Abstract
Licensing of eukaryotic origins of replication requires DNA loading of two copies of the Mcm2-7 replicative helicase to form a head-to-head double-hexamer, ensuring activated helicases depart the origin bidirectionally. To understand the formation and importance of this double-hexamer, we identified mutations in a conserved and essential Mcm4 motif that permit loading of two Mcm2-7 complexes but are defective for double-hexamer formation. Single-molecule studies show mutant Mcm2-7 forms initial hexamer-hexamer interactions; however, the resulting complex is unstable. Kinetic analyses of wild-type and mutant Mcm2-7 reveal a limited time window for double-hexamer formation following second Mcm2-7 association, suggesting that this process is facilitated. Double-hexamer formation is required for extensive origin DNA unwinding but not initial DNA melting or recruitment of helicase-activation proteins (Cdc45, GINS, Mcm10). Our findings elucidate dynamic mechanisms of origin licensing, and identify the transition between initial DNA melting and extensive unwinding as the first initiation event requiring double-hexamer formation.
Highlights
Initiation of DNA replication occurs at genomic sites called origins of replication
Our observations identify a key motif required for stable Mcm2-7-Mcm2-7 interactions and identify the transition to extensive origin DNA unwinding as the first step in replication initiation that requires the doublehexamer form of the helicase
We addressed the impact of the Double-Hexamer Motif (DoHM) mutant on origin DNA unwinding using an assay that detects the formation of supercoiled DNA as a consequence of the unwinding process (Douglas et al, 2018)
Summary
Initiation of DNA replication occurs at genomic sites called origins of replication. Each eukaryotic origin is licensed during G1 phase through loading of the replicative helicase. These two loaded Mcm complexes are proposed to subsequently come together through translocation on the double-stranded origin DNA (Coster and Diffley, 2017; Frigola et al, 2013) This model is supported by the in vivo requirement for two ORC-binding sites at origins of replication and mutations that suggest that both the first and second Mcm complexes interact with ORC during loading (Coster and Diffley, 2017; Frigola et al, 2013). Our observations identify a key motif required for stable Mcm2-7-Mcm interactions and identify the transition to extensive origin DNA unwinding as the first step in replication initiation that requires the doublehexamer form of the helicase
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