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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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Two configurations in the ATPγS–CMG–DNA complex.(a) AAA+, side and cut-through view of the CMG in a relaxed ATPase configuration. Cdc45 topologically locks the Mcm5-2 gate. (b) AAA+, side and cut-through N-terminal view of the CMG in a compact ATPase configuration. DNA density surmounting the AAA+ domain has been removed for visualization purposes (also see Fig. 4).
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f2: Two configurations in the ATPγS–CMG–DNA complex.(a) AAA+, side and cut-through view of the CMG in a relaxed ATPase configuration. Cdc45 topologically locks the Mcm5-2 gate. (b) AAA+, side and cut-through N-terminal view of the CMG in a compact ATPase configuration. DNA density surmounting the AAA+ domain has been removed for visualization purposes (also see Fig. 4).

Mentions: As previously proposed161719, unoccupied density mapping next to GINS is assigned to Cdc45 and indeed matches the secondary structure elements of RecJ20 (PDB entry 1IR6), a distant prokaryotic exonuclease homologue (Fig. 1d and Supplementary Fig. 5)2122. In addition, a C-terminal protrusion projects from the RecJ-like catalytically defunct core of Cdc45 and wedges in between the Mcm5 and Mcm2 A domains16, as previously observed at low resolution19 (Figs 1a and 2a,b).


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)

Two configurations in the ATPγS–CMG–DNA complex.(a) AAA+, side and cut-through view of the CMG in a relaxed ATPase configuration. Cdc45 topologically locks the Mcm5-2 gate. (b) AAA+, side and cut-through N-terminal view of the CMG in a compact ATPase configuration. DNA density surmounting the AAA+ domain has been removed for visualization purposes (also see Fig. 4).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Two configurations in the ATPγS–CMG–DNA complex.(a) AAA+, side and cut-through view of the CMG in a relaxed ATPase configuration. Cdc45 topologically locks the Mcm5-2 gate. (b) AAA+, side and cut-through N-terminal view of the CMG in a compact ATPase configuration. DNA density surmounting the AAA+ domain has been removed for visualization purposes (also see Fig. 4).
Mentions: As previously proposed161719, unoccupied density mapping next to GINS is assigned to Cdc45 and indeed matches the secondary structure elements of RecJ20 (PDB entry 1IR6), a distant prokaryotic exonuclease homologue (Fig. 1d and Supplementary Fig. 5)2122. In addition, a C-terminal protrusion projects from the RecJ-like catalytically defunct core of Cdc45 and wedges in between the Mcm5 and Mcm2 A domains16, as previously observed at low resolution19 (Figs 1a and 2a,b).

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