Structure:Function Correlation in the Human DNA Repair Factor CtIP

Our bodies are composed of billions of cells of many different types that perform all the tasks we need to survive. Every day our healthy cells are constantly being challenged by damage to their DNA. As DNA contains all the information necessary for life, it is crucial that its structure is maintained. DNA damage causes mistakes known as mutations and if these are not repaired properly it can eventually lead to diseases such as cancer. We are interested in a particularly harmful type of damage called double-strand breaks (DSBs), where the DNA is physically broken by damaging agents which come from outside the cell or simply as a result of the many complex processes that occur normally on DNA. When a DSB occurs, it is crucial that the ends are joined back together again without errors. This process requires many different proteins which collaborate to bridge the DNA ends, trim away any bulky adducts at the DNA ends that have arisen when it was damaged, and then unwind the DNA double-helix while cleaving one of the DNA strands. This exposes the genetic code surrounding the damage, allowing the cell to find an equivalent undamaged portion of DNA to use as a template for repair. The overall scheme for this process, which is called Homologous Recombination, is complicated but has been well-studied. However, there is a lack of fine detail in the understanding of the way in which the individual proteins that act as repair factors work together, and this negatively impacts on our ability to treat diseases caused by defective DNA repair pathways, as well as to safely apply new methods for editing human genomes.

We are especially interested in a protein called CtIP, which is especially important as it appears to act as a structural hub for repair of broken DNA by co-ordinating the broken DNA ends with many of the other factors required to fix them. Moreover, when CtIP is not working properly, it has been implicated in cancer and the rare human diseases Seckel and Jawad syndromes which cause dwarfism and neurological disorders. Despite its significance, we know very little indeed about the architecture of the CtIP protein, how it interacts with DNA and other proteins, and what is wrong with CtIP in the disease state. In this project, we have assembled a team of interdisciplinary researchers to apply a range of techniques in biophysics, biochemistry and cell biology to piece together the relationship between the structure and cellular function of this important protein and the complexes it forms with DNA and other partners. This new knowledge will dramatically improve our understanding of human DNA break repair with wide ranging implications for the diagnosis and treatment of cancer and other diseases, as well as the further refinement of modern gene editing technology.

This proposal will build upon our recently published work (Wilkinson at al., eLife) to provide a step change in our understanding of the stucture:function relationships in the important DNA break repair factor CtIP. This protein plays a critical role in the mechanism and regulation of the human resectosome as a hub protein that integrates signalling information and co-ordinates broken DNA with many other repair factors. Despite intense interest in CtIP and DNA break repair more widely, we have a very poor understanding of its function at the molecular level.

Our major objective is to identify and map the interactions that are made between CtIP and other protein partners within the resectosome, and to better understand how CtIP interacts with broken DNA, including though the identification of its unknown DNA binding site. We hypothesise that these interactions may be perturbed by regulatory post-translational modifications or disease-state mutations, and that these changes can impact on DNA break repair pathways. We will test this directly using a range on biochemical and cell biological assays. Therefore, the development of tools to define CtIP structure:function relationships will provide many novel molecular level insights into double-stranded DNA break repair mechanism, regulation and dysfunction in the disease state.

A variety of different stakeholders will benefit from our research into CtIP and its role in homologous recombination which we briefly summarise under four headings below. Further details, including how we will deliver these objectives through specific activities, are available in the Pathways to Impact document.

Academic Impact Objectives: We will gain fundamental new insights into DNA break repair by the homologous recombination (HR) pathway. This is directly relevant to the fields of DNA break repair, cell cycle regulation, mitosis, meiosis, telomere biology, genetic instability, molecular ageing, recombination, gene editing, macromolecular interactions, integrated structural biology and DNA:protein interactions. Our work will also involve technique development in mass spectrometry and single molecule methods.

UK Skills Base and Training Objectives: Our interdisciplinary proposal represents an outstanding training opportunity for the PDRA Wilkinson and other staff members in the collaborating groups. Novel developments in structural-MS will benefit the UK biosciences community more widely.

Biotechnology and Medicine Objectives: Our work is highly relevant to improving molecular level understanding of cancer and other human genetic disease including the dwarfism disorders Seckel and Jawad syndrome that are caused by mutation in CtIP. This protein is also a potential target as a cancer therapeutic based on the concept of synthetic lethality between degenerate DNA repair pathways. Finally, enhancement and/or modulation of DSB repair pathway choice is an important element of improving modern gene editing techniques to a level where they might be routinely used therapeutically.

Education and Public Engagement Objectives: We will inform and inspire the public and explain why our work on DNA repair is important to them and should be funded.