The molecules of life are small to us—billionths of our size. They move fast too, and in the cell they crowd together impossibly. Bringing that strange world into ours is the trick of molecular biology. One approach is to harness many copies of a molecule and iterate a reaction many times to glimpse what happens at that small, foreign scale. This is a powerful way to do things and has provided major insights. But ultimately, the fundamental unit of molecular biology is the individual molecule, the individual interaction, the individual reaction. Single-molecule bioscience is the study of these phenomena. Eukaryotic DNA replication is particularly interesting from the single-molecule perspective because the biological molecules responsible for executing the replication pathway interact so very intricately. This work is based on replication in budding yeast—a model eukaryote. The budding yeast genome harbors several hundred sequence-defined sites of replication initiation called origins.

Origins are bound by the Origin Recognition Complex (ORC), which recruits the ring-shaped Mcm2-7 complex during the G1 phase of the cell cycle. A second Mcm2-7 is loaded adjacent to the first in a head-to-head orientation; this Mcm2-7 double hexamer encircles DNA and is generally termed the Pre-Replicative Complex, or Pre-RC. Mcm2-7 loading is strictly dependent on a cofactor, Cdc6, which is expressed in late G1. Much less is known about the details of downstream steps, but a large number of factors assemble to form active replisomes. Origin-specific budding yeast replication has recently been reconstituted in vitro, with cell cycle dependence mimicked by the serial addition of purified Pre-RC components and activating kinases. This work introduces the translation of the bulk biochemical replication assay into a single-molecule assay and describes the consequent insights into the dynamics of eukaryotic replication initiation. I have developed an optical microscopy-based assay to directly visualize DNA replication initiation in real time at the single-molecule level: from origin definition, through origin licensing, to replisome formation and progression.

I show that ORC has an intrinsic capacity to locate and stably bind origin sequences within large tracts of non-origin DNA, and that ordered Pre-RC assembly is driven by Cdc6. I further show that the dynamics of the ORC-Cdc6 interaction dictate the specificity of Mcm2-7 loading, and that Mcm2-7 double hexamers form preferentially at a native origin sequence. This work uncovers key variables that control Pre-RC assembly, and how directed assembly ensures that the Pre-RC forms properly and selectively at origins. I then characterize replisome initiation and progression dynamics. I show that replication initiation is highly precise and limited to Mcm2-7 double hexamers. Sister replisomes fire bidirectionally and simultaneously, suggesting that previously unidentified quality control mechanisms ensure that a complete pair of replisomes is properly assembled prior to firing. I also find that single Mcm2-7 hexamers are sufficient to support processive replisome progression. Moreover, this work reveals that replisome progression is insensitive to DNA sequence composition at spatial and temporal scales relevant to the replication of an entire genome, indicating that separation of the DNA strands by the replicative helicase is not rate-limiting to replisome function.

I subsequently applied this replication assay to the study replisome-replisome collisions, a fundamental step in the resolution of convergent replication forks. I find that, surprisingly, active replisomes absolutely lack an intrinsic capacity to displace inactive replisomes. This result eliminates the simplest hypothesized mechanism for how the cell resolves the presence of un-fired replisomes and has prompted and guided the development of alternate testable hypotheses. Taken together, these observations probe the molecular basis of eukaryotic inheritance in unprecedented detail and offer a platform for future work on the many dynamic aspects of replisome behavior.