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Review
. 2017 Jun 1;31(11):1073-1088.
doi: 10.1101/gad.298232.117.

From Structure to Mechanism-Understanding Initiation of DNA Replication

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Free PMC article
Review

From Structure to Mechanism-Understanding Initiation of DNA Replication

Alberto Riera et al. Genes Dev. .
Free PMC article

Abstract

DNA replication results in the doubling of the genome prior to cell division. This process requires the assembly of 50 or more protein factors into a replication fork. Here, we review recent structural and biochemical insights that start to explain how specific proteins recognize DNA replication origins, load the replicative helicase on DNA, unwind DNA, synthesize new DNA strands, and reassemble chromatin. We focus on the minichromosome maintenance (MCM2-7) proteins, which form the core of the eukaryotic replication fork, as this complex undergoes major structural rearrangements in order to engage with DNA, regulate its DNA-unwinding activity, and maintain genome stability.

Keywords: CMG; DNA replication; MCM2–7; cryo-EM; pre-RC; replisome.

Figures

Figure 1.
Figure 1.
Eukaryotic initiation of DNA replication. Relevant complexes that have been characterized by electron microscopy are shown in surface view. (A) ORC is chromatin bound throughout the cell cycle (Electron Microscopy Data Bank [EMDB]: 1156). (B) ORC/Cdc6 is the landing platform MCM2–7/Cdt1 (EMDB: 5381). (C) The association of MCM2–7/Cdt1 (EMDB: 6671) with ORC/Cdc6 results in the OCCM (EMDB: 8540) formation with the dsDNA inserted into MCM2–7 hexamer. (D) Cdt1 and Cdc6 are released from the OCCM in an ATP hydrolysis-dependent reaction, and, upon recruitment of another Cdc6, the OCM the complex is formed. The OCM is an essential intermediate in the loading reaction and is responsible for the recruitment of a second MCM2–7/Cdt1 heptamer, although the details of the reaction are currently not known. (E) The final product of the loading reaction is a MCM2–7 DH embracing dsDNA (EMDB: 6338). This inactive complex is a stable DNA replication intermediate, which becomes activated only in S phase. (F) Preinitiation of DNA replication in S phase relies on Dbf4-dependent kinase (DDK)-dependent phosphorylation of the DH and a plethora of factors that interact with the DH. One of the landmarks of preinitiation complex formation is the binding of Cdc45 and GINS (from the Japanese go-ichi-ni-san, meaning 5-1-2-3, after the four related subunits of the complex: Sld5, Psf1, Psf2, and Psf3) to MCM2–7, resulting in formation of the replicative helicase: the Cdc45/MCM2–7/GINS (CMG) complex. (G) During the DNA-unwinding process, the CMG (EMDB: 8518) associates with both polymerases ε and α into a replication fork (RFK) to synthesize the leading and lagging strands. The helicase is propelled by the C-terminal AAA+ motor domains, and the unwinding takes place on the N-terminal face.
Figure 2.
Figure 2.
Origin recognition. (A) The domain and structural organization of Orc and Cdc6 proteins. The initiator-specific motif region of the AAA-RecA domain is indicated in red. (B, left) The OCCM structure in top view and side view (Protein Data Bank [PDB]: 5UDB). (Right) A top view of the ORC/Cdc6–DNA structure is shown enlarged. (C) Archaeal Orc (PDB: 2V1U) and the DNA-binding subunits of S. cerevisiae (Sc) ORC and Cdc6 are shown (PDB: 5UDB). The DNA-binding regions are indicated by red circles. The Orc4 insertion helix is shown in dark blue. (D) An overlay of S. cerevisiae Orc4 (PDB: 5UDB), D. melanogaster (Dm) Orc4 (PDB: 4XGC), and H. sapiens (Hs) Orc4 (PDB: 5UJM). (E) Structural comparison of D. melanogaster Orc1–Orc2 with S. cerevisiae Orc1–Orc2. The arrows show the rotation of D. melanogaster Orc1 AAA+ and Orc2 WHD to fit the S. cerevisiae Orc1 AAA+ and Orc2 WHD positions.
Figure 3.
Figure 3.
MCM2–7 helicase function arises from its architecture. (A) Domain structure of an Mcm protein with its four domains: A, oligonucleotide binding (OB), ATPase, and WHD. The color-coding for specific motifs—zinc finger (ZF), N-terminal hairpin (NtHp), Walker A (WA), Walker B (WB), presensor-1 (PS1), helix 2 insertion (H2i) β hairpin, arginine finger (RF), and WHD—is identical in A and B. (B) Atomic model of an exemplary Mcm subunit (PDB: 5U8S; Mcm2 of CMG bound to a replication fork) outlining its domain organization, with the WHD not shown. (C) Mcm subunits show different N-terminal and C-terminal extensions. (D) The MCM2–7 hexameric ring structure extracted from the CMG bound to a replication fork viewed from the top and side (PDB: 5U8S).
Figure 4.
Figure 4.
Structural changes in Mcm subunits dictate their function. Cartoon-style atomic models of OCCM (A; PDB: 5UDB), MCM2–7 DH (B; PDB: 3JA8), and CMG bound to a replication fork (C; PDB: 5U8S). (D) Comparison of the conformation of the MCM2–7 subunits in the OCCM, DH, and CMG. The CTDs of each Mcm subunit in the OCCM/DH/CMG conformations were aligned with the CTD of Mcm6 in the DH conformation, and this was taken as a fixed common position that was then used to generate the common reference center in the NTD (marked in green in the DH). This common reference point was used to detect the movement of the Mcm NTD in the OCCM and CMG relative to the DH, considering the Mcm NTD as a unit. Movement of the NTD toward the left is shown in red, and movement toward the right in is shown blue; the rotational axis relative to the common reference point is shown as a symbol. The alignment used the atomic structures of the proteins, but the figure depicts 10 Å surface view representations for improved clarity. (E) Schematic representation of the ATPase pockets of MCM2–7 in the OCCM, DH, and CMG. (F) ATPase pockets of ORC/Cdc6 in context of the OCCM.
Figure 5.
Figure 5.
MCM2–7 DNA interactions within the OCCM, DH, and CMG complexes. (A) Detailed structural overlays of selected budding yeast (gray; PDB: 5U8S; Mcm2 of CMG bound to a replication fork) and S. solfataricus (blue; PDB: 3F9V) Mcm regions are shown. A superimposition of the PS1 loop, H2i loop, NtHp, and ZFs is depicted. (B) A schematic cut-through of the CMG with adjacent polymerases is shown with important domains labeled and color-coded as in A. The asterisk denotes that the hairpins of Mcm2/3/5/6 gather on one side of the central channel to interact with the passing ssDNA. (C) Structures of the budding yeast OCCM (PDB: 5UDB), DH (PDB: 3JA8), and CMG (PDB: 5U8S; CMG bound to a replication fork) are depicted. Furthermore, the central channels of the OCCM and CMG and its DNA bending are indicated. In the context of the DH, the interhexamer angle is shown.
Figure 6.
Figure 6.
MCM2–7 protein–protein interactions in the context of OCCM and CMG. Interactions between the Mcm subunits (in gray gradient) and the Orc subunits (Orc1 in green, Orc2 in brown, Orc3 in salmon, Orc4 in cyan, Orc5 in purple, and Orc6 in gray), Cdc6 (light pink), and Cdt1 (blue) in the OCCM (A) and GINS (pale pink) and Cdc45 (red) in the CMG (B). The dashed lines indicate flexible regions not solved in the structures. (C) Summary of the different functions in which the MCM2–7 complex is involved though interactions with other proteins.

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