The structure and function of the SLX4 nuclease complex

Our genetic blueprint is contained within long, chromosomal molecules of DNA in the nucleus of our cells. During our lifetime, the cells in many of our tissues are constantly dividing to replace old and damaged cells. This is part of the natural ageing process. Before dividing, a cell must replicate its DNA accurately to prevent chromosomal changes that could lead to debilitating degenerative diseases including cancer and neurodegeneration, many of which are hallmarks of ageing. DNA replication is performed by DNA polymerases that copy the template strands in the parent cell, as part of full chromosome duplication and cell division. Since the DNA is composed of two strands of DNA, replication involves separation and replication of these two strands, and replication of each strand in a co-ordinated process requiring dedicated factors for each strand. When the DNA strands are separated the structure produced contains unwound regions, and the junction where unwinding and replication are ongoing is named a 'replication fork'. Importantly, during every round of replication the dedicated replication proteins encounter structures or damage within the DNA that block their progress. These structures might be naturally arising regions of the DNA that are 'hard' to replicate, or they might be chemically damaged regions within DNA. Such chemical damage can arise spontaneously as a result of normal cellular processes that are constantly occurring, or they might be inflicted through exposure to external agents, for example solar radiation (sunlight) or a variety of environmental chemical agents. Moreover, this type of damage is produced is also produced by several important medicines used to treat cancer, and a full understanding of how cells respond to this damage might help us improve chemotherapy.

The abnormalities encountered during replication must be repaired, and this frequently involves proteins called endonucleases, that cut abnormal DNA structures. This can occur either during the process of replication, or structures generated during DNA replication can be later resolved after replication is complete. Endonucleases initiate this cascade of repair events, and several key factors known to be required for are the XPF-ERCC1, SLX1 and MUS81-EME1 proteins. However, it has recently become apparent that these factors must be associated with a large 'platform' protein called SLX4 which helps direct it to the damaged replication forks, and other structures associated with the repair of damaged DNA. Solving the three-dimensional structure of the SLX4 complex at high resolution will be key to understanding its mechanism. This in turn will ultimately help in the development of new therapeutics combating a number of degenerative conditions associated with ageing. It should also improve our diagnosis of developmental and malignant disorders and provide important insights that the pharmaceutical and biotechnology sectors could use to generate new medicines and technologies, especially since it appears that targeted inhibition of these repair reactions might help improve cancer therapy.

Loss of genomic stability directly contributes to the initiation of human ageing and disease. During every cell division, structural aberrations in the DNA can block the progression of replication forks. The SLX4 nuclease platform plays a critical role in spatially targeting and modulating the activity of three structure-selective nucleases, XPF-ERCC1, MUS81-EME1 and SLX1 during replication fork repair and homologous recombination. Our recent work with the human SLX4 complex has demonstrated that its constituent proteins are difficult to produce in amounts needed for structural studies, and suffer from aggregation problems during in vitro complex formation. For this reason, we have turned to the Xenopus laevis SLX4/SMX complex, which is functionally homologous to the human complex, but during purification behaves very differently from the human complex. This complex is far less prone to aggregation and can be produced in a soluble single-state form with sufficient yield to permit structural studies.

Here, we propose to solve the structure of the SLX4/SMX complex using cryo-electron microscopy. We will initially structurally characterise several biochemically active SLX4 subcomplexes. Our preliminary data suggest that we will subsequently be able to tackle the full-length SLX4 protein in association with XPF-ERCC1, MUS81-EME1 and SLX1 allowing us reveal the structural basis for nuclease targeting and stimulation by SLX4. Our structural studies will be complemented with functional biochemistry and cellular phenotype analysis, that will allow us to iteratively test the functional importance of the structural features identified during DNA repair and recombination. Together, this will provide an unparalleled insight into the structure and mechanism of one of the most important, yet poorly understood DNA repair complexes, and one which is critical to maintaining health, suppressing the hallmarks of ageing and represents a highly attractive target in oncology.

The work proposed here has broad implications for human health, which could bring significant benefit to the public and also economy through improving suggesting new therapeutic strategies to the pharmaceutical and biotechnology industries, as well providing information and biomarkers that could be useful in disease prevention and public health. The work will characterise a a key molecular target required to maintain genetic stability, helping us to ameliorate the symptoms of developmental disorders associated with genomic instability, staving off the effects of ageing and improving cancer treatment, potentially in combination with DNA-damaging drugs/radiation. Therefore, outside of the more immediate academic field, the following groups should ultimately benefit: (i) Pharmaceutical and Biotechnology Industries, where there is a very strong current interest (especially in the UK) in targeting DNA repair factors to improve cancer therapy. Interventions could also potentially improve fertility and well-being during ageing. Exploitation of our research in this fashion would both benefit human health and be of economic benefit to the UK. (ii) The NHS and related Public Sector, who also have a strong interest in disease prevention, might ultimately employ the factors studied here as biomarkers to improve healthy ageing either through intervention, or through lifestyle choices (see point iii, below). (iii) Policy makers and Government. Those involved in policy making are required to make recommendations and laws that to help protect the population from health risks, established and emerging (for example, from nano-particles). Here, an understanding of the pathways required to combat environmental DNA damage is important as it will help in the assessment of threats that could produce acute or longer-term damage to human cells and tissue. Policy could be modified to reduce human risk from exposure to a minimum. (iv) The UK employment sector - the Post-Doctoral Fellow and Research Assistant working on this project will gain a wealth of experience in cutting-edge structural biology and biochemistry which is directly transferable within the sectors described herein. (v) The general public will benefit as our group, academic department and institute are all heavily committed to public engagement. The applicant and his group regularly communicate their findings to the public at events held within the Oncology Department and Weatherall Institute, including at an annual WIMM Public Lecture evening which is strongly angled towards local schools. Naturally, we maintain an up-to-date website detailing our work and its outputs, and where appropriate the University utilises its very effective media outlets to publicise findings nationally and internationally. Finally, we will publish our work in leading academic journals and disseminate findings at the appropriate academic conferences in a timely manner.