Greiwe, Julia Franziska;
(2023)
DNA replication initiation studied by cryo-EM.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Eukaryotic DNA replication is tightly regulated to ensure faithful duplication of genetic information before cell division. The motor of the replicative helicase is the minichromosome maintenance (MCM) complex, which unwinds duplex DNA and exposes single-stranded DNA to replicative polymerases. The MCM is loaded onto duplex DNA as an inactive double hexamer (DH), in a process named origin licensing. I solved a 3.0 Å resolution structure of the DNA-loaded Saccharomyces cerevisiae DH, showing that Watson–Crick base pairing remains intact. DH phosphorylation by the Dbf4-dependent kinase (DDK) represents the first step towards activation of the helicase, nucleation of DNA melting and replication fork establishment. DDK selectively targets DNA-loaded DHs, but the mechanism is unknown. Using in vitro reconstitution and cryo-electron microscopy (cryo-EM) I discovered that DDK recognises the three-dimensional structure of the DH by docking onto one MCM hexamer and phosphorylating the other hexamer. Truncation of the docking domain of DDK does not affect kinase function per se but abrogates DH phosphorylation. DDK autophosphorylation is known to reduce kinase activity. While this is true for an isolated MCM peptide, I established that DH phosphorylation is virtually unaffected, meaning selectivity for the DH is increased. When DNA damage is detected in S phase, the checkpoint kinase Rad53 targets DDK to stop origin firing. I found that Rad53 phosphorylation of DDK blocks DH engagement and helicase activation. Little is known about DNA replication in humans. In a collaborative effort, I reconstituted human origin licensing in vitro. I then analysed the entire reaction by cryo-EM to identify five protein assemblies that present similarities and differences compared to helicase loading intermediates in S. cerevisiae. Unlike yeast DH, I found that the human DH untwists and melts duplex DNA. My results indicate that nucleation of origin DNA melting occurs via distinct mechanisms in yeast and humans.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | DNA replication initiation studied by cryo-EM |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2022. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences |
| URI: | https://discovery-pp.ucl.ac.uk/id/eprint/10172781 |
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