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Title: Structural and biophysical analysis of human DNA repair protein CtIP
Author: Morton, Christopher Robert
ISNI:       0000 0004 7968 5080
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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The integrity of our genome is constantly threatened by endogenous and exogenous sources of DNA damage. The successful repair of DNA lesions is necessary for cellular survival and prevention of disease such as cancer. Therefore, complex molecular pathways have evolved to allow for accurate and timely DNA repair. It is now well-established that mammalian CtIP (CtBP-Interacting Protein) has an important function in DNA Double-Strand Break repair, by promoting the resection of DNA ends in preparation for homologous recombination (HR) and micro-homology mediated end joining (MMEJ). However, the biochemical basis of CtIP's critical role in DNA double-strand break repair remains poorly understood. The aim of this work is to improve our understanding of CtIP's function in maintaining genomic stability. In my thesis I describe the structural and biophysical characterisation of human CtIP and its two evolutionarily conserved N- and C-domains that are critical for CtIP function. Sufficient quantities of recombinant CtIP were expressed and purified for in vitro analysis. A combination of biophysical approaches, including analytical ultra-centrifugation, size-exclusion chromatography and cryo EM, were used to probe the oligomeric nature of CtIP. All techniques showed the presence of high molecular weight oligomeric CtIP species. The N-terminal region of CtIP has been previously shown to be essential for its role in DNA end resection, HR and MMEJ. Using X-ray crystallography in combination with small-angle X-ray scattering I demonstrate that an extended region of the CtIP N-terminus spanning amino acids 31 to 136 forms an elongated parallel helical dimer with coiled-coil segments flanking a central zinc-binding motif. In combination with previous crystallographic analysis of the N-terminal tetramerisation motif of CtIP, my new structural information allows modelling the overall architecture of the CtIP NTD. I have explored the ability of CtIP to bind DNA, via electrophoretic mobility shift assays and fluorescence polarisation, to determine the affinity and minimum binding site of its interaction with double-strand DNA. Using alanine scanning mutagenesis, I have investigated the zinc-binding ability of putative metal-coordination residues within the C-terminal domain. Furthermore, I provide evidence for the presence of a coiled-coil region N-terminally juxtaposed to the CtIP-CTD, which might mediate its dimerization. My findings advance our current knowledge of the biochemical and structural properties of human CtIP. They represent a useful contribution to our current efforts to improve our limited understanding of the molecular mechanisms presiding over the critical DNA-end resection step of HR.
Supervisor: Pellegrini, Luca Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: DNA Repair ; Homologous Recombination ; CtIP