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Reading, writing and molecular switching in the DNA-damage response

Primary Supervisor: Professor Steve Smerdon

Secondary supervisor: Prof. Joanne Morris

PhD project title: Reading, writing and molecular switching in the DNA-damage response

University of Registration: University of Birmingham

Project outline:

How cells detect and respond to a bewildering variety of chemical, physical and environmental stimuli is a fundamental question in biology. Although much remains to be understood, it is clear that post-translational alteration of protein structure and behaviour through the timely addition of different chemical and macromolecular modifications plays a central role. This is a highly effective strategy since 'writing' of the modification and its removal by a separate class of 'eraser' enzymes provides the desirable property of rapid reversibility whilst the modification itself acts as a molecular 'switch' that initiates downstream signalling events. The ways in which these modifications are generated and regulated, and how the switching mechanism works at the molecular level form the main interests of the lab and we investigate them using a highly multidisciplinary approach that enables us to relate structural and biochemical information to important physiological signalling mechanisms.

Our biological signalling network of choice is the DNA-damage response (DDR) for two main reasons. Firstly, disruption of the DDR is a characteristic of many human diseases including cancer, neurodegeneration, growth abnormalities, immunodeficiency and others. It's complexity also provides an opportunity to study how the core DDR processes of DNA-damage detection and repair are integrated with signalling to and from many other cellular processes such as metabolic state, cell cycle and developmental stage, cell type-specific effects and the execution of transcriptional programmes.

Many types of post-translational modifications (PTM) are now known and vary greatly in complexity ranging from simple phosphorylation events through to addition of entire proteins such as ubiquitin. Whilst tens of thousands of modifications have been identified by proteomic studies etc, the functional significance of most remains unknown. At the molecular level, PTMs can affect target protein activity in many different ways including promotion of conformational changes, and sponsoring protein complex assembly through the activity of modification-specific interaction modules or 'readers'. These effects are not mutually exclusive and our goal is to understand how combinations of multiple same or different modifications add to complexity and therefore precision in signal transduction mechanisms.

Projects in several areas relevant to these overall goals are available which will use a combination of classical structural biology methods (X-ray crystallography, NMR, EM etc) with chemical/synthetic biology and other approaches to answer outstanding questions such as:

  1. How are the enzymes and enzyme complexes that 'write' the modification themselves regulated?
  2. How are molecular barcodes generated from multiple and/or different types of modification interpreted to control cellular activities such as localisation of signalling and repair complexes in different chromatin contexts before, during and after DNA-damage
  3. How, and to what extent do post-translational modifications of specific sites contribute to large-scale conformational rearrangements in repair complexes and how are these effects influenced by interactions with specific 'reader' proteins?

Background references

  1. Fournier, M. et al (2020) ‘KAT2-mediated acetylation switches the mode of PALB2 chromatin association to safeguard genome integrity’ BioRxiv 575910; 735811 doi:https://doi.org/10.1101/735811.
  2. Kyrieleis, O.J.P. et al (2016) ‘Three-dimensional architecture of the human BRCA1-A histone deubiquitinase core complex’ Cell Reports, 17 3099-3106.
  3. Yata, K. et al (2012) ‘Plk1 and casein kinase 2 act in concert to regulate Rad51 during recombinational repair’ Mol. Cell 45, 371-383.
  4. Lloyd, J. et al (2009) 'A supra-modular FHA/BRCT-repeat architecture mediates Nbs1 adaptor function in response to DNA-damage' Cell 139, 100-111.
  5. Li, J. et al (2008) 'Chk2 oligomerisation studied by phosphopeptide ligation: Implications for regulation and phosphodependent interactions' J. Biol. Chem. 283, 36019-36030.

BBSRC Strategic Research Priority: Understanding the Rules of Life: Structural Biology

    Techniques that will be undertaken during the project:

    • Molecular biology (cloning, site-directed mutagenesis etc)
    • Recombinant protein expression and purification
    • Assembly of native and modified mono/di-nucleosomes and chromatosomes
    • Chemical/synthetic biology (protein/peptide ligation, post-translational modifications, orthogonal ribosome technology)
    • Protein-protein/protein-ligand interactions (eg BIACore, ITC, co-immunoprecipitation, mass spectrometry)
    • Structural/biophysical analysis (SEC-MALS, X-ray crystallography, EM)

    Contact: Professor Steve Smerdon, University of Birmingham