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Cryo-EM structures of the eukaryotic replicative helicase bound to a translocation substrate.

Abid Ali F, Renault L, Gannon J, Gahlon HL, Kotecha A, Zhou JC, Rueda D, Costa A - Nat Commun (2016)

Bottom Line: In the predominant state, the ring-shaped C-terminal ATPase of MCM is compact and contacts single-stranded DNA, via a set of pre-sensor 1 hairpins that spiral around the translocation substrate.In the second state, the ATPase module is relaxed and apparently substrate free, while DNA intimately contacts the downstream amino-terminal tier of the MCM motor ring.These results, supported by single-molecule FRET measurements, lead us to suggest a replication fork unwinding mechanism whereby the N-terminal and AAA+ tiers of the MCM work in concert to translocate on single-stranded DNA.

View Article: PubMed Central - PubMed

Affiliation: Macromolecular Machines, Clare Hall Laboratory, The Francis Crick Institute, Blanche Lane, South Mimms EN6 3LD, UK.

ABSTRACT
The Cdc45-MCM-GINS (CMG) helicase unwinds DNA during the elongation step of eukaryotic genome duplication and this process depends on the MCM ATPase function. Whether CMG translocation occurs on single- or double-stranded DNA and how ATP hydrolysis drives DNA unwinding remain open questions. Here we use cryo-electron microscopy to describe two subnanometre resolution structures of the CMG helicase trapped on a DNA fork. In the predominant state, the ring-shaped C-terminal ATPase of MCM is compact and contacts single-stranded DNA, via a set of pre-sensor 1 hairpins that spiral around the translocation substrate. In the second state, the ATPase module is relaxed and apparently substrate free, while DNA intimately contacts the downstream amino-terminal tier of the MCM motor ring. These results, supported by single-molecule FRET measurements, lead us to suggest a replication fork unwinding mechanism whereby the N-terminal and AAA+ tiers of the MCM work in concert to translocate on single-stranded DNA.

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CMG helicase structure at subnanometre resolution.(a) Resolution density (7.4 Å) map of the CMG viewed from the MCM N-terminal face, without or with docked MCM and GINS atomic structures. (b) Detailed view of the MCM N-terminal DNA-interacting collar. Psf3 contacts Mcm3, Psf2 contacts Mcm5 and Cdc45 contacts Mcm2. (c) Detailed view of the GINS assembly with docked human atomic structure (PDB entry 2Q9Q). (d) Density assigned to Cdc45. The Cdc45 core matches the secondary structure elements of the bacterial RecJ exonuclease (PDB entry 1IR6).
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f1: CMG helicase structure at subnanometre resolution.(a) Resolution density (7.4 Å) map of the CMG viewed from the MCM N-terminal face, without or with docked MCM and GINS atomic structures. (b) Detailed view of the MCM N-terminal DNA-interacting collar. Psf3 contacts Mcm3, Psf2 contacts Mcm5 and Cdc45 contacts Mcm2. (c) Detailed view of the GINS assembly with docked human atomic structure (PDB entry 2Q9Q). (d) Density assigned to Cdc45. The Cdc45 core matches the secondary structure elements of the bacterial RecJ exonuclease (PDB entry 1IR6).

Mentions: Catalytically active, baculovirus-expressed Drosophila melanogaster CMG was incubated with a model replication fork in the presence of ATPγS, required for stable DNA binding6. Particles embedded in vitrified ice were imaged on a FEI Polara electron microscope equipped with an energy filter and a K2 Summit direct electron detector (Gatan, Inc.; Supplementary Fig. 1). Following two-dimensional (2D) and three-dimensional (3D) classification, a first structure was refined to 7.4 Å resolution (Supplementary Fig. 2). Atomic docking was employed to interpret the cryo-EM map, using the coordinates of known holo-helicase components. These efforts provide an exhaustive description of the CMG intersubunit interaction network. The structure contains a closed hexameric ring face that matches the N-terminal DNA-interacting collar of yeast MCM4 (Fig. 1a,b, PDB entry 3JA8), albeit with significant inter-domain rearrangements (Supplementary Fig. 3 and Supplementary Movie 1). Combined with previous subunit mapping studies1617, our data confirm that GINS components Psf2 and Psf3 (PDB entry 2Q9Q) interact with the outer perimeter of MCM subunits 5 and 3 (Fig. 1a–c). Remarkably, Psf2 α-helices 3 and 5 (as defined in the human GINS structure18) contact a region of the Mcm5 N-terminal ‘A domain' that is protected by the N-terminal extension of the MCM subunit 7 from the opposing ring in the double hexamer, as described in the atomic resolution yeast structure4 (Supplementary Fig. 4).


Cryo-EM structures of the eukaryotic replicative helicase bound to a translocation substrate.

Abid Ali F, Renault L, Gannon J, Gahlon HL, Kotecha A, Zhou JC, Rueda D, Costa A - Nat Commun (2016)

CMG helicase structure at subnanometre resolution.(a) Resolution density (7.4 Å) map of the CMG viewed from the MCM N-terminal face, without or with docked MCM and GINS atomic structures. (b) Detailed view of the MCM N-terminal DNA-interacting collar. Psf3 contacts Mcm3, Psf2 contacts Mcm5 and Cdc45 contacts Mcm2. (c) Detailed view of the GINS assembly with docked human atomic structure (PDB entry 2Q9Q). (d) Density assigned to Cdc45. The Cdc45 core matches the secondary structure elements of the bacterial RecJ exonuclease (PDB entry 1IR6).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4759635&req=5

f1: CMG helicase structure at subnanometre resolution.(a) Resolution density (7.4 Å) map of the CMG viewed from the MCM N-terminal face, without or with docked MCM and GINS atomic structures. (b) Detailed view of the MCM N-terminal DNA-interacting collar. Psf3 contacts Mcm3, Psf2 contacts Mcm5 and Cdc45 contacts Mcm2. (c) Detailed view of the GINS assembly with docked human atomic structure (PDB entry 2Q9Q). (d) Density assigned to Cdc45. The Cdc45 core matches the secondary structure elements of the bacterial RecJ exonuclease (PDB entry 1IR6).
Mentions: Catalytically active, baculovirus-expressed Drosophila melanogaster CMG was incubated with a model replication fork in the presence of ATPγS, required for stable DNA binding6. Particles embedded in vitrified ice were imaged on a FEI Polara electron microscope equipped with an energy filter and a K2 Summit direct electron detector (Gatan, Inc.; Supplementary Fig. 1). Following two-dimensional (2D) and three-dimensional (3D) classification, a first structure was refined to 7.4 Å resolution (Supplementary Fig. 2). Atomic docking was employed to interpret the cryo-EM map, using the coordinates of known holo-helicase components. These efforts provide an exhaustive description of the CMG intersubunit interaction network. The structure contains a closed hexameric ring face that matches the N-terminal DNA-interacting collar of yeast MCM4 (Fig. 1a,b, PDB entry 3JA8), albeit with significant inter-domain rearrangements (Supplementary Fig. 3 and Supplementary Movie 1). Combined with previous subunit mapping studies1617, our data confirm that GINS components Psf2 and Psf3 (PDB entry 2Q9Q) interact with the outer perimeter of MCM subunits 5 and 3 (Fig. 1a–c). Remarkably, Psf2 α-helices 3 and 5 (as defined in the human GINS structure18) contact a region of the Mcm5 N-terminal ‘A domain' that is protected by the N-terminal extension of the MCM subunit 7 from the opposing ring in the double hexamer, as described in the atomic resolution yeast structure4 (Supplementary Fig. 4).

Bottom Line: In the predominant state, the ring-shaped C-terminal ATPase of MCM is compact and contacts single-stranded DNA, via a set of pre-sensor 1 hairpins that spiral around the translocation substrate.In the second state, the ATPase module is relaxed and apparently substrate free, while DNA intimately contacts the downstream amino-terminal tier of the MCM motor ring.These results, supported by single-molecule FRET measurements, lead us to suggest a replication fork unwinding mechanism whereby the N-terminal and AAA+ tiers of the MCM work in concert to translocate on single-stranded DNA.

View Article: PubMed Central - PubMed

Affiliation: Macromolecular Machines, Clare Hall Laboratory, The Francis Crick Institute, Blanche Lane, South Mimms EN6 3LD, UK.

ABSTRACT
The Cdc45-MCM-GINS (CMG) helicase unwinds DNA during the elongation step of eukaryotic genome duplication and this process depends on the MCM ATPase function. Whether CMG translocation occurs on single- or double-stranded DNA and how ATP hydrolysis drives DNA unwinding remain open questions. Here we use cryo-electron microscopy to describe two subnanometre resolution structures of the CMG helicase trapped on a DNA fork. In the predominant state, the ring-shaped C-terminal ATPase of MCM is compact and contacts single-stranded DNA, via a set of pre-sensor 1 hairpins that spiral around the translocation substrate. In the second state, the ATPase module is relaxed and apparently substrate free, while DNA intimately contacts the downstream amino-terminal tier of the MCM motor ring. These results, supported by single-molecule FRET measurements, lead us to suggest a replication fork unwinding mechanism whereby the N-terminal and AAA+ tiers of the MCM work in concert to translocate on single-stranded DNA.

Show MeSH
Related in: MedlinePlus