Abstract
ORC3 is an essential subunit of the Origin Recognition Complex (ORC), best known for its crucial role in DNA replication initiation. However, emerging evidence suggests ORC3 may have additional functions beyond its canonical role, particularly in post-mitotic tissues such as the brain. Despite its importance, the mechanisms by which ORC3 dysfunction contributes to disease remain poorly understood. This study investigates the cellular consequences of patient-derived ORC3 variants, revealing their effects on protein expression and stability, subcellular localisation patterns, chromatin association dynamics, and potential neuronal specific roles across development. Using a multidisciplinary approach, this thesis investigated a cohort of patients harbouring monoallelic and biallelic ORC3 variants, revealing a broad phenotypic spectrum. Patients with compound heterozygous variants, particularly missense variant p.Val216Asp, presented with severe hypomyelinating leukodystrophy, a neurological disorder characterised by deficient myelin formation. In contrast, individuals with monoallelic variants exhibited diverse developmental abnormalities, including features overlapping with other PreReplication Complex (Pre-RC)-associated disorders such as Meier-Gorlin syndrome (MGORS). Functional characterisation demonstrated that the p.Val216Asp variant impairs ORC3’s nuclear and chromatin localisation, as evidenced by biochemical fractionation and immunofluorescence. This mislocalisation likely compromises replication licensing efficiency and chromatin organisation, consistent with ORC3’s canonical role in DNA replication initiation. Structural modelling further suggested that p.Val216Asp destabilises ORC3’s interaction with HP1α, a critical regulator of heterochromatin maintenance. Intriguingly, ORC3 was also detected in cytoplasmic compartments across experimental models, hinting at potential non-canonical functions beyond DNA replication. Transcriptomic analysis of publicly available datasets revealed sustained ORC3 expression in post-mitotic terminally differentiated tissues, particularly in neural tissues like Purkinje cells, supporting ORC3s putative role in neurodevelopment. Unlike other ORC subunits, ORC3 exhibited neural-specific enrichment, with peak expression coinciding with critical periods of iii myelination. This spatiotemporal regulation aligns with the neurological deficits observed in patients, implicating ORC3 in both DNA replication-dependent and independent processes. The findings of this thesis establish ORC3 as a multifunctional protein whose dysfunction underlies a potentially novel neurodevelopmental disorder with multisystem involvement. The study provides mechanistic insights into how ORC3 variants disrupt chromatin dynamics, neuronal maturation, and potentially Rho/ROCK signalling, offering a foundation for future research into therapeutic interventions. By integrating molecular, cellular, and bioinformatic approaches, this work expands understanding of ORC3’s roles in genome stability and neurodevelopment, bridging the gap between replication biology and neurological disease.