Structure of the pre-initiation complex explains CMGE biogenesis

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DNA replication must occur only once per cell cycle to maintain genome stability. To achieve this, eukaryotes have evolved to temporally separate the loading of the replicative helicase from its activation 1 . During the G1 phase of the cell cycle, two copies of the MCM motor of the replicative helicase are loaded as an inactive double hexamer (DH) onto DNA replication origins 2 , 3 . Activation occurs after S-phase transition and involves the recruitment of Cdc45 and the tetrameric GINS (Go-Ichi-Ni-San) complex to MCM, together forming the Cdc45–MCM–GINS (CMG) helicase 4 , 5 . CMG assembly occurs under the control of three kinases. It is promoted by the Dbf4-dependent kinase (DDK) 6 , 7 , 8 and Cdc28–Clb5 (hereafter, CDK), the activity of which increases when cells enter S phase 9 , 10 , 11 . CMG assembly is inhibited by the checkpoint kinase Rad53, which blocks late origin firing if DNA damage is detected 9 , 10 , 11 , 12 , 13 . DDK selectively phosphorylates DNA-loaded DHs 14 . A heterodimeric firing factor composed of Sld3 (essential) and Sld7 (dispensable) then recognizes the phosphorylated DH and recruits Cdc45 to MCM (refs. 10 , 11 , 15 ). An N-terminal truncation of Mcm4 bypasses the requirement for DDK in cells 7 , 8 and cryo-electron microscopy (cryo-EM) work has shown that phosphorylation causes N-terminal Mcm4 to become unstructured 16 , 17 . Whether Sld3 engages an epitope that is unmasked after N-terminal Mcm4 phosphorylation or whether it merely reads phosphorylated (phospho-) Mcm4 sites remains to be established. Also, although we know that Rad53 prevents CMG formation by targeting Sld3 (alongside DDK) 13 , the mechanism is unclear. The second activating kinase, CDK, targets two firing factors, Sld3 and Sld2. The latter was previously implicated in GINS recruitment. Phospho-Sld2 and phospho-Sld3 are recognized by the Dpb11 ‘phospho-reader’ 10 , 11 . The leading-strand polymerase Pol ε (formed of Pol2, Dpb2, Dpb3 and Dpb4) also contributes to CMG assembly and becomes part of the holohelicase, CMGE. In particular, the N-terminal domain of Dpb2 supports CMG formation in cells 18 , and a complex containing Dpb2 and the C-terminal half of Pol2 achieves GINS recruitment and replication initiation in a reaction reconstituted with purified yeast proteins 19 .

In the inactive DH, an Mcm7-specific N-terminal insertion (NTI) from one MCM hexamer reaches across the DH interface and protects the N-terminal A domain of Mcm5 of the opposing hexamer 20 (Fig. 1a ). The same Mcm5 A-domain site is engaged by GINS in the CMG, implying that Mcm7 must let go of Mcm5 for GINS to bind 21 , 22 . How this happens is unknown. Likewise, it is established that Cdc45 and GINS recruitment occur sequentially 23 , 24 , 25 . Whether recruitment of each component involves only one MCM hexamer or both MCM hexamers at once is debated 25 , 26 , 27 .

Fig. 1: Structures of the double hexamer and the pre-IC. The alternative text for this image may have been generated using AI.

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a , In the double hexamer (DH; Protein Data Bank (PDB) ID: 7P30 ), the Mcm7 NTI element from one MCM ring protects the N-terminal Mcm5 A domain of the opposed ring. b , CDK-prephosphorylated Sld3/7 (ppSld3/7) and the Sld2(8D) phosphomimetic mutant support origin-dependent replication reconstituted in a test-tube, albeit at reduced levels (experiment performed twice). The low levels could be due either to the Sld2(8D) phosphomimetic mutations covering only a subset of CDK sites, or to inefficient phosphorylation of Sld3 in the isolated prephosphorylation reaction, compared with the complete initiation mix. For gel source data, see Supplementary Fig. 1 . c , CMG assembly in the absence of ATP, using ppSld3/7 and Sld2(8D), enables formation of the pre-IC. HSW, high-salt wash. Right, representative 2D classes. d , Cryo-EM structure of the pre-IC. The two MCMs are split on one side, with Mcm2 and Mcm6 functioning as a hinge. ZnF, zinc finger domain.

Origin activation is controlled by ATP binding and hydrolysis. MCM loading, for example, requires ATP hydrolysis 28 , 29 , so that, by the time a DH is formed, eight of its twelve subunits are bound to ADP (ref. 14 ). Firing-factor recruitment to the DH promotes ADP release, and ATP binding by MCM achieves stable double CMG–Pol ε (ATP–dCMGE) complex formation, which nucleates DNA melting 24 , 30 . It has been postulated that a pre-initiation complex (pre-IC) exists 31 , in which Sld2, Sld3–Sld7 (hereafter, Sld3/7) and Dpb11 all bind to the DH at the same time, while also recruiting Cdc45, GINS and Pol ε (refs. 24 , 32 ). However, the pre-IC has not so far been isolated, and we do not know how Sld2, Sld3/7 and Dpb11 are ejected to achieve ATP–dCMGE maturation 24 , 30 . Finally, whether the role of Sld2 is conserved across eukaryotes is unclear. In fact, Sld2 functions in CMGE assembly in yeast, whereas the metazoan orthologue, RecQL4, is involved in a loosely defined downstream activation step. To…