replication factors, and Joe Yeeles, an experienced and talented biochemist, was soon in position to look for DNA replication. Then, one August afternoon in 2014, there it was: a small smudge of radio-labelled DNA whose synthesis required everything we knew it should! After some optimisation, the replication products grew in length and partitioned into leading and lagging strand products (Yeeles et al, 2015) – it was clear we had a minimal DNA replication system up and running!
We continue on our quest to reconstitute the entire DNA replication reaction with purified proteins, and we are already learning a great deal about initiation mechanism. In addition to this, though, I believe we can extend our biochemical approaches to understand how DNA replication interfaces with many nuclear processes, including epigenetic inheritance, chromosome cohesion and post- replication repair. Ultimately, we hope to contribute to the reconstitution of a functional chromosome from constituent parts. The next few years will be fascinating.
Figure 1 A Model for DNA Replication
This model summarises some of our current understanding of how DNA replication initiates. In the first step, which is inhibited by CDK, ORC, Cdc6 and Cdt1 load the MCM helicase and an inactive double hexamer bound around double stranded DNA. In the second step, which is promoted by CDK, the listed firing factors, including the Dbf4 dependent kinase, contribute to activating MCM by generating the Cdc4-MCM-GINS (CMG) holo-helicase. This is followed by assembly of the complete replisome. The DNA damage checkpoint kinase Rad53, when active, inhibits origin firing and stabilises stalled replication forks. Additional detail is found in the text.
Bell SP, Stillman B (1992) ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex. Nature 357: 128-134
Dahmann C, Diffley JFX, Nasmyth KA (1995) S-phase-promoting cyclin- dependent kinases prevent re-replication by inhibiting the transition of replication origins to a pre-replicative state. Curr Biol 5: 1257-1269
Diffley JFX, Cocker JH (1992) Protein-DNA interactions at a yeast replication origin. Nature 357: 169-172
Diffley JFX, Cocker JH, Dowell SJ, Rowley A (1994) Two steps in the assembly of complexes at yeast replication origins in vivo. Cell 78: 303-316
Kriegstein HJ, Hogness DS (1974) Mechanism of DNA replication in Drosophila chromosomes: structure of replication forks and evidence for bidirectionality.
Proc Natl Acad Sci U S A 71: 135-139
Remus D, Beuron F, Tolun G, Griffith JD, Morris EP, Diffley JFX (2009) Concerted loading of Mcm2-7 double hexamers around DNA during DNA replication origin licensing. Cell 139: 719-730
Santocanale C, Diffley JFX (1998) A Mec1- and Rad53-dependent checkpoint controls late-firing origins of DNA replication. Nature 395: 615-618
Seki T, Diffley JFX (2000) Stepwise assembly of initiation proteins at budding yeast replication origins in vitro. Proc Natl Acad Sci U S A 97: 14115-14120
Yeeles JT, Deegan TD, Janska A, Early A, Diffley JF (2015) Regulated eukaryotic DNA replication origin firing with purified proteins. Nature 519: 431- 435
Zegerman P, Diffley JFX (2007) Phosphorylation of Sld2 and Sld3 by cyclin- dependent kinases promotes DNA replication in budding yeast. Nature 445: 281- 285