*EASTBIO* Developing novel techniques for studying protein-DNA interactions using site-specific cross-linking and high-resolution mass spectrometry

While the use of mass spectrometry to study protein-protein interactions is a mature field, the elucidation of the interactions of proteins and protein complexes with DNA, particularly with regard to DNA repair, is still in its infancy. Recently, we have successfully developed a number of mass spectrometry based techniques1 that complement the existing more traditional tools.

Proteins that become covalently linked to the DNA in a cell pose a great risk of DNA damage and cell death. For example, topoisomerases are essential enzymes for relaxing superhelical tension in the DNA that is caused by key cellular processes such as transcription and replication. Under certain circumstances topoisomerases can become covalently stalled at DNA strand breaks on the DNA, for example when exposed to so-called "topoisomerase poisons" like the anti-cancer drug CPT. Human tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a DNA repair enzyme that can specifically remove a CPT-stalled topoisomerase IB enzyme from the DNA, which makes it a promising target for anti-cancer therapy2.

X-ray crystallography has revealed how Tdp1 binds single stranded DNA substrates3. Using site-specific UV cross-linking and mass spectrometry, we have so far confirmed the results for single stranded DNA, and are now investigating the binding of Tdp1 to double stranded DNA. We plan to further extend our studies to other proteins and protein complexes involved in DNA repair.

Our objectives are:

(1) To develop tools that are applicable to the analysis of protein-DNA interactions for any DNA binding protein. Of particular importance are enrichment strategies that are necessary to concentrate the peptide-DNA heteroconjugates before they can undergo analysis by mass spectrometry. Techniques that allow the identification of low abundance cross-linked material, such as differential heavy isotope labelling will also be further developed.

(2) To understand at the molecular level how Tdp1 binds to its various biologically relevant DNA-protein substrates by using site-specific protein-DNA cross-linking and high-resolution mass spectrometry to further map interactions between DNA and protein in solution. We will use site-directed mutagenesis and our panel of biochemical assays to verify the importance of newly identified Tdp1-DNA interactions. In this context we are working closely with structural biologists to complement and contrast our mass spectrometry derived data.

(3) To extend the range of DNA-binding proteins and protein complexes analysed by protein-DNA photo cross-linking and mass spectrometry to other DNA repair proteins and also protein complexes acting on DNA.

While the student will be based in the Interthal lab, they will be expected to spend a considerable part of the time at the mass spectrometry facility based in the School of Chemistry. They will develop a broad range of skills in the Interthal lab which is focussed on the molecular biology of DNA repair. Skills developed will include protein expression and purification, protein-DNA binding assays as well as enzyme activity and in vivo assays. Optimisation of photo cross-linking protocols and protein-DNA heteroconjugate enrichment strategies will be a major part of the project. Importantly, the student will develop expertise in a number of mass spectrometry techniques while working in the facility in the School of Chemistry. The facility is one of the best equipped in Europe, with a wide range of instruments, including a 12T SolariX FT-ICR mass spectrometer (Bruker Daltonics), an Orbitrap (Thermo Finnigan) and a MALDI-TOF-TOF among others. Dr. Mackay will provide training and supervision. The student will also have access to state-of-the-art facilities for protein purification in the Edinburgh Protein Production Facility (EPPF) and the Biophysical Characterisation Facilities.