Hybrid Ensemble and Single-molecule Assay to Image the Motion of Fully Reconstituted CMG

We study the motion dynamics of different components of the eukaryotic replisome at the single molecule level. In order to obtain a deep quantitative understanding of how cells can duplicate their entire genomes, every cell cycle. In a breakthrough in 2015, eukaryotic DNA replication was fully reconstituted in vitro from purified protein components.

And this gave us a lot of control over the system. And since then, it's been used extensively to answer questions about different stages of DNA replication with increasing temporal and spatial resolution. And this was done using complementary approaches such as cryo EM and single molecule biophysics.

In the single molecule field, one of the biggest challenges with this system is the number of proteins involved and also the concentrations at which these components are required in order to maximize the efficiency of the overall reaction because this can complicate the single molecule imaging. The protocol described here introduces a hybrid approach in which a protein complex is first assembled in an efficient manner using ensemble biochemistry before then being introduced and studied in a single molecule setting. This avoids the introduction of two high protein concentrations at the single molecule level.

We illustrate this approach for studying the behavior of the replicative helicase CMG on DNA at the single molecule level following its assembly via the origin based pathway. But this approach can also be used to study the dynamics of many other types of DNA binding protein complexes.