Structural and biochemical characterisation of phospho-regulation in the DNA damage response

Stewart, Matthew James ORCID: 0000-0002-4421-5623 (2025). Structural and biochemical characterisation of phospho-regulation in the DNA damage response. University of Birmingham. Ph.D.

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Abstract

The human phosphoproteome constitutes as many as 90,000 (pSer/pThr/pTyr) phosphorylation sites, however most phosphosites remain functionally uncharacterised. Phosphorylation events drive allosteric protein regulation, recruitment of phospho-binding domains, and assembly of multi- domain complexes that serve as a direct link between phosphorylation, ubiquitination, and SUMOylation pathways. The DNA damage response (DDR) is an essential signal transduction pathway that maintains genome stability and demonstrates the precision and efficiency of phospho- protein regulation. This thesis investigates the structural and biochemical basis of phospho- regulation of MDC1-TQxF motifs, Rad50-T690, and Sae1-S185 in DDR signalling. In the first instance, binding experiments and the crystal structure of the PellinoFHA:MDC1pT765 phosphopeptide complex supported a revised Pellino DNA damage recruitment model, which is dependent on MDC1-TQxF phosphorylation status, rather than gH2AX. We found no consistent evidence for a phosphorylation-dependent interaction between Rad50 and the PLK1 Polo Box domain, despite extensive biophysical binding experiments and attempts at complex purification. Despite this setback, site-specific incorporation of a phosphothreonine residue at Rad50-T690 into a recombinantly purified zinc-hook coiled-coil fragment was successful. This reagent represents a technical breakthrough for further structural and biochemical analysis of phospho-regulation of Rad50 and the MRN. Finally, we found no significant phospho-S185-dependent difference in SUMO E1 activity. Nonetheless, purification of recombinant Sae1 with site-specific incorporation of phosphoserine at S185, and the corresponding Sae1-pS185:Sae2 heterodimer, was successful. Both tools will provide useful for future investigations into alternate modes of phospho-regulation of the SUMO E1.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):