Shining the spotlight on DNA replication
Researchers in the Biochemistry and Physics departments have used single-molecule imaging to show that the precise molecular details of DNA replication in the bacteria E.coli differ from how they have been portrayed in textbooks for years.
The findings, published in Science by David Sherratt and Rodrigo Reyes-Lamothe in Biochemistry and Mark Leake in Physics, demonstrate the power of single-molecule live cell imaging in revealing previously hidden details of molecular events inside the cell1.
DNA replication is a complicated process that requires the co-ordination of many different proteins. Textbooks show that the molecular machinery necessary for replication is contained in a complex called the replisome that couples the activities of more than 11 proteins including DNA polymerase, the enzyme that replicates the two strands of DNA. The replisome is shown with two DNA polymerases, each associated with one strand of DNA to be copied.
Schematic showing the independent tracking of replisomes around the E.coli chromosome
Achieving this level of understanding has taken years of painstaking studies of the process in vitro. 'What we've done historically,' explains Professor Sherratt, 'is try and get things out of a cell and working in a test-tube. One of the great triumphs of molecular biology was to reconstitute DNA replication in a test-tube.'
But such studies have their limitations, says Professor Sherratt. 'The fact that you have something working in a test-tube doesn't necessarily tell you how it's working in the cell. There's the context of the cell to consider - where is it working in the cell and at what times'
A few years ago, whilst Dr Reyes-Lamothe was still working on his PhD, he and Professor Sherratt used conventional microscopy to show that it was possible to visualise individual replisomes in living E.coli cells. But they wanted to understand the replisome's composition which required more sophisticated microscopy and analysis. They turned to Dr Leake for help. 'We knew that Mark had approached similar problems in the past,' says Dr Rodrigo-Lamothe. 'We were very lucky that he was just metres from us in the Department of Physics. So Dave and I explained our aims, Mark found them interesting, and our collaboration started.'
Dr Leake admits that this work stretched the imaging capabilities of his microscope to the limit. 'DNA replication is the most challenging system I've had to look at. It occurs in the cytoplasm where it's very watery. Things move so quickly that they blur out.' The two particular challenges he had to overcome were the sensitivity and also the timing so that the microscope can see a single molecule in a way that is not blurred.
"We're living in exciting times in which the boundaries between cell biology and biochemistry are fading away"
Rather than rebuild his microscope to tackle the problem, Dr Leake increased its complexity. The result is slimfield microscopy in which the light is condensed into a much smaller volume than previously done. This floods the sample with light, thereby pushing down the image rate to milliseconds - fast enough to image components moving in the cytoplasm. 'That's the key,' explains Dr Leake. 'Once you can see individual single molecules on each individual frame, then you can apply image analysis techniques to actually count how many protein components there are.'
The group adopted a very collaborative approach to the work, particularly on development of the image analysis techniques. 'While I concentrated on constructing E.coli strains and doing the microscopy, Mark used his prowess on the analysis,' explains Dr Reyes-Lamothe. 'The three of us would then get together, which was always exciting because Mark would bring the latest analysed results, and we would then plan the next round of experiments.'
Schematic model for replisome components. Two DNA polymerases shown are within the core of the replisome along with many other proteins necessary for replication
Working on Dr Leake's home-built microscope was a unique experience, adds Dr Reyes-Lamothe. 'When I started the experiments, the shutters to stop the laser light that illuminate the sample were not in place, so I had to use some cardboard. To start recording, I had the mouse in one hand and the cardboard in the other. But the data that came out of it were amazing.'
To visualise individual protein components of the replisome, the researchers first had to take those proteins - including DNA polymerase and a protein called a clamp loader which holds the polymerase onto the DNA - and label them with a fluorescent dye which could be detected under the microscope. This allowed them to track each protein, one at a time, inside the E.coli cell.
Using slimfield microscopy, they followed the flourescently-labelled proteins continuously, capturing images every few milliseconds. Once they had collected the raw data, they then applied the image analysis software which they had developed to locate precisely where the signal was coming from and determine its intensity. This allowed them to work out the number of fluorescent molecules in the signal.
They found a replisome architecture that was different from how it appears in the textbooks. 'The big surprise is that we see three DNA polymerases, one on each strand and one somewhere else,' explains Professor Sherratt. They also found three sliding clamps, only two of which were permanently associated with the replisome. The other is attached to the DNA at some distance from the replisome, perhaps ready to load the third polymerase onto the DNA when necessary.
The work shows that we have only an incomplete picture of how DNA replication works in the cell. Using techniques such as slimfield microscopy, says Professor Sherratt, will help us fill the gaps in our understanding. The researchers' current focus is on developing multicolour imaging which will allow simultaneous tracking of several different molecules under the microscope, giving them a powerful extra handle on the molecular machines they are studying.
The replication machinery in bacteria is very similar to that in more complex cells such as mammalian ones. So studying bacteria, which have only a single chromosome to be replicated and which lend themselves to this microscopy technique, will tell us a lot about how the process works in other cells. Ultimately, says Dr Leake, researchers will want to move into other cells. 'If you want to start addressing questions of more biomedical relevance then you would go to a more appropriate cell system and yeast would be a good choice.'
Even a relatively simply organism such as E.coli has proved a formidable challenge. When Mark Leake and Rodrigo Reyes-Lamothe started out on this project 3 years ago, they were not sure whether it would work, but it has proved to be a very fruitful collaboration. 'You have to start small and develop the physics techniques hand in hand with the biological questions,' says Dr Leake. 'Once you can crack those, you're in a good state to move into more complex systems.'
DNA replication is just one of many fundamental processes in the cell which slimfield microscopy can provide a unique insight into, enabling researchers to probe dynamic events in living cells. In principle, the technology can be used for studying any molecular machine and asking how many molecules it is composed of, how these are associated and what happens if components are taken away.
Dr Reyes-Lamothe is excited about the tremendous potential of such imaging techniques.''We're living in exciting times in which the boundaries between cell biology and biochemistry are fading away. In a few years, it will be as common to determine the number of proteins under the microscope as it is now to determine their localisation. But to know the number of proteins is just the first step. The real challenge is to study biochemical reactions as they happen in the cell.'