Single-Molecule Characterization of Prokaryotic and Eukaryotic Replisomes

  • Yujing Ouyang

Student thesis: Doctoral Thesis


DNA replisomes are the replication machinery mainly comprised of DNA polymerases and helicases. The simplest one is the bacteriophage T7 leading-strand replisome with only three proteins, the DNA polymerase gene 5 protein, the DNA helicase gene 4 protein, and the processivity factor thioredoxin from its host Escherichia coli (E. coli). The E. coli leading-strand replisome is more advanced, with the appearance of the sliding clamp β2 and the clamp loader complex. Compared to the T7 and E. coli prokaryotic replisomes, the most drastic change in eukaryotic replisomes is the relocation of the helicase from the lagging strand to the leading strand. Related single-molecule studies have accumulated for years, however, the heterogeneities of the leading-strand replisomes are still under investigation. To explore the existence and physiological meanings of these heterogeneities, we used single-molecule flow-stretching bead assays to characterize T7, E. coli, and Simian Virus 40 (SV40) leading-strand DNA synthesis in vitro. Piecewise linear function and linear clustering algorithm fittings revealed rate changes during no-pausing T7 leading-strand synthesis, reflecting hidden interaction modes between T7 polymerase/thioredoxin and helicase. T7 and E. coli replication fork arrests by E. coli Tus-Ter complexes showed not only that there are independent mechanisms of the C(6)-mousetrap model in Tus-Ter activity, but also how the heterogeneity in the E. coli replisome and between T7 and E. coli replisomes could determine the fates of DNA replication forks. Zooming in on the human polymerase δ holoenzyme using single-molecule imaging demonstrated that the dynamic interactions among the polymerase, the sliding clamp, and the primer/template DNA are one source of heterogeneity. The human polymerase δ-based SV40 leading-strand synthesis displayed that new factors, the human Fork Protein Complex and the Mini-chromosome maintenance protein 10, changed the replication kinetics significantly, as other sources of heterogeneity. The work provides a deepening and comprehensive understanding of prokaryotic and eukaryotic leading-strand replisomes and unravels various mechanisms that lead to their dynamics.
Date of AwardNov 2022
Original languageEnglish (US)
Awarding Institution
  • Biological, Environmental Sciences and Engineering
SupervisorSamir Hamdan (Supervisor)

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