DNA replication initiation and fidelity : a nanoscale view of the code of life

<p dir="ltr">Every time a cell divides it needs to copy its entire genome. This is a fragile and challenging task, involving billions of DNA base pairs, tightly bound proteins, DNA damages and DNA secondary structures. The forced proliferation of cancer cells compromises DNA replication fidelity and causes DNA replication stress. The latter fuels cancer progression but also provides therapeutic opportunities, as DNA replication inhibitors are proven to be potent anti-cancer agents. Therapies that impede replication forks are effective in killing cancer cells but typically cause adverse side effects as they target all dividing cells. Thus, treatments that target replication factors overexpressed in cancer cells could prevent them from initiating DNA replication, resulting in the selective inhibition of tumour cell proliferation while minimising effects on normal cells. While many essential steps involved in the initiation of DNA replication have been extensively studied, key aspects - such as the timing, precise selection, and activation of replication origins - remain incompletely understood, obstructing the design and validation of innovative therapies that act at the level of replication initiation.</p><p dir="ltr">Through three distinct projects, I have developed a novel methodology for studying DNA replication, investigated the molecular mechanisms governing DNA replication initiation and cell cycle regulation, and explored the therapeutic potential of targeting nuclear kinases. Collectively, with this PhD thesis, I have contributed to the advancement of our understanding of DNA replication and cancer therapy.</p><p dir="ltr">A more detailed abstract for each paper continues on the following pages.</p><p dir="ltr">Paper I. Spatial mapping of DNA synthesis reveals dynamics and geometry of human replication nanostructure<br><br>DNA replication is essential to life and ensures the accurate transmission of genetic information, which is significantly disturbed during cancer development and chemotherapy. While DNA replication is tightly controlled in time and space, methods to visualise and quantify replication dynamics within 3D human cells are lacking. Here, we introduce 3D-Spatial Assay for Replication Kinetics (3D- SPARK), an approach enabling nanoscale analysis of DNA synthesis dynamics in situ. 3D-SPARK integrates optimised nucleotide analogue pulse labelling with super-resolution microscopy to detect, classify, and quantify replication nanostructures in single cells. By combining immunofluorescence techniques with click chemistry-based nascent DNA labelling and transfection of fluorescent nucleotide derivatives, we map multi-colour DNA synthesis events in relation to established replication proteins, local RNA-protein condensates or large subnuclear domains. We demonstrate quantitative changes in size, relative abundance and spatial arrangement of nanoscale DNA synthesis events upon chemotherapeutic treatment, CDC6 oncogene expression and loss of chromatin organiser RIF1. The flexibility, precision and modular design of 3D-SPARK helps bridging the gap between spatial cell biology, genomics, and 2D fibre-based replication studies in health and disease.</p><p dir="ltr"><br>Paper II. Temporal control of human DNA replication licensing by CDK4/6-RB signalling and chemical genetics<br><br></p><p dir="ltr">Cyclin-dependent kinases (CDKs) coordinate DNA replication and cell division, and play key roles in tissue homeostasis, genome stability and cancer development. The first step in replication is origin licensing, when minichromosome maintenance (MCM) helicases are loaded onto DNA by CDC6, CDT1 and the origin recognition complex (ORC). In yeast, origin licensing starts when CDK activity plummets in G1 phase, reinforcing the view that CDKs inhibit licensing. Here we show that, in human cells, CDK4/6 activity promotes origin licensing. By combining rapid protein degradation and time-resolved EdU-sequencing, we find that CDK4/6 activity acts epistatically to CDC6 and CDT1 in G1 phase and counteracts RB pocket proteins to promote origin licensing. Therapeutic CDK4/6 inhibitors block MCM and ORC6 loading, which we exploit to trigger mitosis with unreplicated DNA in p53-deficient cells. The CDK4/6-RB axis thus links replication licensing to proliferation, which has implications for human cell fate control and cancer therapy design.</p><p dir="ltr">Paper III. Novel Dihydropteridinone Derivatives As Potent Inhibitors of the Understudied Human Kinases Vaccinia-Related Kinase 1 and Casein Kinase 18/ ¿</p><p dir="ltr">Vaccinia-related kinase 1 (VRK1) and the 8 and & isoforms of casein kinase 1 (CK1) are linked to various disease-relevant pathways. However, the lack of tool compounds for these kinases has significantly hampered our understanding of their cellular functions and therapeutic potential. Here, we describe the structure-based development of potent inhibitors of VRK1, a kinase highly expressed in various tumour types and crucial for cell proliferation and genome integrity. Kinome-wide profiling revealed that our compounds also inhibit CK18 and CK1. We demonstrate that dihydropteridinones 35 and 36 mimic the cellular outcomes of VRK1 depletion. Complementary studies with existing CK18 and CK18 inhibitors suggest that these kinases may play overlapping roles in cell proliferation and genome instability. Together, our findings highlight the potential of VRK1 inhibition in treating p53-deficient tumours and possibly enhancing the efficacy of existing cancer therapies that target DNA stability or cell division.</p><h3>List of scientific papers</h3><p dir="ltr">My PhD thesis is based on three projects which have produced three papers. The status of each of these papers, at time of printing this thesis, is as follows. The first paper is available on BioRxiv and has been reviewed and resubmitted after addressing reviewer's comments. The second paper has been accepted for publication and the third paper has been published.</p><p dir="ltr">I. <b>Michael Hawgood</b>, Bruno Urién, Ana Agostinho, Praghadhesh Thiagarajan, Yiqiu Yang, Xue Zhang, Giovanni Giglio, Gemma Quijada, Matilde Fonseca, Jiri Bartek, Hans Blom, Bennie Lemmens. Spatial mapping of DNA synthesis reveals dynamics and geometry of human replication nanostructures. [Submitted]</p><p dir="ltr">II. Anastasia Sosenko Piscitello, Ann-Sofie Nilsson, <b>Michael Hawgood</b> Abid H Sayyid, Vasilis S Dionellis, Giovanni Giglio, Bruno Urién, Pratikiran Bajgain, Sotirios G Ntallis, Jiri Bartek, Thanos D Halazonetis, Bennie Lemmens. Temporal control of human DNA replication licensing by CDK4/6-RB signalling and chemical genetics. [Accepted]</p><p dir="ltr">III. Fernando H. de Souza Gama, Luiz A. Dutra, <b>Michael Hawgood</b>, Caio Vinícius dos Reis, Ricardo A. M. Serafim, Marcos A. Ferreira Jr., Bruno V. M. Teodoro, Jéssica Emi Takarada, André S. Santiago, Dimitrios-Ilias Balourdas, Ann-Sofie Nilsson, Bruno Urien, Vitor M. Almeida, Carina Gileadi, Priscila Z. Ramos, Anita Salmazo, Stanley N. S. Vasconcelos, Micael R. Cunha, Susanne Mueller, Stefan Knapp, Katlin B. Massirer, Jonathan M. Elkins, Opher Gileadi, Alessandra Mascarello, Bennie B. L. G. Lemmens, Cristiano R. W. Guimarães, Hatylas Azevedo, Rafael M. Couñago. Novel Dihydropteridinone Derivatives As Potent Inhibitors of the Understudied Human Kinases Vaccinia-Related Kinase 1 and Casein Kinase 18/E. Journal of Medicinal Chemistry. 67(11), pp.8609-8629 (2024). <a href="https://doi.org/10.1021/acs.jmedchem.3c02250" rel="noreferrer" target="_blank">https://doi.org/10.1021/acs.jmedchem.3c02250</a></p>