Abstract
DNA replication origins serve as sites of replicative helicase loading. In all eukaryotes, the six-subunit origin recognition complex (Orc1-6; ORC) recognizes the replication origin. During late M-phase of the cell-cycle, Cdc6 binds to ORC and the ORC-Cdc6 complex loads in a multistep reaction and, with the help of Cdt1, the core Mcm2-7 helicase onto DNA. A key intermediate is the ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) complex in which DNA has been already inserted into the central channel of Mcm2-7. Until now, it has been unclear how the origin DNA is guided by ORC-Cdc6 and inserted into the Mcm2-7 hexamer. Here, we truncated the C-terminal winged-helix-domain (WHD) of Mcm6 to slow down the loading reaction, thereby capturing two loading intermediates prior to DNA insertion in budding yeast. In "semi-attached OCCM," the Mcm3 and Mcm7 WHDs latch onto ORC-Cdc6 while the main body of the Mcm2-7 hexamer is not connected. In "pre-insertion OCCM," the main body of Mcm2-7 docks onto ORC-Cdc6, and the origin DNA is bent and positioned adjacent to the open DNA entry gate, poised for insertion, at the Mcm2-Mcm5 interface. We used molecular simulations to reveal the dynamic transition from preloading conformers to the loaded conformers in which the loading of Mcm2-7 on DNA is complete and the DNA entry gate is fully closed. Our work provides multiple molecular insights into a key event of eukaryotic DNA replication.
Highlights
Our work reveals two conformations of the OCCM complex prior to DNA insertion
The semi-attached OCCM is characterized by high flexibility between ORC–Cdc[6] and Cdt1–Mcm[2,3,4,5,6,7]
These interactions guarantee that Cdt1–Mcm[2,3,4,5,6,7] recognizes the ORC–Cdc[6] complex but not ORC alone, since both WHDs contact Cdc[6], providing complex specificity
Summary
Cryo-EM Captures Two OCCM Intermediates Using the Mcm6-ΔC6 Mutant. To examine the structural rearrangements occurring during helicase loading in budding yeast, we employed an Mcm[27] complex containing McmΔC6 and the ATP analog ATPγS, which both slow down the pre-RC reaction cascade (Fig. 1 A) (14). Analysis of 2D class averages showed one set of classes where the density of the helicase loader ORC–Cdc[6] is well resolved while Cdt1–Mcm[2,3,4,5,6,7] is clearly present but is blurred (Fig. 1 B, Upper) This suggests the capture of a very early intermediate in which Mcm[2,3,4,5,6,7] is connected to ORC–Cdc[6] yet the two complexes have not docked onto each other. The Orc[2] WHD appears to play a role in restricting DNA loss from
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
