Molecular Cell
Volume 58, Issue 3, 7 May 2015, Pages 483-494
Journal home page for Molecular Cell

Article
The Dynamics of Eukaryotic Replication Initiation: Origin Specificity, Licensing, and Firing at the Single-Molecule Level

https://doi.org/10.1016/j.molcel.2015.03.017Get rights and content
Under an Elsevier user license
open archive

Highlights

  • Pivotal steps of replication initiation are studied at the single-molecule level

  • Cdc6 dictates the ordered and specific assembly of pre-RC components

  • Initiation is tightly regulated, ensuring that sister forks fire simultaneously

  • Replisome progression is processive and insensitive to DNA sequence composition

Summary

Eukaryotic replication initiation is highly regulated and dynamic. It begins with the origin recognition complex (ORC) binding DNA sites called origins of replication. ORC, together with Cdc6 and Cdt1, mediate pre-replicative complex (pre-RC) assembly by loading a double hexamer of Mcm2–7: the core of the replicative helicase. Here, we use single-molecule imaging to directly visualize Saccharomyces cerevisiae pre-RC assembly and replisome firing in real time. We show that ORC can locate and stably bind origins within large tracts of non-origin DNA and that Cdc6 drives ordered pre-RC assembly. We further show that the dynamics of the ORC-Cdc6 interaction dictate Mcm2–7 loading specificity and that Mcm2–7 double hexamers form preferentially at a native origin sequence. Finally, we demonstrate that single Mcm2–7 hexamers propagate bidirectionally, monotonically, and processively as constituents of active replisomes.

Cited by (0)