Single-molecule protein dynamics during DNA replication

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 continuous interplay of these proteins on the DNA produces unavoidable physical conflicts that have their own impact on genomic stability. Beyond the complexities of the cellular processes that involve DNA as a reaction partner, the duplex is also constantly exposed to DNA-damaging agents as a result of environmental factors such as UV radiation and oxidative stress. It comes as no surprise that replisomes frequently stall and dissociate because of encounters with DNA damage or tightly-bound protein-DNA complexes. In bacteria, such genomic instability can result in the genetic changes that drive antibiotic resistance evolution. Genomic stability is maintained through pathways that ensure continued replication by minimising the frequency or impact of collisions and identifying and repairing stalled forks. The methodologically diverse toolkit of single-molecule biophysics has been used to address a wide range of questions related to complex protein machineries. Specifically, this thesis highlights the application of single-molecule fluorescence methods to visualize and characterize DNA and the proteins that interact with it. In addition, it describes methodological advances that have been made to utilize linear DNA substrates to uncover protein dynamics. The overall goal of the projects described in this thesis was to design protocols and workflows for the production of linear DNA substrates which are (1) easily customizable to adjust for different experimental parameters and (2) which could be utilized to address a diverse range of biological questions, with a key focus on the controlled introduction of specific chemical lesions. This protocol was employed in support of answering a specific question: How do polymerase exchange dynamics affect lesion bypass mechanisms? This thesis focuses on the protein dynamics that occur at the replication fork in the context of roadblocks and lesions. For the first time, we observe replisome collisions with site-specific cyclobutane pyrimidine dimer lesions on linear substrates at the single-molecule level. This assay presents an exciting avenue to unveil further details of replication stalling and restart. Furthermore, this assay can be adapted to introduce a diverse range of roadblocks, to study dynamics of repair proteins at replication forks and observe the behavior of other replisome complexes. Classical biochemical and single-molecule techniques have provided insight into the proteins and macromolecular complexes responsible for rescue of stalled DNA replication forks. While the majority of studies have employed a reductionist approach in focusing on functions of isolated enzymes, recent work has started to explore the reconstitution of multiple-protein complexes of replication and repair pathways on single molecules of DNA. As we gain more knowledge of the dynamics and mechanisms observed at the single-molecule level, we will see emerging a more detailed picture of the molecular steps associated with the rescue stalled forks. This thesis represents an important step towards that more refined understanding