Post-Replicative Resolution of Under-Replication

  • James T. Carrington

    Student thesis: Doctoral ThesisDoctor of Philosophy

    Abstract

    The evolutionary pressure to prevent re-replication by inactivating licensing during S phase leaves higher-eukaryotes with large genomes, such as human cells, vulnerable to replication stresses. Origins licensed in G1 must be sufficient to complete replication as new origins cannot be licensed in response to irreversible replication fork stalling. Interdisciplinary approaches between cellular biology and biophysics predict that replication of the genome is routinely incomplete in G2, even in the absence of external stressors. The frequency of converging replication forks that never terminate due to irreversible stalling (double fork stall), which result in a segment of unreplicated DNA, was modelled using high quality origin-mapping data in HeLa and IMR-90 cells. From this, hypotheses were generated that related an increase in unreplicated segments of DNA to reduced functional origin number. Presented in this thesis is the confirmation of this relation by quantifying chromosome mis-segregation and DNA damage responses when origin number was reduced using RNAi against licensing factors. The number of ultrafine anaphase bridges and 53BP1 nuclear bodies are in remarkable concordance with the theoretical predictions for the number of double fork stalls, indicating that cells are able to tolerate under-replication through such post-replicative cellular responses. 53BP1 preferentially binds to chromatin associated with large replicons, and functions synergistically with dormant origins to protect the stability of the genome. Additional candidates, inspired by common fragile site research, have also been characterised as responders to spontaneous under-replication, and include FANCD2 and MiDAS, which function in early mitosis to facilitate completion of replication before cells enter anaphase. In conclusion, a series of mechanisms that sequentially function throughout the cell cycle protects the stability of the human genome against inevitable spontaneous under-replication brought about by its large size.
    Date of Award2017
    LanguageEnglish
    Awarding Institution
    • University of Dundee
    Add any sponsors of the thesis researchWellcome Trust
    SupervisorJulian Blow (Supervisor)

    Keywords

    • DNA replication
    • DNA damage
    • DNA damage repair
    • Under-replication
    • 53BP1
    • RPA
    • FANCD2
    • MiDAS
    • Mitotic DNA synthesis
    • UFB
    • Ultrafine anaphase bridge
    • BLM
    • H2AX
    • MCM5
    • MCM2
    • CDT1
    • EdU

    Cite this

    Post-Replicative Resolution of Under-Replication
    Carrington, J. T. (Author). 2017

    Student thesis: Doctoral ThesisDoctor of Philosophy