Lolo, Rafal;
(2020)
Molecular characterisation of HELQ helicase’s role in DNA repair and genome stability.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Maintenance of genome stability is a critical condition that ensures that daughter cells acquire an accurate copy of the genetic information from the parental cell. DNA replication stress that arises from blocked replication forks, can be a major challenge to genome integrity. Cells have therefore developed complex mechanisms to detect and deal with the replication-associated DNA damage. Intra-S-phase ATR checkpoint, FA pathway and RAD51 paralog BCDX2 complex together constitute key components of the replication stress response system that is essential to sense, repair and restart damaged forks. Previous studies in D. melanogaster and C. elegans have positioned HELQ as an important factor in DNA damage repair and maintaining genome stability. In this work I develop and combine biochemical assays, proteomic studies, mouse model and molecular biology tools to further characterise HELQ function in DNA repair and genome stability. I establish that HELQ plays a pivotal role in the replication stress response in mammalian cells. By developing a system in which I was able to pull down tagged HELQ and subject it to Mass Spectrometry analysis I identified its molecular partners and showed that HELQ interacts with, and interfaces between, the central FANCD2/FANCI heterodimer and the downstream RAD51 paralog BCDX2 complex. From mechanistical point of view, interaction with BCDX2 complex was the most interesting one and I took comprehensive experimental approach to identify the nature of HELQ-BCDX2 binding. I was able to show that this interaction does not need any mediating factors and is most likely a direct one. Despite many attempts and trying various tools I was not able to identify interacting motif neither in HELQ nor RAD51 paralogs, I identified however a candidate amino acid stretch within RAD51B as potential candidate. This works requires further validation. Additionally, I show that HELQ is actively recruited to chromatin upon exposure to DNA damaging agents and this chromatin enrichment is ATR-dependent. Once present on chromatin HELQ performs its function to promote Homologous Recombination-depended repair as shown by persistence of chromatin RAD51 and RPA32 protein in HELQ deficient cells as well as decreased HR-dependent repair measured by DR-GFP assay. To better understand how HELQ promotes HR-dependent ICL repair I decided to set up immune-depletion based ICL-repair system in Xenopus laevis egg extract (developed by Johannes Walter group). Although important progress has been made and I produced several valuable tools I did not manage to validate the system due to time limitations. Lastly, our data positions HELQ as an ovarian cancer susceptibility gene with low penetrance effect. This makes HELQ and interesting object of familial cancer screens and potentially opens an exciting therapeutic approach based on PARP inhibition.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Molecular characterisation of HELQ helicase’s role in DNA repair and genome stability |
| Event: | UCL (University College London) |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2020. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/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 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 |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10096151 |
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