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postgraduate thesis: Structural and functional characterization of the eukaryotic replisome
Title | Structural and functional characterization of the eukaryotic replisome |
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Authors | |
Advisors | |
Issue Date | 2023 |
Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
Citation | Xu, Z. [许志纯]. (2023). Structural and functional characterization of the eukaryotic replisome. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. |
Abstract | In eukaryotes, DNA replication is tightly regulated to ensure a faithful transmission of genetic information. The machinery responsible for the replication of DNA consists of Cdc45-MCM-GINS (CMG) helicase, DNA polymerases, and multiple accessory factors. To ensure efficient DNA replication, the activities of the CMG helicase and DNA polymerases must be coupled to coordinate DNA unwinding and synthesis in a translocating replisome. However, the precise mechanisms underlying the synchronization of these two engines at the replication fork remain unclear. In this study, we determined a series of cryo-electron microscopy (cryo- EM) structures of a yeast replisome comprising CMG, the leading strand polymerase Pol ε, and three accessory factors, including Ctf4, Tof1, and Csm3, at a forked DNA. Our findings reveal that the DNA-engaged CMG nucleates Pol ε and other accessory factors to target their preferred substrates. Pol ε occupies two alternative positions that engage or disengage with the motor domains of CMG. The coupling between Pol ε and the MCM ring closely correlates with the rotational movement of single-stranded DNA around the MCM pore. Despite the apparent cycling of Pol ε on and off the MCM ring, the polymerase remains stably attached to the helicase using Psf1 as a hinge. Moreover, a concerted rearrangement of six ATPase sites drives DNA translocation, modulating this dynamic process. These findings provide crucial insights into the intrinsic mechanism by which CMG and Pol ε are coordinated to promote efficient DNA replication while navigating replication forks and addressing obstacles such as DNA damage and epigenetic marks. |
Degree | Doctor of Philosophy |
Subject | Chromosome replication DNA replication |
Dept/Program | Biological Sciences |
Persistent Identifier | http://hdl.handle.net/10722/341559 |
DC Field | Value | Language |
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dc.contributor.advisor | Zhai, Y | - |
dc.contributor.advisor | Li, XD | - |
dc.contributor.author | Xu, Zhichun | - |
dc.contributor.author | 许志纯 | - |
dc.date.accessioned | 2024-03-18T09:55:56Z | - |
dc.date.available | 2024-03-18T09:55:56Z | - |
dc.date.issued | 2023 | - |
dc.identifier.citation | Xu, Z. [许志纯]. (2023). Structural and functional characterization of the eukaryotic replisome. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. | - |
dc.identifier.uri | http://hdl.handle.net/10722/341559 | - |
dc.description.abstract | In eukaryotes, DNA replication is tightly regulated to ensure a faithful transmission of genetic information. The machinery responsible for the replication of DNA consists of Cdc45-MCM-GINS (CMG) helicase, DNA polymerases, and multiple accessory factors. To ensure efficient DNA replication, the activities of the CMG helicase and DNA polymerases must be coupled to coordinate DNA unwinding and synthesis in a translocating replisome. However, the precise mechanisms underlying the synchronization of these two engines at the replication fork remain unclear. In this study, we determined a series of cryo-electron microscopy (cryo- EM) structures of a yeast replisome comprising CMG, the leading strand polymerase Pol ε, and three accessory factors, including Ctf4, Tof1, and Csm3, at a forked DNA. Our findings reveal that the DNA-engaged CMG nucleates Pol ε and other accessory factors to target their preferred substrates. Pol ε occupies two alternative positions that engage or disengage with the motor domains of CMG. The coupling between Pol ε and the MCM ring closely correlates with the rotational movement of single-stranded DNA around the MCM pore. Despite the apparent cycling of Pol ε on and off the MCM ring, the polymerase remains stably attached to the helicase using Psf1 as a hinge. Moreover, a concerted rearrangement of six ATPase sites drives DNA translocation, modulating this dynamic process. These findings provide crucial insights into the intrinsic mechanism by which CMG and Pol ε are coordinated to promote efficient DNA replication while navigating replication forks and addressing obstacles such as DNA damage and epigenetic marks. | - |
dc.language | eng | - |
dc.publisher | The University of Hong Kong (Pokfulam, Hong Kong) | - |
dc.relation.ispartof | HKU Theses Online (HKUTO) | - |
dc.rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works. | - |
dc.rights | This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. | - |
dc.subject.lcsh | Chromosome replication | - |
dc.subject.lcsh | DNA replication | - |
dc.title | Structural and functional characterization of the eukaryotic replisome | - |
dc.type | PG_Thesis | - |
dc.description.thesisname | Doctor of Philosophy | - |
dc.description.thesislevel | Doctoral | - |
dc.description.thesisdiscipline | Biological Sciences | - |
dc.description.nature | published_or_final_version | - |
dc.date.hkucongregation | 2024 | - |
dc.identifier.mmsid | 991044781605303414 | - |