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Download StatisticsKennedy, Tristan (2024). Characterisation of a novel hydroxylase complex and its role in replication fidelity. University of Birmingham. Ph.D.
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Kennedy2024PhD.pdf
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Abstract
Protein hydroxylation is an emerging and poorly characterised post-translational modification that is generally catalysed by enzymes called ‘2-oxoglutarate-dependent oxygenases’. JMJD5 is an ‘orphan’ hydroxylase, which has likely contributed to a relatively poor understanding of its cellular role. In this thesis we have attempted to further our understanding of JMJD5 by focusing on an activity-independent interaction between JMJD5 and the Regulator of Chromosome Condensation Domain containing protein 1 (RCCD1). We begin by identifying the specific amino acids in RCCD1 and the JMJD5 N-terminus required for this interaction. We subsequently show that RCCD1 knockdown causes replication stress and that this phenotype is epistatic with respect to JMJD5. Importantly, we demonstrate the importance of the JMJD5:RCCD1 interaction for DNA replication fidelity through reconstitution of the corresponding binding mutants into knockdown cells. We additionally demonstrate that JMJD5:RCCD1 form a stoichiometric 1:1 heterodimeric complex and develop a co-overexpression system for structural characterisation and interactome analysis. Through subsequent proteomic screens we identify a possible activity dependent interaction of the JMJD5:RCCD1 with the CX3 complex of RAD51 paralogs that prescribe the JMJD5:RCCD1 complex with a role in replication fork restart. Overall, our work identifies a novel heterodimeric complex with an important role in faithful DNA replication and genome stability. With genome instability being regarded as a key hallmark of cancer and both JMJD5/RCCD1 being implicated in cancer, our work is helping to understand a potential novel pathway that may contribute to tumorigenesis.
| Type of Work: | Thesis (Doctorates > Ph.D.) | |||||||||
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| Award Type: | Doctorates > Ph.D. | |||||||||
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| Licence: | All rights reserved | |||||||||
| College/Faculty: | Colleges (former) > College of Medical & Dental Sciences | |||||||||
| School or Department: | Institute of Cancer and Genomic Sciences | |||||||||
| Funders: | Medical Research Council | |||||||||
| Subjects: | Q Science > Q Science (General) | |||||||||
| URI: | http://etheses.bham.ac.uk/id/eprint/14889 |
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