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“Molecular Characterisation of Helq Helicase's Role In “MOLECULAR CHARACTERISATION OF HELQ HELICASE’S ROLE IN DNA REPAIR AND GENOME STABILITY” Rafal Lolo University College London and Cancer Research UK London Research Institute PhD Supervisor: Simon Boulton A thesis submitted for the degree of Doctor of Philosophy University College London September 2018 Declaration I Rafal Lolo confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 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- 3 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. 4 Impact statement Genetic material of all cells is constantly subjected to physical and chemical damaging factors. If unrepaired or repaired illegitimately damaged DNA causes genomic instability and accumulation of mutations which constitute the hallmarks of cancer. Organisms from archaea to vertebrates have evolved elaborated mechanism allowing to detect and repair damaged DNA in efficient and timely manner. Research performed in the field of DNA damage response have provided fundamental insights into both processes driving cancer formation, and ways that cancer biology can be exploited for therapeutic purposes. One of the best examples of such approach is Fanconi Anemia, an extremely rare genetic disorder characterised by congenital defects, bone marrow failure and predisposition to cancer. Research done in the field of Fanconi Anemia contributed immeasurably to better understanding of how DNA damage is formed and repaired, in particular to our better understanding of most widely used chemotherapeutics - regimens based on ICL inducing agents. In the presented study we show that HELQ is an important DNA damage repair factor acting in response to replication fork stalling agents such as ICL-inducing drugs and replication inhibitors. Our work led to detailed identification of interacting network of proteins that HELQ work in response to DNA damage. Interestingly almost all of HELQ identified interacting partners, if mutated lead to increased risk of cancer development placing the protein in the cancer suppressor network. Indeed, in case of HelQ mutated mice we also observed increased incidence of cancer, predominantly ovarian carcinoma. This phenotype is in line with our observation that the role of HELQ in maintaining genome stability can be contributed to promotion of efficient Homologous Recombination (HR) repair of stalled replication forks. The link between germline and somatic mutations in genes responsible for HR and increased risk of ovarian and breast cancers is well established and our data indicate that HELQ is a good candidate gene for screening in ovarian cancer. Importantly, HELQ implication in ovarian cancer can potentially open targeted treatment options for patients caring pathogenic mutations in this gene. DNA 5 damage response inhibition is a novel anti-cancer approach which could revolutionise the treatment of cancer. It works by exploiting DNA damage response defects which are specific to cancer cells and kills them whilst sparing normal cells. Cancer cells with a DNA damage response deficiency are heavily reliant on remaining ‘back up’ DNA repaired systems which can be efficiently blocked by small molecule inhibitors causing synthetic lethality specifically in cancer cells. One of first and best studied example of such system is olaparib - PARP-1 inhibitor. In our work we established that HELQ deficient cells are hypersensitive to olaparib which potentially opens treatment option for patients with HELQ-mutated cancers. To summarize, we established that HELQ plays a pivotal role in the replication stress response, interfacing between the central FANCD2/FANCI heterodimer and the downstream RAD51 paralog BCDX2 complex, which is critical for HR repair. Additionally, we showed that HELQ is a tumor suppressor and a good candidate for screening in ovarian cancer patients for clinical trial of PARP-1 inhibitors. 6 Acknowledgement First, I would like to thank my supervisor Simon Boulton for giving me the opportunity to work in his lab and for guiding me throughout my PhD. I am particularly grateful for the patience and openness Simon has given me in times when I needed it most. I would like to thank all members of the Boulton’s Lab for their friendship, guidance and support throughout the years we spent together. My special thanks go to Carrie who I had the great pleasure to share the HelQ story with. Carrie’s patience, experience and advice were invaluable and catalysed my own growth and development. Thank you, Carrie! I would also like to express my gratitude to Costanzo’s Group for their help and guidance in my work with Xenopus model. Many people have made Clare Hall a very special place and the time spent there an important life lesson. Thank you all. Last but not least, I would like to thank my Mother for the endless reserves of faith and confidence in me, in particular in those moments when I deserved it least. 7 Table of Contents Abstract ………………………………………………………………………………3 Impact statement .............................................................................................. 5 Acknowledgement ............................................................................................ 7 Table of Contents .............................................................................................. 8 Table of figures ............................................................................................... 11 List of tables .................................................................................................... 15 Abbreviations .................................................................................................. 16 Chapter 1.Introduction ................................................................................... 19 1.1 Overview of DNA Damage. ................................................................... 19 1.1.1 Cellular consequences of DNA damage ............................................ 19 1.1.2 DNA Repair Mechanisms .................................................................. 21 1.2 Formation and repair of DNA Interstrand Cross-Links (ICLs). .......... 30 1.2.1 ICL-inducing agents. ......................................................................... 31 1.2.2 ICL Repair mechanisms .................................................................... 37 1.2.3 Recognition of ICL ............................................................................. 41 1.3 The role of HELQ helicase in ICL repair .............................................. 67 Chapter 2.Materials & Methods ...................................................................... 70 2.1 Enzymes and reagents .........................................................................
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