Single Molecule Studies of Eukaryotic Replisomes in Xenopus Egg Extracts

Abstract

In eukaryotes, two MCM2-7 helicases are assembled at each origin of replication in the G1 phase of the cell cycle. In S phase, the helicases are activated, leading to assembly of two sister replisomes that replicate DNA in opposite directions. At present, little is known about the spatial arrangement or molecular mechanism of MCM2-7 complexes that are engaged in DNA replication. One scenario is that the two sister MCM2-7 complexes dissociate during initiation and then travel away from one another. Alternatively, the sister helicases might remain physically coupled. To differentiate between such models, we have established a series of single-molecule visualization tools using a nucleus-free replication system of Xenopus egg extracts. We demonstrate that these extracts can replicate lambda phage DNA that is mechanically well-stretched and specifically tethered at both ends to a functionalized surface. Our observation of large replication bubbles from single origins on such doubly-tethered DNA argues that helicases located at sister forks can function independently during replication.In addition, we aim to observe real-time dynamics of different replisome components on doubly-tethered DNAs. For this purpose, we have generated fluorescently tagged Cdc45, an MCM2-7 co-factor that travels with the MCM2-7 helicase. Since the high concentration of labeled Cdc45 needed to support replication causes a high fluorescence background, we tagged Cdc45 with the photoswitchable fluorescent protein mKikGR. The ability to switch on the fluorescence of only those mKikGR proteins that are bound to DNA via Cdc45 enables single-molecule imaging of active replisomes, even at high ambient concentrations of Cdc45-mKikGR. We present the results of initial experiments that prove the feasibility of these techniques as novel ways to study the activity of replication factors in a physiologically relevant environment.