Role of ORC1 in cell proliferation and regulation of organism growth

Abstract

Mutations in five protein components of the pre-replication complex (pre-RC) have been identified in patients diagnosed with Meier-Gorlin syndrome (MGS), a disorder of global extreme growth failure. ORC1, the most commonly mutated protein, is the largest of six subunits in the origin recognition complex which associates with genomic replication origins and initiates the assembly of the pre-RC during G1 of the cell cycle. Mutations in ORC1, therefore, are expected to perturb cell cycle progression resulting in lengthened cell cycle which leads to a reduction in overall cell number generated during embryogenesis. In this thesis I investigate the developmental and cellular consequences of mutations in ORC1. To address the role of ORC1 during mammalian development I utilised CRISPR/cas9 technology to generate an allelic series of Orc1 mutations in mouse embryonic stem cells (mESCs). Mouse embryos generated by tetraploid complementation assays, using hypomorphic Orc1-mESCs, indicates that impaired Orc1 function in mouse embryogenesis results in developmental delay and a reduction in embryo size. While, no change in proliferation dynamics was seen in Orc1-mESCs in vitro, despite the markedly shortened cell cycle in such cells. Similarly, no significant differences in cell cycle or proliferation rate were detectable in primary patient-derived fibroblasts and lymphoblastoid cell lines. In contrast, Orc1-deficient mouse embryonic fibroblasts had a lengthened cell cycle, as a result of a prolonged G1 phase, implicating the role of the replication licensing checkpoint in somatic cells in lengthening of the cell cycle. In addition, the impact of complete loss of ORC1 was investigated in the developing limb bud. Generation of an Orc1 conditional allele provided a tool to analyse how loss of ORC1 would alter cell cycle in the developing limb. ORC1 is required for embryonic development, and homozygous deletion is embryonic lethal. Mesoderm specific deletions in Orc1 in the developing limb bud using the Prx1-cre transgene resulted in complete loss of forelimb structures, and oligodactyl of the hind limb. Finally, a novel disease gene was identified in patients with MGS. Mutations were identified in 9 patients (7 families) with mutations in CDC45. CDC45 acts downstream of the pre-RC and is required for the formation of the pre-initiation complex, origin activation and fork elongation. As CDC45 acts in the same pathway as ORCs, CDC6 and CDT1, it was hypothesised that hypomorphic mutations in CDC45 might also result in MGS due to impaired DNA replication.