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Title: DNA replication and repair in microcephalic dwarfism
Author: Tarnauskaitė, Žygimantė
ISNI:       0000 0004 7969 1579
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2019
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Human height varies greatly between and within populations, and some individuals fall at the extreme ends of this wide spectrum. At the lower end of this distribution, individuals demonstrating extreme prenatal-onset reduction in body size and brain growth are classified as having microcephalic primordial dwarfism (MPD), which encompasses a group of rare single-gene disorders, usually inherited in an autosomal recessive manner. The human brain is particularly susceptible to perturbation during embryonic development, and the inability of neural progenitor cells to complete timely proliferation is thought to be an important contributor to the observed reduction in cerebral cortical size. Studying genes whose disruption leads to severe reduction in human growth can facilitate our understanding of the molecular pathways underlying cell proliferation and organism development. Mutations in many identified MPD genes result in the extended length of the cell cycle and impaired cell division by affecting essential cellular processes, such as DNA replication, DNA damage response (DDR) signalling, centriole biogenesis and mRNA splicing. The ability of cells to efficiently copy DNA and maintain the stability of their genome by promoting error-free repair of various types of DNA damage caused by endogenous and exogenous sources is particularly important for the timely cell cycle completion and cell survival. Therefore, it is not surprising that many MPD genes play a role in DNA replication, DDR and DNA repair. In this thesis, three DNA replication and DDR genes, mutated in MPD, are investigated. DNA2, encoding an ATP-dependent helicase/nuclease, was found to be mutated in four MPD patients. Experiments to confirm pathogenicity of the identified mutations indicated that they are likely to cause disease by affecting DNA2 transcript splicing and its enzymatic activities. My work described here also analyses the cellular role of TRAIP, an E3 ubiquitin ligase, which was linked to MPD by our laboratory (Harley et al., 2016). Cell experiments using TRAIP knockout cell lines, generated with CRISPR/Cas9 genome editing technology, demonstrated the requirement for TRAIP and its E3 ligase activity in DDR and repair of camptothecin (CPT)-induced DNA damage. Additionally, TRAIP was important for cell survival after mitomycin C (MMC)-induced DNA damage, but no epistasis with the Fanconi Anaemia (FA) interstrand crosslink (ICL) repair pathway was demonstrated, indicating an additive effect of TRAIP and FA-ICL pathways to repair these DNA lesions. Finally, generation of a mouse model of MPD caused by mutations in DONSON, a novel replication fork protection factor (Reynolds et al., 2017), is described in this thesis. DONSON MPD mice, harbouring the mouse equivalent of one of the human MPD missense mutations, showed embryonic lethality, with homozygous mutant embryos significantly smaller than their littermates and exhibiting limb abnormalities. Increased levels of spontaneous DNA damage were observed in mouse embryonic fibroblasts established from these embryos, mimicking the cellular phenotype of human DONSON deficiency. In summary, this thesis advances our knowledge of the cellular and developmental roles of MPD genes TRAIP, DNA2, and DONSON, that encode proteins maintaining genome stability.
Supervisor: Jackson, Andrew ; Adams, Ian Sponsor: Medical Research Council (MRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: microcephalic primordial dwarfism ; TRAIP ; DNA2 enzymatic activities ; DONSON ; mouse model ; DNA damage signalling and repair