Mechanism and importance of Mcm2-7 double-hexamer formation during DNA replication initiation

Abstract

All cells must duplicate their genome completely and accurately in each cell cycle. Thus, DNA replication is a highly-regulated multi-step process that ensures the genome is duplicated only once per cell cycle. In eukaryotic cells, initiation of DNA replication begins with loading of two heterohexameric Mcm2-7 helicases around origin DNA during G1 phase. The two helicases are loaded in opposite orientations and interact with each other at their N-terminal domains to form a head-to-head "double hexamer". In S phase, the helicases are activated by helicase-activation proteins to initiate DNA unwinding. Importantly, this event is the committed step of replication initiation. Loading of two helicases in the head-to-head double hexamer ensures DNA unwinding on both sides of the origin and allows the assembly of bi-directional forks essential for complete DNA replication. Two Mcm2-7 helicases are loaded onto the DNA sequentially.

The order of events during the first helicase loading has been established, but the mechanism of double-hexamer formation remains unclear. Because the two helicases interact at their N-terminal domains, these regions represent potential mediators of double-hexamer formation. This thesis outlines the potential mechanism and the importance of double-hexamer formation. A conserved motif within Mcm2-7 N-terminal region is required for stable double-hexamer formation and cell viability. Single-molecule analyses of Mcm2-7 containing a mutation within this motif indicated that this mutant form double-hexamer interactions briefly before the two hexamers come apart. Interestingly, after double-hexamer dissolution, the two mutant helicases do not form subsequent double-hexamer interaction. Both wild-type and the mutant Mcm2-7 exhibit double-hexamer interaction rapidly after the arrival of the second Mcm2-7.

Together, these data support the model that double-hexamer formation is coordinated with loading of the second Mcm2-7. Finally, the requirement of the double hexamer during helicase activation was investigated using Mcm2-7 complex containing the mutant that inhibits double-hexamer formation. The double hexamer is not essential for recruitment of three critical helicase-activation proteins, but it is required for initial origin DNA unwinding. These findings identify a crucial motif for stable double-hexamer formation and suggest that DNA unwinding is the first step in replication initiation that requires double-hexamer form of the helicases.