A real-time single molecule approach to understand how DNA repair proteins locate and remove damage

From microbes to man DNA repair is crucial to the continuance of life. Each cell in the human body accumulates over 10000 sites of DNA damage every day, therefore efficient and rapid repair is essential. Defects in DNA repair result in cell death or continual proliferation, leading to premature ageing or tumour formation respectively. Repair is mediated by proteins; each one performs a small task in a sequence that eventually leads to lesion repair. To date we do not fully understand the physical basis of how these proteins find damage or come together as functional units. In this project we aim to follow the process of nucleotide excision repair (NER) in a model bacterial system. This simpler system involves the interplay of just three dedicated enzymes instead of up to thirty in humans. We will use recent advances in imaging such as fast sensitive cameras, bright fluorescent tags and powerful computers to directly visualise how and when these protein machines operate; this is discussed in more detail below. Our research offers important insights into how proteins find their targets on DNA, form complexes and permits direct visualisation of the mechanistic sequence underlying a protein cascade. It is anticipated that this research will benefit other scientists by introducing new techniques that could be used to investigate a number of other processes and may also impact the design of new anti-bacterial drugs. To study DNA repair we visualise the process one molecule at a time. Normally, systems are studied as 'ensembles' consisting of thousands of billions of protein molecules. By visualising single molecules we are able to extract information much more accurately about both the order and timing of the process being studied. To make it possible to see a single molecule we attach fluorescent beacons called a quantum dots to our proteins. These tagged proteins can then be followed using a state-of-the-art microscope based imaging technique. However to follow the proteins one more important aspect needs to be considered. When DNA is visualised it is not a long stretched out fibre, instead DNA is bundled, making it impossible to follow the behaviour of a single tagged protein. To overcome this we have developed a unique approach: we suspend the DNA between large beads attached to a microscope slide to create 'DNA tightropes'. These tightropes allow us to introduce tagged proteins and watch how they behave on DNA. Since the repair system uses multiple protein machines to carry out its work, we have tagged the proteins with different colours to distinguish them. DNA repair proteins face the enormous 'needle in a haystack' challenge of finding one damage site amongst a vast excess (millions to one) of undamaged DNA. Using our tightrope technology we will watch how they do this, and at the same time make precise measurements to provide us with a physical understanding of this process. Do the proteins slide along the DNA? Detach and reattach elsewhere? Or both? We will also be able to address long held questions in the field such as how many proteins form a complex? And what role ATP, the cellular energy currency, plays? We will also damage the strung up DNA tightropes and attach a quantum dot beacon to the damage site thus providing us with its location. Then we will introduce all three proteins together and, in real time, we will directly observe how they work together to repair the DNA. In this proposal we present a large amount of data to demonstrate the success of the above outlined approach, which uses technology that is at the leading edge of the field and is unique to our laboratory. The system we are developing here will offer a new insight into DNA repair and also provide enabling technology to offer a new way of understanding how many other protein systems interact with DNA.

We aim to directly visualize the process of prokaryotic nucleotide excision DNA repair (NER) in real-time, using state-of-the-art single molecule imaging methods. Prokaryotic NER is comprised of three dedicated enzymes UvrA, UvrB and UvrC which repair a variety of DNA lesions. We have already tagged the first two enzymes with quantum dots (Qdots) to provide long-lived, bright fluorescent markers, and as part of this proposal we intend to label the third. Also, we have already observed that UvrB reduces the dimensionality of UvrA's lesion search, contrary to the current thinking. This was achieved by employing a new approach where single repair proteins are microscopically imaged as they bind to elevated DNA strands known as tightropes. In this proposal we aim to understand the process of lesion search in more detail and also the role of UvrC. By differentially colour labelling our proteins we can assess how many proteins comprise a functional UvrABC complex. Both the oligomeric and functional properties of the complexes may be affected by the presence of ATP. Therefore we will systematically study the effect of various ATP concentrations on complex mobility and interaction with the DNA. Additionally, we aim to introduce targeted DNA damage that will enable us to directly observe changes in behaviour of the Uvr system, addressing many questions including: Does damage trigger complex breakdown? Recruit enzymes? Finally, to precisely determine the physical nature of the search process we will use a high-speed dark-field microscope to directly measure whether the proteins hop or slide along the DNA in search for their targets. The NER system is ideal for this important observation, due to the slow diffusion rate of the UvrAB search complex. Our ultimate goal is, for the first time, to label the repair proteins and directly observe them working in concert to process the targeted damage in real time.

We are developing a new approach to understand how proteins interact with DNA; therefore there will be beneficiaries across a number of other fields from chromatin remodelling to intracellular-trafficking. The problem of how proteins translocate to binding sites has interested mathematicians, physicists and biologists. Our research offers a direct experimental view of this problem and therefore will appeal to theoreticians and scientists studying other systems that apply random walk theory from nanotechnology to ecology. The process of NER underpins severe human diseases including Xeroderma Pigmentosum and Cockayne syndrome. We will provide an understanding of the equivalent DNA repair process in prokaryotes to uncover principles that will be directly transferable to the more complex human disease systems. Additionally, since DNA repair is crucial to survival; understanding the prokaryotic system may offer new avenues for anti-bacterial drug development. This research will have technological spin-offs to benefit the public/commercial private sector. For example single molecule DNA fingerprinting is used by US genomics (Mass, USA) for enhanced biometric security and also pathogen detection. Current methods rely on amplification which takes time; utilisation of single molecule techniques will permit direct on-site highly sensitive DNA detection, i.e. 'lab on a chip' technologies. US genomics achieves this by differentially colour labelling restriction enzymes and allowing them to bind but not cut the target DNA. Passing single labelled strands through a detector produces a restriction map, providing a genetic fingerprint from just a single molecule of DNA. The technology being developed in this proposal offers an alternative approach to fingerprint single DNA molecules, since so many DNA molecules can be simultaneously examined it becomes possible to screen larger segments of DNA, potentially an entire genome. Such advances may make it possible to achieve genome sequencing for individuals. A single molecule approach for this is being pursued by Pacific Biosciences (Ca, USA); however here we can offer sequencing for much longer lengths of DNA. The academic beneficiaries laid out in the section above will benefit in terms of improved research facilities, knowledge and understanding. Translation of the basic biological knowledge to the development of new therapeutics will result in the improved disease treatment and therefore lower long-term health costs. With concerns over pandemic spreads of infection, the development of pathogen detection technologies may offer much better confirmation of the presence of particular pathogens thus will have an enormous impact on the control of infection spread. Biometric security is also of concern; rapid genetic fingerprinting of individuals at ports for example will permit targeted control at port entry. In this proposal we develop new single molecule technologies. To remain internationally competitive as a knowledge economy on both a research and commercial footing it is important to invest in such research. Single molecule techniques are becoming more widely applied (e.g. biosecurity and DNA sequencing) and applications are expected to be realised over the next decade. As a nation our quality of life will benefit from such technologies, jobs and spin-offs of single molecule research; which is particularly important since nations like India and China are beginning to resource such research at very high levels. All staff involved in this research will receive specific project related training and transferable skills from conference networking, giving seminars, and writing reports. To publicise the impacts outlined above, we will be posting online 'key facts' for our research on our soon to be updated website. This will convey our research message at a level that almost anyone will be able to understand, however more technical information will continue to be available.