John Diffley's 30-year mission to understand DNA replication and his place in the universe.
“If you took the DNA created by your body every minute, and stretched it end to end, it would reach to the moon and back”, says John.
Every minute of your life, something remarkable is occurring inside you. The dividing cells in your body make complete copies of their entire two-metre-long DNA genome, over and over, with incredible accuracy.
The sheer scale at which they do this, says the Crick’s John Diffley, is almost unimaginable. Of your body’s several trillion cells, several hundred billion divide each day. “So if you took the DNA created by your body every minute, and stretched it end to end, it would reach to the moon and back,” he says.
“That is an extraordinary amount of DNA. If you add it up, by the time you’re 50 years old, your body has made at least a light-year of DNA."
And if that seems extraordinary, John says, what’s even more remarkable is how accurate this process is.
“Among the tens of thousands of genes in your genome, there are hundreds that, if mutated, can trigger cancer. But despite continually churning out so much DNA, most people don’t get cancer in their lifetime,” he says. “That shows how incredibly accurate this is.”
The puzzle of how a cell copies its DNA so faithfully has been occupying the minds of researchers since 1953, when Watson and Crick suggested that each strand of a DNA double helix could act as a template, allowing a cell to make two new DNA molecules where only one had existed before.
Famously, Watson and Crick were right – but working out the details of how a cell’s inner machinery copies DNA with such fidelity, and in such a tightly coordinated fashion, has taken many decades. And it’s this question that has fascinated John for his entire career, starting as a young PhD student in the 1980s, and continuing today in his lab at the Crick.
To understand how a molecular machine works, the best solution is to purify its individual components from within the cell and reconstitute them in a test tube. But this is not trivial. Back in the day, old-fashioned ‘bucket biochemistry’, as it was nicknamed, required litres and litres of cells to be bust open and turned into a molecular slurry in which to fish for proteins of interest – a laborious and often frustrating process.
By the 1970s, researchers studying DNA replication in bacteria had used this approach to identify the two classes of enzymes that sit at the heart of DNA replication.
The first, known as DNA helicases, perform a feat of topological genius to unwind the double helix, forcing the two strands apart. The second class, DNA polymerases, copies these separated strands to form two new DNA molecules.
Bacteria, being rather simple life-forms, have small, circular chromosomes, and their DNA replication enzymes have a relatively easy job. Starting in one specific place – a unique stretch of DNA called the ‘origin of replication’ – they just continue round the circle until the DNA is all copied.
It’s a bit different for more complex, ‘eukaryotic’ organisms, such as ourselves. Rather than just a single origin of replication, our huge linear chromosomes each have thousands of origins dotted along their lengths. This poses a logistical challenge: the decision to start replication must be precisely coordinated across different origins on multiple chromosomes, and crucially, must also be synchronised with the machinery that governs cell division itself. Clearly, in addition to a DNA helicase and DNA polymerase, many more enzymes and layers of regulation must be in play.
A decade after the discovery of bacterial DNA replication enzymes, most scientists remained daunted by the sheer complexity of DNA replication in complex organisms, and only a handful of them wanted to study it. John, then a 20-year-old hippy starting a PhD at New York University (NYU), was one of them.
Growing up near New York in the 1960s, John had been fascinated by the space race and Star Trek; as an 11-year‑old on holiday upstate he’d followed the moon landing on the radio in his father’s car. “When I was a kid, it just felt like such an optimistic time, like anything was possible,” he says.
The son of “moderately religious” Irish Catholic parents, John quickly moved away from any kind of faith. “But I think in a funny way it made a mark on me”, he says. “Although the answers on offer weren’t satisfying, the questions remained important. And the idea of asking big questions about why we exist stayed with me.”
At the age of 16, John moved to New York City to go to university. It was great, “for all the wrong reasons – and all the right reasons,” he remembers. “I went from an all-boys Catholic seminary school to Greenwich Village in the 70s – and that was the making of me”. He’d intended to study medicine, but quickly realised it wasn’t for him after his mother, a nurse, arranged for him to spend a revelatory summer working in a hospital.
Instead, after graduating John signed on to NYU’s PhD programme to do a heavy-duty ‘bucket biochemistry’ project – purifying the main DNA polymerase from fruit fly cells. “I found the idea that you could understand life through studying how DNA was replicated completely intoxicating,” he recalls.
It was during his PhD that John read a research paper that not only made him realise the sheer scale of the replication problem, but inspired his lifelong fascination with it.
The paper, from the lab of renowned fruit fly biologist David Hogness, contained an electron micrograph picturing long, snaking chains of replicating fruit fly DNA. Each chain was peppered with bubbles – the opened origins of replication – and something struck John: there were no bubbles-within-bubbles.
“The origins fired in a very strict once-per-cell-cycle way, and I remember thinking that, a few seconds before that image was taken, something was happening that we knew nothing about, which led to the formation of the bubble,” he says.
It meant that the initial step, before replication was allowed to begin, must be tightly restrained – but how? This was far more intriguing to him than replication itself, and it was a question that set the stage for what would become a three-decade odyssey.
John’s PhD project didn’t go entirely as planned. His supervisor left NYU early on, and he washed up in a lab with colleagues who had little interest in what he was doing. Nevertheless, he was successful, and it was a crash course in independent research: “There was nobody to tell me to do this or that, or to give me any guidance. I had to do it on my own, figure out what I wanted to do,” John says. “But I completely loved it. I guess biochemistry is really puzzle solving – what so many of us love about science – figuring out how something works when you don’t know.”
John turned out to be very good at meticulous laboratory work, and in 1984, he successfully applied for a postdoctoral position at Cold Spring Harbor Laboratory, in the lab of Bruce Stillman – a young faculty member who ran one of the few groups in the world interested in what controlled the initiation of DNA replication. Although Bruce’s lab focused mainly on viruses, John decided to strike off in a different direction. “I started working with yeast and, again, I taught myself,” John recalls.
Science is hard, but choosing to be a maverick makes it even harder. What was it that drove John that way? “I guess there’s just a level of contrariness in my personality,” he says. “And my PhD was difficult, but I managed to do it, so it gave me confidence that I could do things independently”.
John’s switch to yeast wasn’t entirely contrary: it’s one of the simplest eukaryotic organisms, and its genome is relatively easy to manipulate. And by the time John started in Bruce’s lab, researchers had identified its replication origins and developed a range of mutant strains, any of which, he reasoned, might be able to shed light on DNA replication.
Sadly, his foray wasn’t as initially successful as John had hoped. With hindsight, his strategy – to use cell extracts to try to recreate initiation in a test tube – was doomed from the get-go: “It turned out to be the worst thing you could possibly do,” he remembers. “There was something crucial about replication initiation that we didn’t understand at the time, that we had to understand in order to get to the next level”.
Undeterred, in 1990 John moved to the UK to take up a position at the Imperial Cancer Research Fund’s Clare Hall Laboratories near London – then one of the best places in the world to work on DNA replication. He set out with a mind-bogglingly ambitious aim: to purify every single protein involved in replication, get the process working in the test tube, and thereby understand it.
It takes a particular sort of person to pursue a project knowing full well that it could take decades to complete – the ‘unknown unknowns’ propel it into the realm of science fiction. However, John’s upbringing – the triumphs of the space race, and all the surrounding positivism – had given him an innate optimism that scientific problems, however difficult, were solvable.
He also wanted to do something meaningful with his life. “Every time I’ve learnt something new about DNA replication, I’ve understood more about the universe, the way it works and my place in it. I’m not an observer – I’m a participant,” he says.
Unquestionably, John has a scientific vocation, but he doesn’t see his career as a noble quest for knowledge. “There’s an analogy my colleague Tim Hunt uses that I’ve always liked, which is that doing science is a bit like gardening,” he says. “If you only go into gardening to make a beautiful garden, you’ll never succeed. But if you do it because you actually like getting your hands dirty and digging up plants, you’ll be fine. And I think that’s exactly true. If you take pleasure in the day-to-day stuff on the way to the big thing, then you’ll be okay.”
"A minor thing", John's current project is reconstructing the replication machinery in the last common eukaryotic ancestor, around two billion years ago. Credit: Dave Guttridge.
In his new lab at Clare Hall, John decided that rather than carrying on fruitlessly trying to purify proteins involved in initiation of replication, it might be more informative to get a picture of what was actually going on at the replication origins in growing yeast cells. To do this, his team turned to a horribly fiddly technique called genomic footprinting, which identifies the precise regions, or ‘footprints’, on DNA where proteins are bound.
This proved much more successful, and by 1994 he and his lab had detected footprints of two differently sized protein machines at yeast replication origins. The smaller, which permanently sat on the origins, turned out to be a collection of six different proteins working together – in biochemical parlance, a “complex”. It corresponded to an entity that had just been identified by Steve Bell and Bruce Stillman at Cold Spring Harbor, which they had called the Origin Recognition Complex (ORC).
Finding the ORC was pleasing, but the other, larger footprint, which John had christened the pre-Replicative Complex (pre-RC), was far more interesting. For a start, its existence hinted that initiating DNA replication involved more than just the proteins that recognised the origins. But most excitingly to John and his lab, this larger complex appeared and disappeared in a highly ordered way that was exactly synchronised with the phases of cell division.
As a cell grows and divides, it cycles through four distinct phases. First is an initial growth phase, known as G1, which is followed by an ‘S’ phase during which the cell replicates its DNA.Then a second growth phase, G2, precedes a final mitosis, or M, phase, during which the cell physically divides in two.
John’s newly discovered pre-RC footprint first showed up in cells just after they had divided, and persisted through the first growth phase, G1. But then, as cells began to replicate their DNA in S phase, the pre-RC disappeared, leaving just the smaller ORC footprint.
This cyclical pattern, John realised, was strikingly similar to that of a poorly understood phenomenon observed a few years earlier in a wildly different species: frogs. Named ‘Licensing Factor’, it had been proposed to regulate the once-per-cycle firing of frog origins of replication.
The implications were huge: first, it suggested that DNA replication might be controlled in the same way in all complex organisms. And secondly, since John could see the pre-RC footprint, he reasoned that it would be a short (if arduous) step to identifying its components.
To everyone’s satisfaction, the pre-RC and Licensing Factor were indeed one and the same, and over the next few years, John led an effort to purify and define all the individual proteins making up the complex. The pre-RC turned out to contain all the six proteins of the ORC complex, along with two other proteins called Cdt1 and Cdc6, and another complex called MCM – the replicative DNA helicase itself, poised to unwind DNA but held in check until the time was right.
Having identified its components, John and his lab spent the next decade working out how the pre‑RC connected to the cell cycle, revealing an elegant two‑step process choreographed by changing levels of an enzyme called cyclin-dependent kinase (CDK).
In the first step, the pre‑RC, including the MCM helicase, is loaded onto origins; this can only occur from just after M phase until the end of G1 phase, when CDK levels are low. Once bound, the complex simply sits there until the cell enters S-phase, when CDK levels rise; this triggers the second step, in which the MCM helicase shifts into gear, leaves the origin and its ORC companions behind, and starts to move along the double helix, unwinding it as it goes so its DNA can be copied. After the entire genome has been replicated, the cell divides, CDK levels drop, and the whole process starts again.
“It’s that two-step reaction that allows the cell to regulate lots of replication origins so that each only gets activated once in a cell cycle,” John says. “Because the cell can only do the first step at the beginning of the cell cycle, it can do it across hundreds of thousands of sites in the genome. And the crucial thing is that when it goes into the second state, where now it can start replication, it can’t do that first step anymore.”
John with the evidence his lab detected in 1993 showing the two steps of DNA replication occurring at replication origins during the cell cycle.
John has always taken seriously a maxim put forward by theoretical physicist Richard Feynman: “What I cannot create I do not understand”, so it was inevitable that eventually he would return to his ambition to reconstitute replication in the test tube.
The thought of purifying a large number of often fragile proteins, then re-assembling them into multiple complexes, is John’s idea of the ultimate good time. “Ever since I was a kid I took things apart,” he says. “I broke every toy I ever got for Christmas within a week, because I took it apart and couldn’t put it back together.” However, he concedes that taking apart, and remaking, DNA replication in a test tube was “a slightly mad thing to do”, and it’s true that in the end it did indeed take a very long time and an awful lot of work.
Knowing that the process happened in two steps meant that he could work on them one by one. In 2009, he accomplished the first challenge: assembling, then loading, the pre-RC onto DNA in the test tube.
Then, in 2015, 30 years after John started working on initiation, his lab published a paper hailed as one of the greatest advances ever achieved in the DNA replication field. It described how they had purified 42 proteins, which formed 16 different complexes, and assembled them on DNA in two steps – pre-initiation and initiation – exactly as happens in a cell. John remembers the moment they realised his postdoctoral researcher, Joe Yeeles, had got DNA to replicate in a test tube as “just one of those great moments. We could only see a tiny bit of DNA synthesis, but all the controls were there, so we knew we’d got it right, and all we had to do was tune up the experimental conditions.”
What do you do when your 30-year odyssey is complete, and the next unknown unknowns are beyond your reach? John says one of the most gratifying things about his career are the people he’s trained and mentored who are now running their own labs, and can take the problem forward far beyond his own scientific lifespan.
John with postdoctoral researcher Evelyn Eastwood, who is working to understand how DNA replication initiates in human cells by reconstituting this process in the lab. Credit: Dave Guttridge
“What this work has done is opened many new avenues”, he says, “and I’m very happy that there are other great people around to explore them”.
John hasn’t been idle since his 2015 paper. “You could spend the last bit of your career dotting i’s and crossing t’s”, he says, “and it’s hard to get the balance right between that and doing new work so that the energy doesn’t go out of the lab”. He’s managing this admirably. The 2016 move to the Crick from Clare Hall meant that he found new collaborators, most notably the cryo-electron microscopy expert Alessandro Costa, and Frank Uhlmann – who studies how replicated chromosomes negotiate the fraught period when a cell splits in two.
John and Alessandro’s labs have worked out in exquisite detail how origins ‘melt’ to allow binding of the pre‑RC, and how MCM helicases are switched on and do their unwinding job. And in yet another feat of biochemical bravura, they can now do test-tube replication not just with purified DNA, but of DNA packaged into chromatin, the complex of DNA and proteins that chromosomes are made of.
Meanwhile Frank and John are collaborating with others to reveal how the replication machinery interacts with the molecules that hold chromosomes together as a cell gets ready to divide – a complicated dance that can go catastrophically wrong in diseases such as cancer.
With his group as productive as ever, John himself is currently back at the lab bench, getting stuck into his own project. It’s only a “minor thing”, he jokes: he wants to reconstruct what the replication machinery looked like in the last common eukaryotic ancestor, around two billion years ago.
Clearly as keen on ambitious questions as he’s always been, he has a message for today’s PhD students and postdocs: “the right questions are interesting, meaningful, and answerable, but don’t look for just the known unknowns”, he says. “Find a place like the Crick where 30-year questions are welcomed, believe in yourself, be optimistic, and who knows what you’ll discover.”
