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Development of novel nano-agents to target DNA replication forks and modulate cell cycle and activity

Principal Supervisor: Professor Mike Hannon - School of Chemistry

Co-supervisor: Dr Nik Hodges, Professor Grant Stewart

PhD project title: Development of novel nano-agents to target DNA replication forks and modulate cell cycle and activity

University of Registration: University of Birmingham

Project outline:

We have developed a novel class of metal-based cylinders that bind to DNA Y-shaped junctions (Figure 1). These cylinders show an exciting activity to push rapidly dividing cells into cytostasis (in cell line studies) but we want to further understand the basic cell and molecular biology of these agents in more detail specifically: (i) how they enter the cell and where (and how quickly) they localise, (ii) how they interact with key biomolecules in the cell such as genomic DNA and the downstream cellular effects of those interactions and (iii) the resultant properties in in vitro cellular systems.

Pic1

Figure 1: Crystal structure of the cylinder bound to a DNA 3-way junction

To facilitate this we will we will use existing cylinders in combination with development of newly modified cylinders (e.g. His-tagged, fluorescently labeled and cylinders with different metal centres) to image and further study cylinder cellular interactions and develop novel imaging tools to directly study biologically important DNA transactions (e.g. DNA replication forks) inside of cells by measuring incorporation of fluorescent base analogues and also using isolated DNA under laminar sheer flow. This technique will allow us to analyze the impact of cylinders on rates of replication fork progression, frequency of fork stalling or collapse, and also will provide insights into the cellular response to cylinder exposure (compensatory origin firing, effect on nearby replication structures). In parallel, we will use a modified version of the iron cylinder, which will be amenable to cross-linking by ‘click’ chemistry by virtue of the presence of several alkyne groups, to identify proteins bound to the DNA within the vicinity of the cylinder.

We will complement this with biochemical studies – mechanism of induction of apoptosis and detection of direct evidence of interference with DNA replication by measuring DNA strand breaks using the comet assay and biophysical studies of interactions with individual biomolecules to build up a picture of the molecular interactions occurring in the cell. Synchrotron X-ray studies at Diamond (XRF and XANES, using the properties of the metals) on the cylinders in cells and on cylinders bound to key biomolecules will afford further information on localisation and biomolecular targets: TEM on cryo-sectioned cells and soft X-ray tomography on whole cells at the new facility at Diamond will provide detail on effects on organelles (through our ongoing collaborations at Diamond with Drs Tina Geraki and Elizabeth Duke)

References:

  1. L. Cardo, V. Sadovnikova, S. Phongtongpasuk, N. J. Hodges, M. J. Hannon, Chem. Commun., 2011, 47, 6575-6577
  2. C. Ducani, A. Leczkowska, N. J. Hodges, M. J. Hannon, Angewandte Chemie International Edition, 2010, 49, 8942--8945
  3. A. C.G. Hotze, N. J. Hodges, R. E. Hayden, C. Sanchez-Cano, C. Paines, N. Male, M. Tse, C. M. Bunce, J. K Chipman, M. J. Hannon, Chemistry & Biology, 2008, 15, 1258 - 1267.
  4. J. Malina, M. J. Hannon, V. Brabec. Iron(II) Supramolecular Helicates Condense Plasmid DNA and Inhibit Vital DNA-related Enzymatic Activities. Chem. Eur. J., 2015, in press
  5. Higgs MR, Reynolds JJ et al, Stewart GS. BOD1L Is Required to Suppress Deleterious Resection of Stressed Replication Forks Molecular Cell, 2015, 59, 462-477.

BBSRC Strategic Research Priority: Molecules, cells and systems

Techniques that will be undertaken during the project:

Key experimental skills involved:

  • Molecular probe design and synthesis
  • Ligand and Compound characterisation
  • Bimolecular Binding studies using spectroscopies
  • Growing and handling mammalian cells and treating them with metallo-cylinders
  • Fluorescent microscopies
  • Quantification of fork arrest in cells using labelled DNA bases
  • Cell Uptake and localisation studies
  • Quantification of DNA damage by the comet assay
  • Synchrotron X-ray imaging

Contact: Professor Mike Hannon, University of Birmingham