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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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The MCM ATPase centres.(a) Segmented density and docked atomic structures of the six Mcm2-7 protomers in the two conformers. The NTD and AAA+ domains tilt and rock with respect to one another. (b) ATPase sites in the relaxed ATPase conformer. (c) ATPase sites in the compact ATPase conformer. One side of the MCM ring is more static and the corresponding active sites are dispensable for viability (−means Walker A mutation kills DNA unwinding, +means tolerated mutation).
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f3: The MCM ATPase centres.(a) Segmented density and docked atomic structures of the six Mcm2-7 protomers in the two conformers. The NTD and AAA+ domains tilt and rock with respect to one another. (b) ATPase sites in the relaxed ATPase conformer. (c) ATPase sites in the compact ATPase conformer. One side of the MCM ring is more static and the corresponding active sites are dispensable for viability (−means Walker A mutation kills DNA unwinding, +means tolerated mutation).

Mentions: To accurately describe ATPase motor dynamics, the yeast Mcm2-7 AAA+ hexamer was fitted into the CMG structure and further energy minimization was allowed for the individual docked ATPase modules into the two refined 3D classes. This docking exercise highlights a remarkable plasticity of the Mcm2-7 protomers, with relative tilting and rocking of the AAA+ domains, occurring both within the six Mcm2-7 ring subunits and between the two CMG conformers (Figs 2 and 3a). As previously proposed1619, we can assign a discontinuity in the AAA+ domain to the Mcm5-2 ATPase interface (Fig. 2a,b). By comparing the compact and relaxed structures, a conformational transition can be modelled in the ATPase tier. This reveals two types of ATPase interfaces that behave differently. Movement mainly depends on the loosening and tightening of the Mcm5-2 interface (root mean squared deviation (r.m.s.d.) 8.2 Å between the open and closed configurations; Fig. 3b,c). Mcm5-2 loosening is chaperoned by compensatory tightening of the neighbouring Mcm3-5 and 2-6 interfaces (r.m.s.d.: 5.3 and 8.3 Å, respectively; Fig. 3b,c), while the rest of the subunits undergo more subtle reconfigurations (with Mcm4-7 and Mcm7-3 r.m.s.d.: 3.1 and 3.3 Å, respectively; Fig. 3b,c). Coherent with these asymmetric rearrangements, in vitro studies of the Drosophila CMG have shown that certain Mcm2-7 ATPase sites (found at the static hinge in our structures) can be inactivated with minimal effects on DNA unwinding by the CMG, while other sites (the active Mcm5-2 ATPase and its immediate neighbors) are required for translocation6 (Fig. 3c).


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)

The MCM ATPase centres.(a) Segmented density and docked atomic structures of the six Mcm2-7 protomers in the two conformers. The NTD and AAA+ domains tilt and rock with respect to one another. (b) ATPase sites in the relaxed ATPase conformer. (c) ATPase sites in the compact ATPase conformer. One side of the MCM ring is more static and the corresponding active sites are dispensable for viability (−means Walker A mutation kills DNA unwinding, +means tolerated mutation).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: The MCM ATPase centres.(a) Segmented density and docked atomic structures of the six Mcm2-7 protomers in the two conformers. The NTD and AAA+ domains tilt and rock with respect to one another. (b) ATPase sites in the relaxed ATPase conformer. (c) ATPase sites in the compact ATPase conformer. One side of the MCM ring is more static and the corresponding active sites are dispensable for viability (−means Walker A mutation kills DNA unwinding, +means tolerated mutation).
Mentions: To accurately describe ATPase motor dynamics, the yeast Mcm2-7 AAA+ hexamer was fitted into the CMG structure and further energy minimization was allowed for the individual docked ATPase modules into the two refined 3D classes. This docking exercise highlights a remarkable plasticity of the Mcm2-7 protomers, with relative tilting and rocking of the AAA+ domains, occurring both within the six Mcm2-7 ring subunits and between the two CMG conformers (Figs 2 and 3a). As previously proposed1619, we can assign a discontinuity in the AAA+ domain to the Mcm5-2 ATPase interface (Fig. 2a,b). By comparing the compact and relaxed structures, a conformational transition can be modelled in the ATPase tier. This reveals two types of ATPase interfaces that behave differently. Movement mainly depends on the loosening and tightening of the Mcm5-2 interface (root mean squared deviation (r.m.s.d.) 8.2 Å between the open and closed configurations; Fig. 3b,c). Mcm5-2 loosening is chaperoned by compensatory tightening of the neighbouring Mcm3-5 and 2-6 interfaces (r.m.s.d.: 5.3 and 8.3 Å, respectively; Fig. 3b,c), while the rest of the subunits undergo more subtle reconfigurations (with Mcm4-7 and Mcm7-3 r.m.s.d.: 3.1 and 3.3 Å, respectively; Fig. 3b,c). Coherent with these asymmetric rearrangements, in vitro studies of the Drosophila CMG have shown that certain Mcm2-7 ATPase sites (found at the static hinge in our structures) can be inactivated with minimal effects on DNA unwinding by the CMG, while other sites (the active Mcm5-2 ATPase and its immediate neighbors) are required for translocation6 (Fig. 3c).

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