Structure of the eukaryotic MCM complex at 3.8 Å

Li, Ningning; Zhai, Yuanliang; Zhang, Yixiao; Li, Wanqiu; Yang, Maojun; Lei, Jianlin; Tye, Bik Kwoon; Gao, Ning

doi:10.1038/nature14685

Cited by 241 publications

(305 citation statements)

References 72 publications

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“…For example, studies on the CMG helicase and its subcomplexes have established that the MCM is a six-member ring with an N-terminal domain that serves as a processivity collar (15) and a C-terminal ATPase motor domain that provides the DNA unwinding function (16)(17)(18)(19)(20)(21). High-resolution cryo-EM analysis has shown that the ATPase motor translocates on the leading-strand template (22), in agreement with work on Xenopus embryo extracts (23).…”

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confidence: 50%

CMG–Pol epsilon dynamics suggests a mechanism for the establishment of leading-strand synthesis in the eukaryotic replisome

Zhou

Janska

Goswami

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

109

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The replisome unwinds and synthesizes DNA for genome duplication. In eukaryotes, the Cdc45-MCM-GINS (CMG) helicase and the leading-strand polymerase, Pol epsilon, form a stable assembly. The mechanism for coupling DNA unwinding with synthesis is starting to be elucidated, however the architecture and dynamics of the replication fork remain only partially understood, preventing a molecular understanding of chromosome replication. To address this issue, we conducted a systematic single-particle EM study on multiple permutations of the reconstituted CMG-Pol epsilon assembly. Pol epsilon contains two flexibly tethered lobes. The noncatalytic lobe is anchored to the motor of the helicase, whereas the polymerization domain extends toward the side of the helicase. We observe two alternate configurations of the DNA synthesis domain in the CMG-bound Pol epsilon. We propose that this conformational switch might control DNA template engagement and release, modulating replisome progression.DNA replication | CMG helicase | DNA polymerase | single-particle electron microscopy D NA replication is catalyzed by the replisome, a molecular machine that coordinates DNA unwinding and synthesis (1). These two functions must be tightly coordinated to prevent the rise of genome instability, which is a major cause of cancer. DNA unwinding by a replicative helicase involves single-strand translocation of a hexameric motor, whereas DNA synthesis requires template priming by a primase and extension by dedicated replicative DNA polymerases (2). In eukaryotes, the helicase function is performed by the Cdc45-MCM-GINS (CMG) complex (3, 4) and the primase function is played by Pol alpha (5), whereas DNA synthesis is catalyzed by two specialized DNA polymerases, Pol epsilon and delta. According to the consensus view, Pol epsilon synthesizes the leading and Pol delta the lagging strand (6-11). However, recent studies indicate that the division of labor between replicative polymerases might be more promiscuous than originally thought (12, 13). In in vitroreconstituted DNA replication reactions, Pol delta can support leading-strand duplication (11, 14), but switching from Pol delta to epsilon is necessary for efficient establishment of leadingstrand synthesis (14). The mechanism of substrate handoff between the two polymerases is currently unknown.Recent breakthroughs in structural biology begin to provide an architectural framework to understand the interaction between helicase and polymerases at the replication fork. For example, studies on the CMG helicase and its subcomplexes have established that the MCM is a six-member ring with an N-terminal domain that serves as a processivity collar (15) and a C-terminal ATPase motor domain that provides the DNA unwinding function (16-21). High-resolution cryo-EM analysis has shown that the ATPase motor translocates on the leading-strand template (22), in agreement with work on Xenopus embryo extracts (23). The GINS and Cdc45 components of the CMG bind to the side and stabilize the N-terminal domain of the...

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mentioning

confidence: 50%

CMG–Pol epsilon dynamics suggests a mechanism for the establishment of leading-strand synthesis in the eukaryotic replisome

Zhou

Janska

Goswami

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

109

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“…Three of the complexes, representing prominent stages of DNA replication, have been structurally characterized recently by cryo-EM at high resolution: the OCCM, the MCM2-7 DH, and the CMG ( Fig. 4A-C; Costa et al 2011Costa et al , 2014Sun et al 2013Sun et al , 2014Li et al 2015;Yuan et al 2016Yuan et al , 2017Georgescu et al 2017). Interestingly, the Mcm subunit conformations are different in these three complexes, reflecting distinct functional states.…”

Section: Mcm2-7 Conformations In the Occm Dh And Cmg Reveal Mcm2-7 mentioning

confidence: 99%

From structure to mechanism—understanding initiation of DNA replication

et al. 2017

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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.

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“…In contrast, activated CMG complexes at replication forks contain a single Mcm2-7 complex and encircle ssDNA (Fu et al 2011;Yardimci et al 2012;Sun et al 2015;Georgescu et al 2017). Structural studies have captured Mcm2-7 at multiple stages during helicase loading and in the CMG complex (Sun et al 2013;Li et al 2015;Abid Ali et al 2016;Yuan et al 2016;Georgescu et al 2017). These structures have provided important insights into Mcm2-7 loading and the interactions of Mcm2-7 with Cdc45 and GINS.…”

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confidence: 99%

Mcm10 regulates DNA replication elongation by stimulating the CMG replicative helicase

2017

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Activation of the Mcm2-7 replicative DNA helicase is the committed step in eukaryotic DNA replication initiation. Although Mcm2-7 activation requires binding of the helicase-activating proteins Cdc45 and GINS (forming the CMG complex), an additional protein, Mcm10, drives initial origin DNA unwinding by an unknown mechanism. We show that Mcm10 binds a conserved motif located between the oligonucleotide/oligosaccharide fold (OB-fold) and A subdomain of Mcm2. Although buried in the interface between these domains in Mcm2-7 structures, mutations predicted to separate the domains and expose this motif restore growth to conditional-lethal MCM10 mutant cells. We found that, in addition to stimulating initial DNA unwinding, Mcm10 stabilizes Cdc45 and GINS association with Mcm2-7 and stimulates replication elongation in vivo and in vitro. Furthermore, we identified a lethal allele of MCM10 that stimulates initial DNA unwinding but is defective in replication elongation and CMG binding. Our findings expand the roles of Mcm10 during DNA replication and suggest a new model for Mcm10 function as an activator of the CMG complex throughout DNA replication.

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Structure of the eukaryotic MCM complex at 3.8 Å

Cited by 241 publications

References 72 publications

CMG–Pol epsilon dynamics suggests a mechanism for the establishment of leading-strand synthesis in the eukaryotic replisome

CMG–Pol epsilon dynamics suggests a mechanism for the establishment of leading-strand synthesis in the eukaryotic replisome

From structure to mechanism—understanding initiation of DNA replication

Mcm10 regulates DNA replication elongation by stimulating the CMG replicative helicase

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