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The structural basis for MCM2–7 helicase activation by GINS and Cdc45

Abstract

Two central steps for initiating eukaryotic DNA replication involve loading of the Mcm2–7 helicase onto double-stranded DNA and its activation by GINS–Cdc45. To better understand these events, we determined the structures of Mcm2–7 and the CMG complex by using single-particle electron microscopy. Mcm2–7 adopts two conformations—a lock-washer-shaped spiral state and a planar, gapped-ring form—in which Mcm2 and Mcm5 flank a breach in the helicase perimeter. GINS and Cdc45 bridge this gap, forming a topologically closed assembly with a large interior channel; nucleotide binding further seals off the discontinuity between Mcm2 and Mcm5, partitioning the channel into two smaller pores. Together, our data help explain how GINS and Cdc45 activate Mcm2–7, indicate that Mcm2–7 loading may be assisted by a natural predisposition of the hexamer to form open rings, and suggest a mechanism by which the CMG complex assists DNA strand separation.

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Figure 1: Mcm2–7 exists in two states.
Figure 2: Subunit mapping of Mcm2–7.
Figure 3: CMG contains a notched, planar Mcm2–7 ring that is sealed upon nucleotide binding.
Figure 4: Mcm-subunit mapping in the CMG.
Figure 5: Structure docking into CMG reconstructions.
Figure 6: GINS contacts within the CMG.
Figure 7: CMG interactions.
Figure 8: Model for Mcm2–7 activation and function.

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Acknowledgements

The authors would like to thank A. Lyubimov and F. Bleichert for comments and help with the manuscript; and G. Lander, P. Grob, R. Hannah, R. Hall, M. Cianfrocco and C. Ciferri for technical help. This work was supported by a European Molecular Biology Organization long-term postdoctoral fellowship (to A.C.), a PhD fellowship from the Boehringer Ingelheim Fonds (to T.P.), the Human Frontier Science Program (RPG0039, to E.N.), the National Institute of General Medical Sciences (GM071747, to J.M.B.) and the National Cancer Institute (CA R37-30490, to M.R.B.). E.N. is a Howard Hughes Medical Institute investigator.

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Contributions

A.C., I.I., M.R.B. and J.M.B. conceived the general ideas for this study. All authors planned experiments. A.C. did all electron microscopy single-particle reconstruction and molecular modeling supervised by J.M.B. and E.N. I.I., N.T. and T.P. did cloning, baculovirus construction and protein purification supervised by M.R.B. A.C., M.R.B. and J.M.B. wrote the manuscript. All authors provided editorial input.

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Correspondence to Michael R Botchan or James M Berger.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 1436 kb)

Supplementary Movie 1

The Mcm2-7 complex morphing between a planar-notched and a spiral-lockwasher configuration. (MOV 4475 kb)

Supplementary Movie 2

The CMG complex morphing between the apo and nucleotide-bound state (MOV 3815 kb)

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Costa, A., Ilves, I., Tamberg, N. et al. The structural basis for MCM2–7 helicase activation by GINS and Cdc45. Nat Struct Mol Biol 18, 471–477 (2011). https://doi.org/10.1038/nsmb.2004

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