DNA double strand breaks (DSBs) - where the backbone of the DNA is cut on both strands simultaneously - are one of the most toxic DNA lesions in cells: if left unrepaired huge amounts of genetic material can be lost. As a result, the cell has evolved multiple elegant and highly controlled pathways to detect, signal and repair DSBs. The response to DNA damage reshapes the local chromatin environment and is largely orchestrated by the deposition, removal and detection of a complex set of chromatin post-translational modifications. Nucleosome modifications act as a central signalling hub in this network to organise responses to a neighbouring damage. While many factors are known to localise to modified damage-adjacent chromatin, how these factors bind to modified chromatin and perform their function is unclear.
Using a wide variety of techniques, principally cryogenic Electron Microscopy (cryo-EM), We aim to characterise the chromatin environment at the sites of DNA damage and understand the molecular mechanisms of deposition, reading and removal of DNA damage histone marks. Focusing on histone ubiquitylation after DNA damage this project will involve:
1) Protein expression and purification of chromatin proteins and histones
2) Chemical biology approaches to generate modified proteins
3) Protein biochemistry and biophysical techniques to understand binding and enzymology of DNA damage proteins to modified nucleosome templates
4) Structural biology approaches, in particular cryo-EM, to understand the molecular mechanisms of chromatin interactions
5) Mass spectrometry approaches to identify novel DNA-damage responsive chromatin interactors
6) Cell biology to extend our in vitro observations into the cell
This would be an ideal project to suit a passionate, motivated student interested in how molecular understanding can help explain fundamental biological processes. A background in biochemistry, structural biology or chemical biology is preferable. Applicants must have, or expecting to be awarded, at least an upper-second class degree (or equivalent qualification) in an area of biochemical-sciences.
Wilson MD, Durocher D, Reading chromatin signatures after DNA double-strand breaks. (2017) Phil Soc Trans B, 372 20160280
Wilson MD, Benlekbir S, Fradet-Turcotte A, Sherker A, Julien JP, McEwan A, Noordermeer SM, Sicheri F, Rubinstein J, Durocher D. (2016) The structural basis of modified nucleosome recognition by 53BP1. Nature, 536 Aug 4 pg 100-103
If you wish to apply for this project, please go to this link.