Abstract
Eukaryotic cells in the process of multipying need to faitfully duplicate the DNA. This process of DNA replication involves a number of molecular machines that act in concert, including the Mcm2-7 protein complex that forms the core of the eukaryoric replicative helicase. Two Mcm2-7 helicases are loaded at every potential replication initiation site in a head-to-head orientation. This orientation ensures that the DNA helicases are poised to initiate bidirectional replication. However, the mechanism by which the helicases are loaded in this fashion had remained unclear prior to our study.
This article presents experiments that investigate interactions between the Mcm2-7 helicase and other helicase-loading proteins to build a detailed model to explain how the oppositely-oriented helicases are loaded on DNA. In a biochemically reconstituted helicase-loading reaction, we exposed tethered DNA to purified proteins that are labeled with fluorescent molecules. We used TIRF microscopy to track the labeled proteins and monitor how they interact with one another, and with DNA. These detailed experiments revealed remarkable gymnastics that a helicase-loading protein perfoms to guide loading of the oppositely-oriented Mcm2-7 helicases.