The control of cell division underpins the multicellular basis of mammalian development. To avoid deleterious re-replication events, the process of DNA replication is divided into two non-overlapping steps, first the “licensing” of potential replication origins during G1 and then their sequential “firing” during only S phase. The “pre-replicative complex” (CDC6, CDT1and ORC proteins), license origins in late G1 phase, loading MCM2-7 (mini chromosome maintenance) double hexamers onto the DNA.
However, due to the size of their genome, mammalian cells have a high risk that a replication failure occurs somewhere in the genome. MCM-driven replication forks can irreversibly stall when they encounter DNA damage or tightly bound protein-DNA complexes, resulting in gross chromosomal defects and ultimately, cell death. To minimise the probability of these catastrophic events, two mechanisms are in place: the licensing of “dormant origins” and the existence of a “licensing checkpoint” in late G1. At the moment, the exact regulation of the licensing system and these two safety mechanisms in primary cells is largely unknown. Therefore, a greater understanding of the threshold value of licensing that is needed to activate the licensing checkpoint is important to identify new highly selective anticancer targets.
Meier-Gorlin syndrome (MGS) is a human syndrome characterised by developmental defects, microcephaly and dwarfism but without evidence of chromosomal instability. It is caused by mutations in pre-RC proteins, especially in the ORC complex. However, a number of different cellular and phenotypic abnormalities have been observed in mouse, in vitro human models and patients, with reduced licensing (MCM mutants). These show proliferation defects, genome instability and cancer susceptibility. Therefore, a deeper understanding of pre-RC mutations in primary cell models is required to clarify how these proteins interact with other regulatory systems. This knowledge has significant potential to uncover cell-specific differences in replication licensing and the maintenance of mammalian genome stability. Such findings are vital to the development and refinement of anti-cancer therapeutics.
In this project I investigated how to generate recombinant human iPS cells (hiPSCs) using Crispr/Cas9 technology for MGS-relevant ORC1 mutations and more broadly the licensing system in MGS primary cells versus cancer cell lines.
In this context, the objectives of this project were:
1) To determine if MGS mutations impair origin licensing and licensing checkpoint activation in hiPSCs
2) To clarify different effects of mutant ORC and MCM on the molecular pathways of the licensing checkpoint in genetically modified hiPSCs
3) To perform comparative analyses of these effects on origin licensing and licensing checkpoint activation in hiPS-derived clinically-relevant lineages (neural, cardiac, pulmonary) and mammalian cancer cell lines.
