Grab, manipulate and watch single DNA molecule replication

DNA replication is a critical process intrinsic to the sustenance and propagation of life, involving a symphony of enzymes including helicases, polymerases, and single-stranded DNA binding proteins (SSBs). These enzymes collaboratively ensure the precise duplication of genetic information, a process that, despite significant research, has elements yet to be fully elucidated, especially intermediary steps and the dynamic engagements between replicative proteins. Single-molecule techniques have recently blossomed, offering enhanced insights into the real-time dynamics and interactions of individual molecules in their natural settings, unveiling obscured intermediate steps and enzyme conformational changes. Chapter 1 outlines the thesis's primary aim, elucidating DNA replication mechanisms. Starting with fundamental biological notions, this chapter transitions to discuss the non-equilibrium nature of living systems, emphasizing the role of single-molecule investigations. Such studies have enhanced our understanding of non-equilibrium systems, revealing cellular mechanisms and influencing factors. SSBs are crucial for maintaining genome integrity as they bind to ssDNA and coordinate with various proteins involved in DNA replication, recombination, and repair. Chapter 2 offers a comprehensive overview of recent advances in our understanding of SSBs, as elucidated by single-molecule assays such as optical tweezers, magnetic tweezers, Förster resonance energy transfer, and their combinations. These techniques have provided novel insights into the dynamics of SSB binding to ssDNA and its interactions with other proteins, emphasizing the central role of SSB in modulating the activities of other proteins. Chapter 3 presents the single-molecule observations of the T7 bacteriophage single-stranded DNA-binding protein (gp2.5) binding to ssDNA. Our experiments demonstrate the significant influence of the base sequence, ssDNA conformation, and the acidic terminal domain of T7 gp2.5 on DNA binding parameters. Moreover, we observe a unique template-catalyzed recycling behavior of T7 gp2.5, facilitating efficient spatial redistribution during the synthesis of successive Okazaki fragments. Despite extensive knowledge of DNA replication, the exchange of individual DNA polymerases at the replication fork remains poorly understood. The prevailing hypothesis suggests that this exchange is coordinated by other proteins, such as helicase. In Chapter 4, I propose an alternative model in which DNA polymerases undergo rapid, independent, and uncoordinated exchange. Our observations reveal fast unbinding and rebinding of DNAp, a force-dependent memory effect, and potential assistance from diffusive DNA polymerase in the exchange process. Chapter 5 investigates the interaction between T7 DNA polymerase and T7 gp2.5. We employ single-molecule force spectroscopy to demonstrate that T7 gp2.5 regulates replication rate potentially by controlling secondary structure formation. Dual-color imaging reveals that gp2.5 remains stationary while DNAp advances toward it, suggesting that DNAp displaces gp2.5 in a sequential manner. In Chapter 6, the regulation of T7 helicase (gp4) by its associated T7 gp2.5 is explored. Utilizing optical trap measurements and fluorescence microscopy, I present preliminary data revealing that single-stranded DNA-binding proteins, such as gp2.5, can facilitate helicase unwinding of "sticky-end" DNA, which is typically a suboptimal substrate for helicases. These findings suggest that gp2.5 promotes helicase unwinding by destabilizing the duplex structure, as evidenced by the colocalization of gp2.5 and helicase.