The Incredible Ways of DNA Replication Professor Michael O’Donnell THE INCREDIBLE WAYS OF DNA REPLICATION For over 30 years, Professor Michael O’Donnell, based at the Rockefeller University in New York, has focused on the mechanisms involved in the duplication of genetic material in cells, a process known as DNA replication. Professor O’Donnell’s work spans from the early 1990s, when his team was the first to discover a ring- shaped protein that encircles DNA and clamps the replication machine to DNA. Most recently, the team has been studying the proteins involved in mammalian DNA replication. The ability to reproduce is one of the In the DNA helix, adenine bases only most fundamental mechanisms needed pair with thymine, and the cytosine by every cell. In order to reproduce, cells bases only pair with guanine. The DNA must be able to make a copy of their polymerase recognises each base along own genetic information and pass it on the template sequence and finds the to the daughter cells. This is known as correct base pair match from a pool DNA replication. of free bases. The replicating machine contains two DNA polymerases, one The genetic material present in for each unzipped strand, resulting in every cell – DNA – is made of two two new duplex copies and providing complementary strands, organised in all the instructions needed for two the classic double helix. Each strand new living cells. Indeed, Dr Arthur is a long chain with four different Kornberg (Stanford University), who was bases, adenine, cytosine, guanine awarded a Nobel Prize in 1959 for the and thymine, and the sequence of discovery of DNA polymerases, provided these bases forms a code needed to postdoctoral training to Dr (now make every protein required for life. Professor) Michael O’Donnell. It is therefore essential that the DNA For Professor O’Donnell, now at the sequence is copied with great precision At first glance, matching two sets of Rockefeller University in New York, this and accuracy. bases may seem an easy process, but creates an interesting conundrum: How when you take into account that it can the DNA polymerase stay bound The DNA helix is duplicated by many needs to be repeated 6 billion times to to DNA and accurately replicate DNA proteins that work together as a make a new copy of DNA in a human while at the same time move along machine. One protein, called a helicase, cell, then it is inevitable that mistakes the new strand at a relatively high unzips the two strands of the double can happen and must be minimised. speed? ‘It seems a contradiction, that helix. This is where an enzyme called This susceptibility to error may suggest a DNA polymerase could achieve an DNA polymerase comes in. DNA the need for a slow and careful enzyme, extremely tight grip to DNA, yet move polymerase works by using each strand but surprisingly DNA polymerases are very fast during synthesis,’ explains as a template to make a new strand, quite fast and in bacteria can go 1000 Professor O’Donnell. ‘The finding resulting in a new copy of the double bases every single second. came as a complete surprise, which is helix. actually quite typical of most scientific discoveries. This puzzle has a beautiful solution.’ WWW.SCIENTIA.GLOBAL ‘The finding came as a complete surprise, which is actually quite typical of most scientific discoveries. This puzzle has a beautiful solution.’ Eukaryotic PCNA Bacterial ẞ Gulbis, Kelman, O’Donnell and Kuriyan Kong, Onrust, O’Donnell and Kuriyan (1996) Cell 87, 297-306. (1992) Cell 69, 425-437 Human (left) and Bacterial Clamps (right). Credit Michael O’Donnell. It’s a Doughnut! special DNA cutting enzyme, the protein Exploring Ways to Load the Clamp did not stay on the cut DNA, implying it The first clue came quite unexpectedly was sliding off the new and unblocked Professor O’Donnell very quickly in the early 1990s. Professor O’Donnell DNA end. Professor O’Donnell proposed realised that the clamp can’t get onto had started a set of experiments with the circular structure of the protein and DNA by itself, but it needs a ‘clamp circular DNA (typical of bacteria) but named the protein a ‘sliding clamp’. loader’ to open the clamp and then ran out of this half-way through his close the ring around DNA. In fact, their experiments and decided to try linear Many scientists did not accept the studies identified that a five-protein DNA instead. This lucky decision would interpretation of a ring protein, so machine was needed to put the clamp turn out to be key in solving the mystery. Professor O’Donnell enlisted the help of onto DNA. But understanding the clamp ‘I ran out of circular DNA, but there was Professor John Kuriyan at Rockefeller loader mechanism proved more difficult a linear DNA in the freezer so I tried this University, a world-leading expert to achieve. ‘I used to think that each instead,’ said Professor O’Donnell. ‘To in X-ray crystallography (a method of the five different clamp loader units my dismay, the protein did not bind the to determine the atomic structure must have one function, and therefore linear DNA. I tried it again, thinking I′d of a protein) to determine the exact there must be five functional steps made a mistake, but still the protein did structure of this enigmatic protein. to the clamp loading process. This not bind the linear DNA.’ ‘When John called me up to take a way of thinking did make progress in look at the results, I′ll never forget his understanding clamp loader function, Professor O’Donnell realised that the words “Mike – you wanted a doughnut, but…in the end, it was the wrong way simplest explanation was that the and we give you a doughnut,”’ joked to think about the clamp loader,’ says protein is also a circle, and encircles Professor O’Donnell. ‘Indeed, it was a Professor O’Donnell. the DNA, and therefore slides off the ring, but never would I have expected end of linear DNA but stays on circular it to look so very beautiful.’ It turned Eventually, the team realised the DNA because it has no ends from which out the sliding clamp protein has a clamp loader cannot be thought of as to slide off. The researcher knew this wonderful structure, built from two separate components with separate was a crazy idea at first because there subunits, each having three identically functions, but as a single multi- were no other proteins known to do shaped domains that together formed a component machine that opens and this. Professor O’Donnell confirmed this beautiful six domain circle. closes the clamp. Professor O’Donnell’s provocative idea when linear DNA with biochemical studies, in collaboration blocks at the ends behaved the same with Professor Kuriyan’s structural work, way as circular DNA, but when the end- revealed a beautifully orchestrated blocked DNA was cut in two pieces by a mechanism in which the clamp loader WWW.SCIENTIA.GLOBAL A Broader Research Focus After solving the clamp mystery, Professor O’Donnell’s research interests expanded to include not only the clamp/clamp loader but also all the components involved in DNA replication, in both eukaryotic cells (like mammals) and bacteria. After a painstakingly slow and complex process, the team was the first to purify and reconstitute a functional eukaryotic replication machine involving 31 different proteins with each component individually cloned and purified. The team knows this is only the first step and there remains binds to the enzyme, warping the clamp in such a way that a long road ahead of them, along with the work of many the ring opens up, allowing DNA to enter into the centre. Once other laboratories, to fully understand DNA replication. They the DNA is inside, the clamp loader changes its conformation, may not know exact details yet, but they have no doubts this allowing the clamp to close. will be a highly regulated and complex mechanism. After all, these proteins must be able to cope with errors during Moving to Mammals replications as well as orchestrate numerous encounters with multiple enzymes and unrelated proteins. Professor At the same time as this work in bacteria was being carried out, O’Donnell is very excited about this field, stating ‘Many labs several research groups were also working on a mammalian worldwide work on this complex replication machinery, and system to study replication. Among the proteins identified, we look forward to all of our labs contributing pieces of the there were two that caught Professor O’Donnell’s attention: one puzzle that finally, someday, understand the whole picture. called PCNA and one called RFC. ‘John Kuriyan and I proposed It is important to human health and disease, because when that PCNA would be a ring of six domains arranged on three gone awry, the process makes mistakes that can cause cancer. subunits and that RFC would be a clamp loader.’ So, understanding exactly how it works will shed light on new medical applications to help people.’ In continued collaboration with Professor Kuriyan, Professor O’Donnell’s team showed that the bacterial and mammalian Professor O’Donnell adds, ‘the mammalian system is much clamp and clamp loader have a strikingly similar structure. more complicated than viral or bacterial systems and very ‘The nearly identical structure of human and bacterial sliding little is presently known about how its functions. Many of its clamps supports the hypothesis that humans and bacteria components are present to regulate the replication machinery, evolved from a common ancestor cell that lived billions of or simply known to be important for replication through years ago.