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

Abstract

SUMMARY Loading of the ring-shaped Mcm2-7 replicative helicase around DNA licenses eukaryotic 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 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.

Structure of eukaryotic CMG helicase at a replication fork and implications to replisome architecture and origin initiation

“…In the S phase the Mcm2-7 double hexamer matures into head-tohead CMGs at the origin before ssDNA is generated (22)(23)(24)(25). The two CMGs at the origin eventually separate because individual replication forks contain just one CMG (39,40). If the C-tier moved ahead of the N-tier during CMG translocation, the head-to-head CMGs could simply motor away from one another in opposite directions, the current prevailing view.…”

Replication fork instability and the consequences of fork collisions from rereplication

“…This complex includes the origin recognition complex (ORC; which directly binds to the DNA) as well as Cdc6, Cdt1, and the Mcm2-7 complex (Tanaka and Araki 2013). Cdt1 recruits Mcm2-7 to ORC and Cdc6-bound origins, and in vitro studies show that both Cdc6 and Cdt1 quickly disassociate once Mcm2-7 is stably loaded (Ticau et al 2015).…”

“…The Mcm2-7 complex is sequentially loaded as a head-tohead double hexamer onto the dsDNA origin to facilitate bidirectional fork movement (Tanaka and Araki 2013;Ticau et al 2015). Once assembled, the origin is said to be licensed.…”

Insights into the Initiation of Eukaryotic DNA Replication

“…10,11 The details of this reaction have recently been elucidated by a number of labs. [12][13][14] This reaction is restricted to G 1 phase in the cell cycle. 15 Since Mcm2-7 complexes cannot load during S phase, this restriction of Mcm2-7 loading to G 1 phase prevents re-replication or over-replication of the parental duplex DNA.…”