Determining Consequences of Mitotic DNA Entanglements, from Molecules to Cells

Background and Importance Growing cells must replicate and segregate metres of DNA. Failure to faithfully segregate DNA results in genome damage and instability, contributing to human diseases including cancer and rare genetic disorders, such as fragile-X syndrome. In extreme cases genome damage leads to cell death, and this is exploited by an increasing repertoire of drugs that target the enzymes involved, as cancer cells divide at an accelerated rate. Ultra-fine bridges (UFBs) are a type of DNA segregation defect, which involve minimal DNA entanglements of one or two strands. Depending on underlying DNA structure, they are resolved by different resolvases, namely TOP2A, MUS81/EME1 and GEN1, and additional resolution pathways are activated particularly if these resolvases fail. UFBs have largely been studied in a cellular context by examining the protein complexes that localise and act upon them, and which DNA processes, when targeted, promote their formation. However, in the complex environment of a cell, determining the underlying DNA structure and deconvoluting enzymatic contributions to the formation and resolution is challenging. Hence, reconstitution of UFB resolution pathways allows a clearer understanding of UFB resolution, ultimately enabling targeting. Our understanding of the DNA structure of UFBs stretched by cell division, is primarily derived from mechanical force response of naked DNA. However, we currently have a limited knowledge of the structural changes induced when DNA entanglements are subjected to force and how this affects enzymatic processing. My preliminary data suggest direct interactions are generated between the entangled DNAs in vitro, indicating significant structural changes. It is vital to understand the molecular basis of these structural changes and the consequence this has on resolvases and cell segregation. This will be achieved by three key work packages (WP): WP1. Determine the structural changes when entangled DNA experiences force. WP2. Determine how crucial resolvase activity and specificity is influenced by force. WP3. Generate systems to individually isolate effects of specific resolvases, cell-cycle stages and DNA sequences. Outcomes and benefits WP1 will use cutting-edge single-molecule and computational approaches, which I have unique expertise in, to establish the molecular detail of entangled DNA structure, allowing the development of hypotheses about how this affects resolvases and chromosome segregation in cells. DNA structural changes will be explored in the context of human disease sequences and the presence of clinical chemotherapy drugs, providing new knowledge of the molecular mechanism of disease and treatments. This work could provide insights into other DNA processes such as homology search, crucial for repairing DNA damage and maintaining genome integrity. WP2 will determine the impact of force-induced DNA structural changes on resolvases by developing single-molecule approaches. Given UFB resolvases are current or potential human drug targets, WP2 will result in system to test such drugs, as well as provide details of the molecular mechanism, helping to identify novel drug targets in the form of off pathway processes and new DNA structures. WP3 puts findings back into the complexity of the cell, to demonstrate effects observed in vitro hold in cells. This cellular system will enable further translational work. Collectively this work will yield new insights into the fundamentals of DNA mechanics and the mechanism of human disease, helping to aid design of future therapeutic approaches that could benefit cancer patients and the wider health care community.