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Faithful DNA replication is essential for all life. A multi-protein complex called the replisome contains all the enzymatic activities required to facilitate DNA replication, including unwinding parental DNA and synthesizing two identical daughter molecules. Faithful DNA replication can be challenged by both intrinsic and extrinsic factors, which can result in roadblocks to replication, causing incomplete replication, genomic instability, and an increased mutational load. This increased mutational load can ultimately lead to a number of diseases, a notable example being cancer. A key example o...

Observing the processes of life that occur in all cellular organisms at the level of single molecules has allowed a deeper understanding of the dynamic processes taking place in complex biological systems. There has been a strong growth in the application of molecular biophysics to visualize in real time the behaviour of single molecules within a reaction, transforming our perception of the molecular processes that occur within a cell. A multitude of proteins participates across the genome to support the processes of replication, transcription, translation, repair and recombination. The contin...

The molecular mechanisms that support life depend on the fate of small populations of molecules. The functions of biomolecules, however, are traditionally characterised through bulk-phase biochemical methods that only report on ensemble averages. Such methods are not sensitive to population heterogeneity or short-lived reaction intermediates. Single-molecule methods that allow for the direct observation of individual molecules have significantly advanced our understanding of biology in the past two decades. The goal of the work described in this PhD thesis was the development and use of single...

R-loop proteins present a stable and robust blockade to the progression of a DNA replication fork during S-phase. The consequences of this block can include mutagenesis and other irreversible chromosomal catastrophes, causing genomic instability and disease. As such, further investigation into the molecular mechanisms underlying R-loop protein resolution is warranted. The critical role of non-replicative accessory helicases in R-loop protein resolution has increasingly come into light in recent years. Such helicases include the Pif1-family, monomeric helicases that have been studied in many di...