| DNA replication is a fundamental cellular process carried out by a multiprotein complex known as the replisome. The coordination of multiple components, activities, and interactions in the replisome involves transient intermediates and dynamic conformational changes that are difficult to observe with ensemble experiments. In this dissertation, single-molecule techniques are used to examine DNA replication by the E. coli replisome. Replication activities of single polymerase holoenzymes and leading-strand replisomes are measured using flow-stretching of DNA. Novel single-molecule fluorescence methods are developed and applied to observing intact replisomes carrying out coordinated leading- and lagging-strand synthesis by observation of DNA product growth and fluorescently labeled replisome components. Together, the single-molecule experiments described here enable real-time measurement of individual, active E. coli replisomes and provide information on replisome composition, activity, and dynamics. |
