The eukaryotic CMG helicase pumpjack and integration into the replisome

Abstract

The eukaryotic replisome is α multiprotein machine that contains DNA polymerases, sliding clamps, helicase, and primase along with several factors that participate in cell cycle and checkpoint control. The detailed structure of the 11-subunit CMG helicase (Cdc45/Mcm2-7/GINS) has been solved recently by cryoEM single-particle 3D reconstruction and reveals pumpjack motions that imply an unexpected mechanism of DNA translocation. CMG is also the organizing center of the replisome. Recent in vitro reconstitution of leading and lagging strand DNA synthesis has enabled structural analysis of the replisome. By building the replisome in stages from pure proteins, single-particle EM studies have identified the overall architecture of the eukaryotic replisome. Suprisingly leading and lagging strand polymerases bind to opposite faces of the CMG helicase, unlike the long-held view that DNA polymerases are located in back of the helicase to act on the unwound strands.

Keywords: 3D reconstruction; CMG; DNA helicase; DNA polymerase; cryoelectron microscopy; primase; replication fork; replisome.

Figure 1.

CMG helicase structure and translocation mechanism. (a) Low resolution stucture of yeast CMG obtained by negative stain and single particle 3D EM reconstruction. The top view (left) shows the central channel through Mcm2-7 with the Cdc45/GINS located to one side of the ring, and the side view (right) shows the CTD-NTD tiers of the Mcm2-7 ring. Adapted from Fig. 1b of ref. (b) High resolution cryoEM structure of 2 conformers of yeast CMG, oriented similar to the side view in panel A. The 2 conformers differ in the CTD tier of the Mcm2-7 ring. Conformer II (left): all intersubunit interfaces of the CTD tier are closed, and the CTD and NTD tiers are roughly parallel. Conformer I (right): The Mcm2-5 interface is open, and the CTD tier tilts upward. Adapted from Fig. 2f,g of ref. (c) Proposed inchworm mechanism of translocation. CMG binds DNA at 2 sites (illustrated here as the CTD and NTD tiers). Cycles of ATP hydrolysis cause CMG to alternate between compact (conformer II) and extended (conformer I) forms, driving CMG translocation along DNA like an inchworm. See text for details.

Figure 2.

Structure of CMGE and architecture of the replisome. Class average side view images of: (a) CMG, and (b) CMGE. Panel (c) is the 3D single-particle EM reconstruction of CMGE. Panels (d-h) are class average side-view images of: (d) CMG-Ctf4, (e) CMG-Ctf4-Pol α, (f) CMGE-Ctf4 in the presence of forked DNA. Panels (g) and (h) are CMGE and CMGE-Ctf4-Pol α, respectively, in the presence of a primed fork DNA. Illustrations to the right of the class averages are the interpretation of proteins in the images. Adapted from Figs. 2,5 of ref.

Figure 3.

DNA threading through the replisome. (a) CTD-to-NTD model. Studies of Drosophila CMG and archaeal Mcm indicate the leading strand ssDNA enters the CTD tier of CMG. In this model, Pol ε is on “top” of CMG. When the unwound leading strand (red) exits the bottom of Mcm2-7 NTD, it must make a U turn to loop back to Pol ε at the top. The lagging strand (blue) must traverse the outside perimeter of CMG to reach Pol α at the bottom of CMG. Adapted from Fig. 6 of ref. (b) NTD-to-CTD model. If the leading strand enters the NTD tier of Mcm2-7, Pol ε will be positioned to immediately accept the unwound leading strand as it exits CMG. Pol α could act at the top of CMG to prime the unwound lagging strand.