“Single-Molecule Studies of Origin Licensing Reveal Mechanisms Ensuring Bidirectional Helicase Loading”

Fig 7finalIn this study, a collaboration between the Gelles lab and Stephen P. Bell’s lab at MIT, Simina Ticau and co-authors studied the mechanism of “licensing”, the initial process in setting up DNA replication in eukaryotic cells. “Licensing is loading of the ring-shaped Mcm2-7 helicase around DNA origins of replication. During loading, Cdc6, Cdt1, and the origin-recognition complex (ORC) assemble two heterohexameric Mcm2-7 complexes into a head-to-head double hexamer that facilitates bidirectional replication initiation. Using multi-wavelength single-molecule fluorescence to monitor the events of helicase loading, we demonstrate that double-hexamer formation is the result of sequential loading of individual Mcm2-7 complexes. Loading of each Mcm2-7 molecule involves the ordered association and dissociation of distinct Cdc6 and Cdt1 proteins. In contrast, one ORC molecule directs loading of both helicases in each double hexamer. Based on single-molecule FRET, arrival of the second Mcm2-7 results in rapid double-hexamer formation that anticipates Cdc6 and Cdt1 release, suggesting that Mcm-Mcm interactions recruit the second helicase. Our findings reveal the complex protein dynamics that coordinate helicase loading and indicate that distinct mechanisms load the oppositely oriented helicases that are central to bidirectional replication initiation.”

Ticau, S., Friedman, L.J., Ivica, N.A., Gelles, J. & Bell, S.P.
Single-Molecule Studies of Origin Licensing Reveal Mechanisms Ensuring Bidirectional Helicase Loading.
Cell 161, 513-525 (2015).

This article was featured in a Cell Preview “Single-Molecule Visualization of MCM2-7 DNA Loading: Seeing Is Believing” by Gheorghe Chisto and Johannes C. Walter. Cell 161, 429-430 (2015) 10.1016/j.cell.2015.04.006.  It was also discussed in
Single Molecule Enzymology Finds its Stride” by Jeffrey Perkel. BioTechniques 59:183–187 (2015) 10.2144/000114337.