DNA replication and repair in microcephalic dwarfism
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Date
06/07/2019Author
Tarnauskaitė, Žygimantė
Metadata
Abstract
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.