For the last quarter of a century, the Clare Hall Laboratories have been a highly productive focal point for research into DNA repair and replication. Never numbering more than 10 small groups, scientists at Clare Hall look out over a landscape of fields, farms, and riding stables, shortly after the last suburbs of North London give way to the placid Hertfordshire countryside. The muddy tranquillity of their surroundings is only slightly disturbed by the distant hum of traffic from the M25 and the A1 motorways, which pass nearby. It is a far cry from the urban buzz surrounding Clare Hall’s metropolitan big brother in Lincoln’s Inn Fields.

By rights, Clare Hall should not have been a success. It was isolated, too small, too focussed­—any of these factors could have been a death sentence. Although the importance of the steady financial support of the ICRF and, latterly, Cancer Research UK, should not be underestimated, the many achievements of those working there are a testament to motivated, smart science, fierce collegial loyalty, and a dedicated and exacting Director.

Unfortunately, a chapter extolling the talents and virtues of all of the inhabitants of Clare Hall would be impossibly long and most likely very tedious; a litany of success can be as good a soporific as any other list. The four protagonists in the pages that follow have therefore been selected for two reasons. Firstly, the two men responsible for the birth and development of Clare Hall were both in the vanguard of the DNA revolution, and their work marches with the evolution of research into replication and repair. Secondly, the stories that complete the chapter are perfect illustrations of what Clare Hall does best: hiring very good people, and then trusting their abilities to turn ambitious, long-term projects from ugly ducklings into swans.

John Cairns and Mill Hill, ‘That Awful Place’

John Cairns, 2002.

(Photograph courtesy of CRUK London Research Institute Archives.)

John Cairns is a fascinating figure in the landscape of 20th century biology. Switching fields with the ease of a small boy scrambling over a fence, he left his mark on everything to which he turned his hand, leaving a trail of significant papers in each discipline before moving on to the next. As an added bonus, he is one of the most thoughtful and elegant chroniclers of the molecular biology revolution, in which he was both a distinguished participant and an observer, and his subsequent writings on the biology and epidemiology of cancer are still relevant and thought-provoking today. The esteem and affection in which he and his wife Elfie are held by their many friends and acquaintances are a tribute to their unique combination of charm, intelligence, and hospitality.

In 1957, John left Elfie and his young family in Canberra for a four-month sabbatical at Caltech, and by a combination of good luck and poverty, found himself at the epicentre of the DNA revolution. Unable to afford the exorbitant room rates at the Caltech Faculty Club, John was rescued from homelessness by Jan Drake, one of Renato Dulbecco’s PhD students, and as a result found himself eating dinner most nights in an apartment shared by Drake, Matt Meselson, and Howard Temin during the period in which Meselson and Frank Stahl were doing what Cairns subsequently christened “the most beautiful experiment in biology.”

Meselson and Stahl’s 1958 paper, “The Replication of DNA in Escherichia coli,” was the first big step towards the wider acceptance that DNA really was the hereditary material of the cell, and not just a boring structural component of chromosomes, as many of the biochemists and physical chemists who worked on it had previously decided. Oswald Avery, Colin MacLeod, and Maclyn McCarty’s 1944 experiment showing that the “transforming principle” of Streptococcus pneumoniae was DNA, and Al Hershey and Martha Chase’s more recent demonstration in 1952 that the infectious material of a phage was also DNA, had not made much of an impact outside the world of molecular biology, perhaps being regarded with suspicion as new-fangled genetic sleights of hand. Watson and Crick had suggested in 1953 that the invariant base pairing of the rungs of the DNA ladder, with cytosine (C) always with guanine (G), and adenine (A) always with thymine (T), provided a great mechanism by which one strand could produce a complementary copy of itself, but it was just another theory until Meselson and Stahl gave it reality. (It should be noted that the invariant C:G and A:T ratio between the bases was originally observed by Erwin Chargaff, but he failed to realise the significance. He was, consequently, very grumpy about DNA and Watson and Crick for much of the rest of his life.) Although they were careful not to overinterpret their experiment, referring cautiously to two DNA subunits, Meselson and Stahl’s work showed that the two strands of the DNA double helix came apart and were each used as a template to make a new strand. The result was a duplication of the original molecule, with each duplicate containing one old and one newly synthesised strand; in other words, DNA was replicated semi-conservatively.

Kornberg, as the host of stories lovingly propagated by his friends and colleagues testifies, was in his own way as charismatic as Delbrück but was an implacable dragon in the laboratory. His allegiance to biochemistry meant that anything other than total commitment and experimental rigour was unacceptable, encapsulated in the maxim that he adopted from his fellow biochemist Efraim Racker: “Don’t waste clean thinking on dirty enzymes”; and the first of his famous Ten Commandments of Enzymology: “Thou shalt rely on enzymology to resolve and reconstitute biologic events.” Kornberg’s genius was to realise that everything, even the most complex molecular machine, could be purified out of the cell, and that reconstituting such machines in the relative tranquillity of the test tube was the way to understand how they worked. His interest in DNA arose because he wanted to know how nucleotides, which he and others had shown were the building blocks of DNA chains, were put together. The fact that DNA might be the hereditary material and that Watson and Crick had proposed a model for its double-helical structure was interesting, but the possibility of proving that DNA synthesis was not one of life’s untouchable mysteries, but as much an enzymatic process as the fermentation of yeast, was what initially drove him. From 1954, when he was appointed Chair of the Microbiology Department at Washington University in St Louis, Kornberg worked towards detecting the polymerisation of nucleotides, beginning first with RNA and then widening his studies to include DNA synthesis.

Having heard that Roy Curtis at Oak Ridge National Laboratory had isolated a mutant of E. coli that made miniature daughter cells containing no DNA but lots of Kornberg polymerase, John set Paula the difficult task of finding the reciprocal mutant of E. coli, one that lacked Kornberg’s polymerase but was perfectly viable. This she did, and the mutant, which John christened polA in homonymic tribute to Paula’s technical virtuosity, caused a flurry of biochemical activity resulting in the isolation of two further E. coli DNA polymerases, one of which, DNA polymerase III, was the real replicative enzyme. Arthur Kornberg’s chagrin that a mere molecular biologist had managed to show something so important without so much as partially purifying a protein, was no doubt mollified by the fact that both new polymerases were identified by his son Tom, whilst a graduate student in Malcolm Gefter’s lab at Columbia.

The business of bacterial DNA replication was not to occupy John for much longer. Keeping Cold Spring Harbor afloat had comprehensively emptied the coffers of the Cairns family, and in 1972, John started job-hunting back in the United Kingdom, where his children could finish their educations for rather less than in the States. In order to be on the spot, he arranged a year’s sabbatical with Michael Stoker at Lincoln’s Inn Fields. It was not long before Stoker realised that John was the perfect solution to a problem that had been dogging him for some time—whom to appoint as the new head of “that awful place” Mill Hill.

The mere existence of DNA repair had come as a big surprise to many people. In the first half of the 20th century, genes were a concept, rather than a fact, and working in such an information vacuum meant that some interesting misconceptions had arisen. One such, in part propagated by the 1935 paper by Max Delbrück, Nikolai Timoféeff-Ressovsky, and Karl Zimmer that had inspired Schrödinger’s What Is Life?, was that it was incredibly hard to mutate genes. To create any kind of change, exposure to extreme stress such as high-intensity ionising radiation was thought to be necessary, because genes were viewed as extremely stable entities, heavily protected from the outside world in order to keep their precious cargo of genetic information intact. This was backed up by the very low rate of spontaneous mutation seen in the organisms, such as the fruit fly, in which the early geneticists did most of their work.

What everybody had missed, and could probably not have predicted before DNA came onto the scene, was that far from being swaddled tenderly in the warm, cosy environs of the nucleus, DNA was continually buffeted by storms of passing mutagens. The reason for the low rate of spontaneous mutation was not that genes existed behind a firewall, but that they were dynamically stable. Like a circus plate-spinner, the cell keeps its genetic information intact because it continually surveys its DNA, detects any damage, and repairs it. Jumping ahead in time, David Lane and Lionel Crawford’s p53 protein is a sophisticated gatekeeper of this determination to preserve an unblemished genome.

Because sunlight and life go hand-in-hand, enzymatic photoreactivation of DNA after ultraviolet (UV) irradiation is probably the oldest repair mechanism in existence. It was also the first identified, stumbled on inadvertently by Albert Kelner at Cold Spring Harbor, and Renato Dulbecco during his time in Salvador Luria’s lab in Bloomington, Indiana. Both Kelner and Dulbecco had been greatly frustrated by their attempts to examine the survival of, respectively, bacteria and phage, following UV irradiation. Both men found that their experiments were completely irreproducible. Sometimes, irradiation resulted in perfect survival curves, where colony number was inversely correlated with the intensity of UV treatment, but sometimes, the curves were all over the place, with seemingly random numbers of survivors cropping up even after punitive UV treatment. After months of checking and rechecking his experimental protocol, Kelner in desperation moved his bacterial plates into a cold room, so that he could work out, using a series of water baths, whether temperature played any part in the process. To his surprise, all of the survival curves began to behave perfectly, but shortly afterwards, the penny dropped: The cold room was windowless, in contrast to his previous lab, and the more sunlight the plates saw, the more colonies grew on them. Dulbecco, with the same problem in his phage experiments, and knowing of Kelner’s work, came to the same conclusion, that daylight could induce repair of UV damage, and their two papers on the subject were published in 1949.

Photoreactivation was just the tip of the repair iceberg. In 1964, Dick Boyce and Paul Howard-Flanders at Yale, and Bill Carrier and Dick Setlow at Oak Ridge National Laboratory, with several other labs hot on their heels, independently discovered an alternative mechanism for ridding the cell of pyrimidine dimers: nucleotide excision repair. Rather than rescuing the bases by remonomerising them, cells could simply cut them out, together with a short stretch of the sequence in which they were embedded, leaving a gap that could be filled in using the opposite strand of the double helix as a template.

There were a few lone voices. Back at Mill Hill, John Cairns had not joined the molecular biology stampede into tumour virology but was bucking the trend to think instead about the unfashionable topic of somatic mutation and cancer. In 1973, Bruce Ames had devised his eponymous test, in which potential carcinogens were assayed for mutagenicity in bacteria rather than in animals. Ames’s work brought into focus the long-standing observation that mutagenesis and carcinogenesis are not always linked; the incidence of cancer in a particular species is not solely determined by exposure to mutagens, and the most powerful carcinogens are not always powerful mutagens. Clearly, something else was going on. In addition to the obvious, that there might be a disparity in DNA repair efficiency, John came up with the Immortal Strand Hypothesis, the idea that all stem cells carry an original, error-free copy of the body’s DNA and that they achieve this by asymmetric replication, passing on newly synthesised strands, into which the error-prone polymerases might have inserted mistakes, to their less important progeny. The theory, although probably wrong, greatly stimulated thinking about how stem cells might retain their identity, and it was in its pursuit that John and his PhD student Leona Samson fell into the DNA repair field, where they were to discover an entirely new enzymatic process.

Using a method originally designed to determine whether bacteria replicated asymmetrically (they didn’t), Leona started testing whether the mutation rate of bacteria was proportional to the mutagen concentration, using an alkylating agent, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), which works by sticking methyl groups onto DNA. Depending on where the methyl group is put, the effect of MNNG ranges from mild to severe; in the worst-case scenario, a guanine residue can be turned into O⁶­-methylguanine, which a DNA polymerase can mistake for adenine, meaning that a thymine instead of a cytosine residue is incorporated into the opposite strand of DNA during replication.

By the time the Robins and Cairns paper was published, some unsettling nonscientific events were also occupying John’s time. By the end of the 1970s, it was clear that there was no future for the ICRF in Mill Hill, because the MRC were determined to take the building back upon the lease’s expiry in 1986. Walter Bodmer, who took over from Michael Stoker as ICRF Director in 1979, remembers that when he “talked to the MRC about the end of the lease, they insisted on having it back as agreed, and so got a bargain” (the original agreement stipulated that the MRC would pay the ICRF a mere £10,000 upon the reversion of the lease). The unit’s scientists were understandably greatly dismayed. John Cairns had succeeded in a very short time in rekindling the Mill Hill laboratory’s reputation as a place where good science was done, and his developmental biology initiative was blossoming spectacularly, so everyone there was determined to try to stick together, come what may. Unfortunately, the price for this decision was the loss of the unit director. John recalls:

John left for the Harvard School of Public Health in 1980, in retrospect a bad idea, because he has described it as “something of a hell hole.” He could have stayed in England: “the other option was to go to Cambridge and be with Sydney [Brenner]. And I had a date to go and talk to Sydney about this, and I just damn well forgot!” British science lost the opportunity for a memorable double act.

John’s strategy of falling on his sword may not have provided an optimal outcome for him, but it had the desired wider effect, saving the jobs of his younger colleagues and paving the way for his successor. Very luckily for the ICRF, romantic necessity had already managed to recruit an eminently suitable candidate as John’s replacement: Tomas Lindahl, the biochemist who was in the process of nailing how John’s Ada protein worked, had fallen in love with Beverly Griffin, still working on the Fifth Floor at Lincoln’s Inn Fields, and was looking to move from Gothenburg in Sweden to join her in London.

Phoenix Rising—Tomas Lindahl and the Birth of Clare Hall

Tomas’s discovery, that the tRNA, rather than being chewed up by ribonucleases, was spontaneously decomposing as a result of being heated during the experimental protocol, prompted him to wonder whether DNA might also be prone to such damage:

I thought that if a small molecule like RNA actually decomposes at a rate that you can see in the lab, what about DNA? It’s supposed to be a bit more stable but still, perhaps DNA is less stable than people have been thinking about. Nobody was working on that so I put that aside, and just to show the slow pace of research in those days, I went to Rockefeller and was a postdoc there for two years, and then I went back to Sweden and set up my own lab there and started doing some enzymology on enzymes like DNA ligases. Three years after I’d done those initial experiments in the US, I thought: “I’ll go back and have a look at the stability of DNA because nobody else is doing it.”

In tracking down how the cytosine-to-uracil error was fixed, Tomas and his lab unmasked an entirely new class of repair mechanism: base excision repair. Uracil is detected and chopped out of the DNA chain by a glycosylase, an enzyme able to cleave the bond between the errant base and the backbone, leaving the double-stranded backbone intact. A gang of other enzymes then takes over, chopping out the sad remains of the original nucleotide together with its nearest neighbours, mending the gap using the opposite strand as a template, and finally stitching up the break.

Tomas’s strategy of looking at repair as a response to intracellular rather than extracellular insults was an innovative approach that threw up numerous other ways in which DNA can be damaged just by the cell going about its normal daily life. However, these attacks by the toxic by-products of metabolism are valiantly fought off by an arsenal of DNA repair mechanisms that keeps the genome in surprisingly good shape; a recent paper comparing the genomes of parents and offspring showed that of the 3 billion base pairs of human DNA, only around 70 had been mutated between the generations.

Somewhat remarkably, whilst Tomas was making a name for himself working on the enzymology of DNA repair, he had also managed to become a big noise in the tumour virus world. As he recalls: “I was doing two careers in parallel because I was doing the work I liked, DNA repair and DNA metabolism, and my chairman in Stockholm, Peter Reichard, came and said ‘That’s all very well, but nobody else in Sweden is doing that, so it would be good if you did something else so you can talk with people.’ ” Reichard suggested a collaboration with the eminent virologist Georg Klein, a fellow Karolinska inmate, who worked on Epstein-Barr Virus (EBV), and thought the time was ripe for someone to start some EBV molecular biology. The collaboration quickly bore fruit, with Tomas making the major discovery that EBV replicated in cells as a discrete circle, rather than by the SV40 and polyoma strategy of integrating into host DNA.

Coming to London to be with Beverly turned out to be a good career decision as well as the start of a life partnership that has endured to this day. Because the ICRF Director Walter Bodmer was a human geneticist, he was more clued up than most cancer biologists about the importance of somatic mutation and therefore DNA repair, and willingly subscribed to the idea that it was a good area for a cancer charity to fund. After a short time on the Fifth Floor with Beverly, Tomas set up a lab at Mill Hill, taking over John Cairns’s space there. He also reverted to monotheism, stopping his EBV projects to work entirely on DNA repair: “For a happy year or two, we discovered about one important DNA repair enzyme every year, and I thought: ‘This won’t last forever, this actually creates a new branch of the DNA repair field that will hopefully become important.’ So I decided rather than getting an ulcer and trying to be on top of two fields at the same time, to put viral work aside.”

Beverly Griffin and Tomas Lindahl, 2002.

(Photograph courtesy of CRUK London Research Institute Archives.)

Tomas clearly being a good researcher, Walter was very anxious to keep his new recruit happy and to involve him in the ongoing problem of what was going to happen to the scientists at Mill Hill. In 1982, Walter cut a deal that allowed the Mill Hill developmental biologists to move into a new, ICRF-funded unit at Oxford University, but the problem remained of what to do with Tomas. Shoehorning him into Lincoln’s Inn Fields was not an option that Walter, always an enthusiastic expansionist, favoured, and the healthy economic climate meant that there was money to spare for something more ambitious. The animal and service facilities at Mill Hill and Lincoln’s Inn Fields were going to be unified and expanded on the site of Clare Hall, an empty and decaying ex-smallpox and TB hospital near the newly built M25 motorway, so perhaps it would be possible to persuade Tomas to go there too?

Clare Hall laboratories.

(Photograph courtesy of CRUK London Research Institute Archives.)

Having achieved the building he wanted, Tomas, now officially appointed Director of the Clare Hall Laboratories, decided that the labs would be very focused: “I was very concerned it wouldn’t be like the classical biochemistry or cell biology department that you see in universities, where because of the teaching, you have to be very broad like an old fashioned zoo—one zebra, one lion, one giraffe and one turtle and so on! That’s obviously an exaggeration, but if you have as your next door neighbour an expert on lipid metabolism it’s not perhaps somebody you easily share a seminar series with if you do nucleic acid or molecular biology work.” Not surprisingly, the focus that Tomas chose was his own—the biochemistry and metabolism of DNA, and the three Rs of Repair, Recombination, and Replication.

Sustained institutional success in science is a rare and interesting beast, seemingly dependent on a single factor: appointing the right person to be in charge. This certainly seems to have been the case with Clare Hall. Matched only by Cold Spring Harbor in terms of the scientific quality and impact of its publications, with its occupants clanking around under the weight of their many awards and honours, Clare Hall’s success revolved around its Director’s abilities. Tomas had an extremely good eye for hiring stars in the making, but turning Clare Hall into a place where the stars wanted to stay long term was an even greater feat.

Intellectual powerhouses cannot thrive long term unless their occupants are reasonably happy with their lot, and although Tomas’s egalitarian management style was greatly appreciated, he had two other invaluable assets, in the persons of Brenda Marriott and Frank Fitzjohn. With Tomas, Brenda and Frank formed a triumvirate that dealt with anything getting in the way of doing good science, and their importance to Clare Hall’s success cannot be overstated.

Frank’s job of Clare Hall lab manager was dreamt up by Tomas after his time on the Fifth Floor at Lincoln’s Inn Fields. In the frenetic atmosphere there, communal equipment was frequently broken, radioactive, or both, because nobody was prepared to take responsibility for fixing it. Anyone who has experienced the homicidal rage generated by standing in front of a broken centrifuge, holding a rack full of laboriously prepared and rapidly disintegrating samples, can understand the importance of having someone to ensure the smooth running of a lab, and Frank, in the nicest way possible, was a combined troubleshooter and vigilante without parallel. Bill House, who had hired him into the position, had simply described his job as: “Look after Lindahl,” and Frank took him exactly at his word. Whatever was required, from guiding fundraisers round the labs (elderly lady: “How many people work here?” Frank: “About half of them.”), to climbing into the drains to check them for radioactivity, fell within Frank’s remit. The move from Mill Hill to Clare Hall was seamlessly arranged and executed by Frank (Tomas: “Move us to Clare Hall, Frank, I’ll see you there in three weeks”), and thereafter, successive generations of scientists benefited from Frank’s efficiency (David Lane: “The good thing about Frank was, you asked him a question and he always said yes!”).

Tomas describes his two lieutenants thus:

If Clare Hall became a success I can take some of the credit for that, but some of the credit is due to Brenda and Frank. They created [a] very efficient buffer, so when people wanted something, including lab heads, they would first go to Brenda and Frank, [who] had a very good sense when a scientist tried to take selfish advantage or when there was a real request.

Brenda is a very outgoing person, and people came and told her all kinds of things. She may have told me something like 5% or 10%, and she sifted the information I needed to know, because if she had told me everything, people would quickly find out and nobody would talk with her any more! There were things that perhaps I should have known that I didn’t know about, but more importantly, if there was something, she would come and say, do you know this, do you know that? And Frank was the same, and he also kept a good eye on the place, so if you found a dirty ultracentrifuge he would backtrack and find who had done that. Both he and Brenda had the sort of personalities that meant nobody on the site was keen to pick a fight with them or be told off by them. So they ran the place, and I was more the last instance when there was real disagreement.

Despite the stresses and strains of being Clare Hall Director, Tomas managed to go on working very productively. Uncovering the multitude of weird and wonderful enzymes involved in base excision repair was to take up much of his subsequent career, although he also made some notable diversions. One of these came to fruition in 1988, the year Tomas was elected to the Royal Society, when he and his postdoc Rick Wood made the first breakthrough towards identifying the UV-repair enzymes defective in xeroderma pigmentosum. Tomas promoted Rick into an independent position, and in 1995, Rick’s lab purified and reconstituted in vitro the human enzymes responsible for nucleotide excision repair. This tremendous feat gave Clare Hall the double in the excision repair biochemistry championships, because in 1994 and 1996, Tomas’s lab completed the equally laborious task of reconstituting base excision repair in vitro, using, respectively, the E. coli and human enzymes, some of which were remarkably conserved across the yawning evolutionary gulf.

In 2010, in recognition of his seminal contributions to the understanding of the biochemistry of DNA repair, Tomas was awarded the Royal Society’s Copley Medal, the oldest scientific prize in the world, whose other recipients include Michael Faraday, Charles Darwin, and, latterly, Stephen Hawking. However, by this time, the other inhabitants of Clare Hall were rapidly catching him up in the scientific eminence stakes. Two in particular, Steve West and John Diffley, have used the enlightened environment of Clare Hall to undertake ambitious, long-term projects, the anathema of the normal three-to-five-year grant cycle. Their stories, which complete this chapter, illustrate everything good about Tomas and the lab he built with such success.

Steve West and Genetic Recombination

Steve West, the last group leader to make up the new Clare Hall intake, had not intended to like Clare Hall at all, having only come for an interview from his then workplace, Yale, because he fancied a free trip to visit his mum in Yorkshire. However, almost three decades on, he is still an inmate, one of the big beasts of the DNA repair world, and probably Tomas’s most inspired hiring. As well as all the normal attributes that a great scientist requires, Steve’s reputation rests on his eye for major problems, his extraordinary skill as a biochemist, his phenomenal workrate, and his tenacity. All of these are exemplified in a knotty problem that Steve brought back with him from the United States, and that in the end took 25 years to solve: how does the cell cut through the tangled products of genetic recombination to regenerate two functional double helices?

Until the 1960s, genetic recombination was viewed by most biologists as an esoteric problem worked on by a small number of fungal geneticists. The exchange of information between two strands of DNA is essential for evolution and happens every time two people get together to reproduce, but pre–Watson and Crick, its study was caught in an intimidating briar patch of statistical analysis and complicated hypotheses. However, like everything else in biology, the DNA revolution meant that the geneticists’ ideas could be reformulated in terms of the interaction of DNA molecules, opening the way for biochemists and molecular biologists to start thinking about exactly how recombination worked.

Early on in the history of recombination research, it became evident, from work performed by Dick Boyce and Paul Howard-Flanders, the codiscoverers of excision repair, that homologous recombination was also very important for repairing DNA damage. The most dangerous hit a DNA molecule can take is one that breaks both its strands simultaneously, because there is then no template from which to repair the lesion. Like a snapped rope, the flailing strands can be spliced to anything else that happens to be around, or simply left dangling, and the consequences to the cell can be disastrous: Large- and small-scale mutation and problems with replication lead inexorably to the catastrophes of cell death or (in multicellular organisms) cancer. Homologous recombination is one of two mechanisms for fixing double-strand breaks and averting these unpleasant fates.

In 1965, Alvin Clark and Ann Dee Margulies, working in Berkeley, published a set of E. coli mutants defective for both recombination and repair of UV damage, one of which they named RecA. By the mid-1970s, it had become clear that whatever the RecA protein did, it was central to both recombination and repair. A great deal of effort was expended to discover its identity, culminating in 1977 with the publication of three papers detailing the purification of RecA protein. One of this trio was an early appearance in print of Steve West, then a young lad from Hessle, near Hull, doing a PhD whilst supplementing his income DJ-ing round the student haunts of Newcastle.

Steve’s project was to work on a mystery protein, unoriginally called Protein X, which appeared in vast quantities following irradiation of E. coli. It was not a subject of great interest to his supervisor: “Peter was famous for saying: ‘Protein X is for the birds—why are you bothering with this stupid thing?’ ” but Steve hung on, reckoning that if a cell made something in such large amounts, it had to be important. He was more than vindicated when he discovered that Protein X was in fact RecA, and the 1977 paper describing this finding set Steve well on his way in the world of DNA metabolism. He moved to Yale to the lab of Paul Howard-Flanders, who turned out to be an understated Englishman with an almost pathological disinclination to dictate his new postdoc’s research direction. Under the circumstances, the easiest path for Steve was to simply continue with his RecA work, which in any case was still very interesting, so this he duly did.

Steve West, 1978.

(Photograph courtesy of Steve West.)

Life at Yale was very happy for a single-minded biochemist: “I stayed all night, worked fifteen hours a day, seven days a week. I used to take my girlfriend to a Greek restaurant near the lab, and the nice thing was, I could nip out between courses and change dialysis buffers, and she could stay there and go for a dance—it was one of those little restaurants where they do Greek dancing—until I came back from changing the buffer. She thought the mad scientist thing was cute.” Much to Steve’s good fortune, this amazingly tolerant person eventually agreed to marry him.

I came in on Christmas morning, and half the tritium was still in the ³²P, and half the ³²P was still in the tritium. So I called Paul at home at lunchtime on Christmas Day, and I said: “I’ve got this really weird result and I just don’t understand it.” … And there was this absolute silence on the phone, like a minute. Paul was actually a little strange in that he would have these speech gaps, he would stop talking for some time, but this was abnormally long. So I’m sitting there, and a minute goes by, and he said: “Well Steve, if it’s a recombination protein, maybe that’s what it’s supposed to do.” And at that point I realised it had done strand exchange! And then we were both kind of silent to each other on the phone, because it had done strand exchange—it had done something which was miraculous. And that was cool—a cool moment.

At this point, having been at Yale for five years, Steve knew it was time to go on the job market, for which he needed an interesting project that was entirely his own. The mechanism of Holliday junction resolution was a complete black box, so, being nothing if not ambitious, Steve decided that he would go hunting for the enzymes involved, with the goal of getting the whole of homologous recombination to work in vitro. In his last two years in Paul’s lab, he set up the basics for the resolvase project, doing the lab three-week preps to make figure-of-eights, cracking open bacteria to make protein extracts, and seeing if any of the extracts could resolve the figure-of-eights in vitro. Much to his credit, Paul funded all of the work, but wanted none of the glory, a practice that Steve maintains: “He was fabulously generous and I try to do the same with people in the last year they’re with me—do what you want, I don’t care, and I don’t want to be on the paper. And it’s good, it allows them to have their own independence.”

Despite having some good job offers from places in the States, Steve was seduced into coming to Clare Hall after meeting Tomas at an end-of-conference banquet in Cambridge:

Steve duly arrived in his empty lab space in 1985 and got on with his resolvase-hunting experiments. Things had become a little easier since he’d begun them, because the three-week figure-of-eight preps had vanished into the past, in favour of sticking together four radioactively labeled artificial oligonucleotides (short lengths of DNA, in this case, 60 nucleotides long) to create a cruciform structure that was a perfect in vitro mimic of a Holliday junction. With this innovation, introduced by Nev Kallenbach at the University of Pennsylvania in Philadelphia, experiment times were reduced to afternoons, rather than weeks, and you could tell whether the junction had been cut simply by running the reaction products on a gel and seeing whether any of the original oligonucleotides had changed size from 60 bases long to 30. By 1989, Steve and his first postdoc, Bernadette Connolly, could definitely get resolution of the cruciforms by adding E. coli extracts to them, but try as they might, they simply could not purify the responsible enzyme from the bacterial protein soup. At this point, having exhausted the heavy guns of biochemistry, Steve decided to deploy the more subtle weapon of genetics, calling up his mate Bob Lloyd, who was one of the best geneticists in the field. Steve and Bernadette had already dabbled with making protein from known bacterial recombination mutants, to see if any of them were defective in resolvase activity, but all had been able to resolve cruciforms with no problem, showing that they must still contain functional resolvase. However, Bob Lloyd had four new mutant E. coli strains—ruvA, ruvB, ruvC, and orf26—which he was happy to send down from his lab in Nottingham to Clare Hall.

Steve’s lab was now on a roll and showed very quickly that the RuvA and RuvB proteins were responsible for branch migration, acting together as a molecular motor that could move Holliday junctions along DNA until the RuvC protein, squatting on top of them, found a sequence that it liked, and cut the DNA to resolve the junction. Thanks to his work, and that of a few other labs, notably that of Hideo Shinagawa in Osaka, the mechanics of homologous recombination in bacteria were pretty much sorted out, and not a moment too soon; research into DNA repair and homologous recombination was about to become extremely trendy.

The sudden upturn in interest came about after two key findings. Firstly, the machinery of DNA repair and recombination was shown to be evolutionarily conserved to a remarkable degree, such that close relatives of many of the bacterial enzymes were found in mammals, and, secondly, mutations in the repair machinery started to crop up in cancers, beginning with the discovery in 1993 that a form of hereditary colon cancer was caused by defects in the repair of mismatched nucleotides. The tentative connection between DNA repair and human health, first made by Jim Cleaver in his xeroderma work, had finally clicked firmly and irrevocably into place.

Steve, somewhat unwillingly, was dragged out of bacteria into mammalian cells, although the Clare Hall facilities made the transition less alarming:

As well as making a start on the search for the mammalian version of the RuvC resolvase, Steve had not forgotten his first love, RecA. In 1994, his lab was the first to purify its human homologue, RAD51. RAD51 coats single-stranded DNA to create a nucleoprotein filament that invades a homologous double helix, starting the process of recombination in a very similar way to RecA. The subsequent discovery that RAD51 was both positively and negatively regulated by the BRCA2 protein in response to DNA damage brought this work to centre stage in cancer biology. BRCA2, like p53, is a tumour suppressor, essential for guarding the cell from possibly oncogenic mutations. Without it, homologous recombination is defective, and double-strand breaks can only be repaired by a more error-prone mechanism called nonhomologous end-joining, leaving the cell extremely vulnerable; that mutation of BRCA2 is found in some 10% of inherited breast cancers is therefore no surprise.

As the West lab grew and diversified, they continued to take on and meet multiple biochemical challenges in mammalian cells, all relating to defects in DNA repair and their effects on disease. They worked out that the huge BRCA2 protein, a nightmare to handle because it is so big and likes to fall apart, controls the ability of RAD51 to bind DNA, and they sit at the forefront of research into repair-linked diseases such as Bloom’s syndrome, Fanconi Anaemia, and the unpleasant progressive neurological condition Ataxia with Oculomotor Apraxia 1. For all these achievements, Steve has won many awards and has been a fellow of the Royal Society since 1995. However, through all the years of success, Steve carried another project close to his heart, a failure-ridden enterprise that, despite its promising start, was foundering in a sea of dashed hopes and postdoctoral frustration: the quest for the mammalian resolvase.

The century turned over, and Steve was elected to the Academy of Medical Sciences and won the Leeuwenhoek Prize of the Royal Society, capping that in 2008 with Europe’s most prestigious scientific award, the Louis-Jeantet Prize for Medicine. The resolvase project still languished in the doldrums and had even lost its sad little entry in the London Research Institute Annual Report. The project was so unsuccessful and such hard work for people that it gained an unhealthy reputation as a postdoctoral graveyard. In the end, Steve had to let it go fallow for two or three years because he simply couldn’t talk anybody into trying it.

Uli slowly worked his way through the TAP-tagged yeast library, whilst Ip got on with the brute-force purification. And, finally, after 1100 TAP-tagged protein assays and Ip spending so long in the cold room running samples that he could have walked to the Antarctic and back, there came an afternoon in 2008 when Steve looked up from his computer and saw his heroic resolvase postdocs framed in the doorway of his office: “Uli, Ip and Miguel stood at my door and there were tears running down Ip’s cheeks, because they knew they had it!” Almost simultaneously, Uli had isolated the yeast resolvase, Yen1, and Ip had found the human one, GEN1. Amazingly, they were homologous to each other but were entirely unrelated to RuvC, their bacterial counterpart. The 20-year journey from E. coli to humans had ended at last.

The three resolvase postdocs.

(Photograph courtesy of Steve West.)

There would be many more twists and turns to come in the resolvase story, but for Steve, finding Yen1 and GEN1 was one of the biggest thrills of his career, easily up there with his Christmas morning realisation that RecA could do strand exchange: “It was a fantastic little story. It’s fantastic just in terms of the ICRF and Cancer Research UK, because you could not have done this anywhere else in the world. You could not find any other funders who would fund the same project for twenty years. You had to be able to do this sort of thing without justifying it—it was at the point where it was becoming unjustifiable just to keep doing it.”

John Diffley and DNA Replication

In 1990, David and Birgit Lane were lured away from Clare Hall by the University of Dundee, giving Tomas an opportunity to hire new faculty, but also a problem. The loss of the Lanes triggered an outbreak of mingled pessimism and Schadenfreude in some parts of the scientific community; how would such a small, quiet place recover from the departure of two of its biggest guns? The notion that Clare Hall was doomed was soon scotched, however, by the surprise announcement that one of biochemistry’s most visible and popular figures was moving there. Tim Hunt, who, most people thought, would only leave his beloved Cambridge when carried out feet first in a wooden box, had decided to abandon the creaky environs of the university Biochemistry Department for somewhere more congenial and better equipped, where he could concentrate on research rather than teaching. Tomas was delighted with his coup: “I was obviously very happy to take on somebody with Tim’s high reputation. It gave me some personal satisfaction when people said to me, ‘Oh dear, who are you going to find to replace David Lane?’ and I was able to say ‘Tim Hunt!’ That really shut them up—none of them expected that answer!” Tim was equally pleased with his decision: “It was just wonderful suddenly to be surrounded by all these biochemists. Steve West was a total revelation to me when I went there. I was amazed—here was this guy putting recombination on the biochemical map. Sydney [Brenner] was always interested in recombination, but it was always phage genetics and that sort of thing—very very hard to understand.”

Having had to figure out his next career move by himself as well, John got into Bruce Stillman’s lab on the strength of the two extremely competent papers on polymerase purification that he published during his PhD. Any normal person, especially one coming out of John’s situation, would probably have taken advantage of the new lab’s expertise and done a solid biochemical project on DNA replication. However, John is nothing if not unusual: “I started doing yeast genetics, and again I taught myself. Bruce is a great biochemist but he doesn’t do yeast, so I set up yeast in the lab. Everyone else was doing SV40 replication and I was doing yeast—I guess there’s just a level of contrariness in my personality!”

Unsurprisingly, in the 30-odd years since Kornberg had found the first DNA polymerase, an awful lot had been discovered about the enzymes responsible for replicating DNA: Helicases unwound the DNA double-helix; primases started the new strands off; multiple types of polymerase, tailored to particular tasks, and each equipped with an editing function to cut out mismatches, got on with elongation; sliding clamps held the replicating strands of DNA steady; and ligases sewed the new pieces together to make the final product. The whole process resembled a busy factory production line, and although there was still considerable interest in the individual components of the process, attention was shifting to finding out how it was regulated. What was responsible for triggering replication, and how does the cell know to switch the replication machinery on once, and only once, during the cell cycle?

John Diffley, ca. 2010. (Photograph courtesy of CRUK London Research Institute Archives.)

Like Steve West before him, John applied to Clare Hall for less than scientific reasons; he fancied a trip to England, where he’d never been, and the ICRF job advert he’d seen in the back of Nature would pay his airfare. However, once he got here, the Clare Hall magic took over. Tomas remembers the stringent recruitment process very well: “I think I took him to the local pub and it was Sunday afternoon, and he was sitting looking out at the slopes of the ridge up there, a very nice British early summer day.” John met the other group leaders, liked them, liked their work, liked the extremely generous starting offer, and decided to take the job: “The similarity to Cold Spring Harbor in terms of isolation is what appealed to me when I came here. I saw a lot of the same things in it and it either suits you or it doesn’t. I did miss the people passing through Cold Spring Harbor at first—I started having to read the literature again! When you’re at Cold Spring Harbor you don’t have to read anything as you find out about it.”

Following John’s arrival, Tomas was slightly dismayed to find that his new recruit had, temporarily at least, abandoned biochemistry. Taking his example from the transcription field, where people were having a lot of success in reconstituting transcription in vitro by simply adding cell extracts to DNA templates, John had spent the end of his postdoc trying to do the same with replication but had completely failed. It was a perfectly sound strategy, but what he didn’t know at the time was that replication is so tightly controlled that most of the proteins involved are either absent or held in an inactive state until immediately before they’re required; adding random extracts was never going to work. In the face of this defeat, John decided to develop some approaches to look at what was sitting on the yeast origins of replication in vivo, in the cell itself, and for this, he had to use an extremely fiddly technique called genomic footprinting.

The result of John’s departure from biochemistry was a Nature paper in 1992, identifying a complex, called the origin recognition complex, or ORC, that was bound to yeast origins of replication in real time in the cell, as well as in a test tube. However, there was a lot more to be done. Although ORC was clearly sitting on origins of replication, it was unlikely to be what determined when those origins fired during the cell cycle; it was bound to DNA almost all of the time, and the protein that flicked the “on” switch was likely only to be there right before it was needed.

John’s lab expanded their footprinting operations and found that one particular DNA sequence in yeast replication origins was only occupied by proteins during a phase of the cell cycle called G₁, which occurs immediately before S phase, the point where the genomic DNA is replicated. The new data appeared in a major paper in Cell that showed that this protein complex, dubbed the Pre-Replicative Complex (preRC), was very similar to Xenopus Licensing Factor, an activity in frog eggs that was required for initiation of replication. That similar complexes were found in two such dissimilar organisms suggested that the control of replication regulation might be broadly the same in all eukaryotes, and this has, indeed, turned out to be the case.

The preRC is the thread running through all of John’s subsequent research, and 18 years on from the first in vivo footprint, John’s lab finally achieved its in vitro reconstitution. This scientific and technological tour de force put a vital piece into the puzzle of how replication is limited to a one-time event during the cell cycle, because regulation of the preRC turns out to be the most important thing. The cell has evolved an elegant system for preventing origins from firing more than once: only allow the preRC to be assembled at a particular time in the cell cycle, and only allow its subsequent activation during S phase if the cell is confident that all systems are go.

Such complexity of regulation might appear daunting, but to a biochemist of John’s calibre and ambitions, the thought of purifying 60–70 proteins and then assembling them into somewhere around 25–30 complexes is his idea of the ultimate good time:

This may be a bit of a boy thing, but ever since I was a little kid I took things apart. I broke every toy I ever got for Christmas within a week because I took it apart and couldn’t put it back together. So now that I’m finally putting things back together, it’s great! There are really interesting questions to ask about how these things work. What’s particularly interesting is that there isn’t a precedent in viruses or in bacteria—it really works completely differently from replication in any other realm of life as far as we know, so the things we’re going to learn are really new. Replication is the centre of everything in the nucleus, because everything that happens is coupled somehow with replication, whether it’s chromosome inheritance or sister chromatid cohesion, so I think having an in vitro replication system is really going to open up a lot of stuff for biochemical analysis.

In the short term, the loading of the Mcm2–7 helicase onto the origin is currently occupying John’s thoughts because persuading a multisubunit helicase to wrap itself round DNA is an intriguing topological problem. In addition, his lab has just found that ORC, which together with its accessory protein Cdc6 is the ATP-burning enzyme that loads the helicase up, can tell whether the helicase is intact or not, and unload it if components are missing. The ORC ATPase, unsurprisingly, also has a further level of regulation, by CDKs.

Nobel Prize celebrations, Clare Hall, 2001. (Left to right) Steve West, Frank Fitzjohn, Tim Hunt, Tomas Lindahl, Brenda Marriott. (Photograph courtesy of Steve West.)

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