Mechanism and inhibition of the DNA repair enzyme Tdp1

Supervisors: Julia Richardson, Heidrun Interthal

Project description:

DNA topoisomerases have been described as “magicians of the DNA world”. They allow DNA strands and double helices to “pass through each other” thereby resolving topological strain created during DNA replication and transcription. To work their magic topoisomerases form a covalent complex with DNA. But if this complex is stalled, DNA damage ensues. This is the mode-of-action of the anti-cancer drug campothecin which intercalates at the topoisomerase IB (Topo IB)-DNA junction thereby stabilising the stalled nucleic acid-protein complex and ultimately leading to potentially lethal DNA double strand breaks.  

However, the cell has a back-up plan: it can process stalled Topo IB-DNA complexes using the DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 (Tdp1).  Tdp1 hydrolyses the phosphodiester bond between a tyrosine side-chain on Topo IB and a DNA 3’ phosphate allowing the damaged DNA ends to be repaired.  Blocking DNA repair by targeting human Tdp1 with small molecule inhibitors could be a useful adjunct to anti-cancer chemotherapy. 

Our objectives are: 

(1) To understand at the molecular level how human Tdp1 recognises, binds and processes its physiological Topo IB-DNA substrate. To address this we will use complementary structural and biochemical approaches. 

(2) To identify small molecules that bind to and inhibit Tdp1.

We will use both in silico and high-throughput in vitro approaches (beads coupled with chemical compounds). Potential inhibitors will be tested in vitro and in vivo in assays developed in the Interthal lab. 

The student will develop a broad range of skills in structural biology and biochemistry (in the Richardson lab) and in the molecular biology of DNA repair (in the Interthal lab). Skills developed will include protein expression and purification; protein-DNA cleavage, binding and foot printing assays; structure determination by X-ray crystallography; fluorescence spectroscopy and high-throughput screening.