Application of photoactivated localisation microscopy to visualising eukaryotic DNA replication processes

DNA replication is a crucial process that ensures duplication of the genome prior to cellular division. The fidelity of this process is of upmost importance for ensuring genomic stability and the integrity of subsequent generations. Obstruction of the replication machinery by DNA damage, protein barriers or other impediments can cause replication stress, a phenotype often observed in cancer cells. Studying the underlying molecular mechanisms of DNA replication and the repair processes involved during replication arrest is thus critical to ensure a complete understanding of the process and the role it plays in cancer development and progression. A key technique used to study DNA replication and repair proteins is fluorescence microscopy, which allows researchers to visualise the expression and spatial organisation of cellular components. Until recently, the information that could be extracted from fluorescence images was restricted by limited resolution, a consequence of the diffraction of light. Recent advancements in fluorescence microscopy have yielded techniques that can break this diffraction barrier and achieve nanometre scale resolution. One such technique is Photoactivated Localisation Microscopy (PALM), which relies on the detection and high precision localisation of single fluorescent molecules. The work presented in this thesis outlines the development of an adaptation to PALM that can be used to study the chromatin association of proteins inside unfixed cells. This technique was subsequently used to study the role of ubiquitination of the replication-sliding clamp during unperturbed DNA synthesis and characterise the global DNA binding of the Smc5/6 complex during replication stress.