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DNA replication inevitably encounters topological impasses created from the double helical structure of double stranded DNA. Incomplete resolution of the supercoils generated during fork progression leads to intertwining, or catenation, of the two sister chromatids which in turn leads to deleterious downstream effects such as aneuploidy. While biochemical aspects of regulation of catenation prevention and topoisomerase II, the enzyme primarily responsible for resolving supercoils during replication, activity on naked DNA have been deeply explored, the role of mechanical properties of chromatin and topoisomerase II activity on eukaryotic chromatin in its highly compacted form has been largely overlooked. Here I present single-molecule studies that provide insight into the effect of chromatin compaction via formation of nucleosomes in eukaryotic DNA replication and topoisomerase activity. First is the characterization of torsional mechanics of eukaryotic chromatin and its consequences followed up by observation of topoisomerase II activity on nucleosomal DNA. Secondly, I suggest a methodology to dissect the kinetic details of topoisomerase II activity and use it to observe response of the enzyme in physiologically relevant magnesium concentrations.

DNA replication is a fundamental cellular process. However, the structure and dynamics of the eukaryotic DNA replication machinery remain poorly understood. A soluble extract system prepared from Xenopus eggs recapitulates eukaryotic DNA replication outside of a cell on a variety of DNA templates. This system has been used to reveal many aspects of DNA replication using a variety of ensemble biochemical techniques. Single-molecule fluorescence imaging is a powerful tool to dissect biochemical mechanisms. By immobilizing or confining a substrate, its interaction with individual, soluble, fluorescently-labeled reactants can be imaged over time and without the need for synchrony. These molecular movies reveal binding parameters of the reactant and any population heterogeneity. Moreover, if the experiments are imaged in wide-field format, the location or motion of the labeled species along the substrate can be followed with nanometer accuracy. This dissertation describes the use and development of novel single-molecule fluorescence imaging techniques to study eukaryotic DNA replication. A biophysical characterization of a replication fork protein, PCNA, revealed both helical and non-helical sliding modes along DNA. Previous experiments demonstrate that the egg extracts efficiently replicate surface-immobilized linear DNA. This finding suggested replication of DNA could be followed as motion of the replication fork along the extended DNA. However, individual proteins bound at the replication fork could not be visualized in the wide-field due to the background from the high concentration of the fluorescent protein needed to compete with the extract’s endogenous protein. To overcome this concentration barrier, I have developed a wide-field technique that enables sensitive detection of single molecules at micromolar concentrations of the labeled protein of interest. The acronym for this method, PhADE, denotes three essential steps: (1) Localized PhotoActivation of fluorescence at the immobilized substrate, (2) Diffusion of unbound fluorescent molecules to reduce the background and (3) Excitation and imaging of the substrate-bound molecules. PhADE imaging of flap endonuclease I (Fen1) during replication revealed the time-evolved pattern of replication initiation, elongation and termination and the kinetics of Fen1 exchange during Okazaki fragment maturation. In the future, PhADE will enable the elucidation of the dynamic events at the eukaryotic DNA replication fork. PhADE will also be broadly applicable to the investigation of other complex biochemical process and low affinity interactions. It will be especially useful to those researchers wishing to correlate motion with binding events.