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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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DNA engagement and deformation by the CMG helicase.Single-molecule FRET analysis of (a) isolated DNA and (b) DNA+ATPγS CMG. Top to bottom: cartoon schematic of the single-molecule FRET experiment indicating FRET between Cy3 (Donor, blue sphere) and Cy5 (Acceptor, red sphere, 7-nt separation) attached to biotinylated 3′-tailed duplex DNA, immobilized on a neutravidin-coated biotin-PEG surface; donor (blue) and acceptor (red) intensity trajectories are anti-correlated until single-step photobleaching of the acceptor. FRET trajectories (black) between Cy3 and Cy5 exhibit a sharp drop to zero FRET when the acceptor photobleaches; histogram of FRET values collected from all molecules with a Gaussian fit are shown in red, where x0 is the mean FRET and σ is the distribution width. ATPase controlled ring rotation and DNA stretching by the CMG helicase. (c) The AAA+ tier of the Mcm2-7 motor rotates clockwise as Mcm2 moves towards the Mcm5 protomer, to close a gap in the motor domain. The DNA-interacting NTD rotates anticlockwise as a rigid body, together with GINS and Cdc45.
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f6: DNA engagement and deformation by the CMG helicase.Single-molecule FRET analysis of (a) isolated DNA and (b) DNA+ATPγS CMG. Top to bottom: cartoon schematic of the single-molecule FRET experiment indicating FRET between Cy3 (Donor, blue sphere) and Cy5 (Acceptor, red sphere, 7-nt separation) attached to biotinylated 3′-tailed duplex DNA, immobilized on a neutravidin-coated biotin-PEG surface; donor (blue) and acceptor (red) intensity trajectories are anti-correlated until single-step photobleaching of the acceptor. FRET trajectories (black) between Cy3 and Cy5 exhibit a sharp drop to zero FRET when the acceptor photobleaches; histogram of FRET values collected from all molecules with a Gaussian fit are shown in red, where x0 is the mean FRET and σ is the distribution width. ATPase controlled ring rotation and DNA stretching by the CMG helicase. (c) The AAA+ tier of the Mcm2-7 motor rotates clockwise as Mcm2 moves towards the Mcm5 protomer, to close a gap in the motor domain. The DNA-interacting NTD rotates anticlockwise as a rigid body, together with GINS and Cdc45.

Mentions: Comparison of the DNA-engaged MCM ATPase tier with other structures of nucleic acid-bound hexameric helicases highlights a mixture of similarities and differences (Fig. 5). A universal feature appears to be the presence of a right-handed spiral formed by the ATPase pore loops, which follows the helical character of the nucleic acid substrate15. Whereas in previously reported helicase structures (Rho, E1 and, to a lesser extent, DnaB) the translocation substrate is more compressed than that of canonical B-form DNA243132 (Fig. 5a–c), the ATPγS–CMG–DNA complex matches the structure of one single strand extracted from a B-form double helix (Fig. 5d). Noticeably, the Rho and E1 helicases, which significantly compress the translocation substrate as they encircle it, contact one base per ATPase protomer, while DnaB contacts two bases per protomer. These observations are compatible with an unwinding step size of 1- (Rho and E1) and 2-bp (DnaB) per ATP hydrolysed. Although the resolution of our compact ATPγS–CMG–DNA complex is not high enough to count bases in the single-stranded nucleic acid, the more extended state of the DNA substrate suggests that the CMG might unwind at least two base pairs per ATP molecule hydrolysed. To confirm the notion that single-stranded DNA is stabilized in an extended configuration on CMG binding, we performed single-molecule experiments, in the presence of either ATPγS or ATP. CMG–DNA binding was monitored by measuring FRET efficiency between a Cy3 donor and a Cy5 acceptor fluorophore, separated by seven nucleotides in surface-immobilized single-stranded DNA. This is an established method to measure DNA stretching by multi-subunit, DNA-encircling ATPases33 (Fig. 6a,b). CMG binding to DNA in the presence of ATPγS results in a dramatic decrease from 0.94±0.01 (naked DNA; Fig. 6a) to 0.44±0.01 (DNA+CMG; Fig. 6b and Supplementary Fig. 12) in mean FRET (x0), due to both a reduction in molecular flexibility relative to naked single-stranded DNA and CMG-mediated single-stranded DNA stretching. Similar results were obtained with ATP (x0=0.96±0.01 for naked DNA and x0=0.48±0.01 for CMG), although binding/stretching events occurred with lower frequency (Supplementary Fig. 12). These data agree with the observation that use of a slowly hydrolysable ATP analogue is required to stabilize the interaction between the MCM AAA+ domain and single-stranded DNA612, and support the notion that CMG binding stabilizes and stretches single-stranded DNA. We note that the lower frequency of DNA binding/stretching observed when CMG is ATP incubated correlates with the predominance of the relaxed ATPase form observed in the ATP–CMG cryo-EM data set.


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)

DNA engagement and deformation by the CMG helicase.Single-molecule FRET analysis of (a) isolated DNA and (b) DNA+ATPγS CMG. Top to bottom: cartoon schematic of the single-molecule FRET experiment indicating FRET between Cy3 (Donor, blue sphere) and Cy5 (Acceptor, red sphere, 7-nt separation) attached to biotinylated 3′-tailed duplex DNA, immobilized on a neutravidin-coated biotin-PEG surface; donor (blue) and acceptor (red) intensity trajectories are anti-correlated until single-step photobleaching of the acceptor. FRET trajectories (black) between Cy3 and Cy5 exhibit a sharp drop to zero FRET when the acceptor photobleaches; histogram of FRET values collected from all molecules with a Gaussian fit are shown in red, where x0 is the mean FRET and σ is the distribution width. ATPase controlled ring rotation and DNA stretching by the CMG helicase. (c) The AAA+ tier of the Mcm2-7 motor rotates clockwise as Mcm2 moves towards the Mcm5 protomer, to close a gap in the motor domain. The DNA-interacting NTD rotates anticlockwise as a rigid body, together with GINS and Cdc45.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f6: DNA engagement and deformation by the CMG helicase.Single-molecule FRET analysis of (a) isolated DNA and (b) DNA+ATPγS CMG. Top to bottom: cartoon schematic of the single-molecule FRET experiment indicating FRET between Cy3 (Donor, blue sphere) and Cy5 (Acceptor, red sphere, 7-nt separation) attached to biotinylated 3′-tailed duplex DNA, immobilized on a neutravidin-coated biotin-PEG surface; donor (blue) and acceptor (red) intensity trajectories are anti-correlated until single-step photobleaching of the acceptor. FRET trajectories (black) between Cy3 and Cy5 exhibit a sharp drop to zero FRET when the acceptor photobleaches; histogram of FRET values collected from all molecules with a Gaussian fit are shown in red, where x0 is the mean FRET and σ is the distribution width. ATPase controlled ring rotation and DNA stretching by the CMG helicase. (c) The AAA+ tier of the Mcm2-7 motor rotates clockwise as Mcm2 moves towards the Mcm5 protomer, to close a gap in the motor domain. The DNA-interacting NTD rotates anticlockwise as a rigid body, together with GINS and Cdc45.
Mentions: Comparison of the DNA-engaged MCM ATPase tier with other structures of nucleic acid-bound hexameric helicases highlights a mixture of similarities and differences (Fig. 5). A universal feature appears to be the presence of a right-handed spiral formed by the ATPase pore loops, which follows the helical character of the nucleic acid substrate15. Whereas in previously reported helicase structures (Rho, E1 and, to a lesser extent, DnaB) the translocation substrate is more compressed than that of canonical B-form DNA243132 (Fig. 5a–c), the ATPγS–CMG–DNA complex matches the structure of one single strand extracted from a B-form double helix (Fig. 5d). Noticeably, the Rho and E1 helicases, which significantly compress the translocation substrate as they encircle it, contact one base per ATPase protomer, while DnaB contacts two bases per protomer. These observations are compatible with an unwinding step size of 1- (Rho and E1) and 2-bp (DnaB) per ATP hydrolysed. Although the resolution of our compact ATPγS–CMG–DNA complex is not high enough to count bases in the single-stranded nucleic acid, the more extended state of the DNA substrate suggests that the CMG might unwind at least two base pairs per ATP molecule hydrolysed. To confirm the notion that single-stranded DNA is stabilized in an extended configuration on CMG binding, we performed single-molecule experiments, in the presence of either ATPγS or ATP. CMG–DNA binding was monitored by measuring FRET efficiency between a Cy3 donor and a Cy5 acceptor fluorophore, separated by seven nucleotides in surface-immobilized single-stranded DNA. This is an established method to measure DNA stretching by multi-subunit, DNA-encircling ATPases33 (Fig. 6a,b). CMG binding to DNA in the presence of ATPγS results in a dramatic decrease from 0.94±0.01 (naked DNA; Fig. 6a) to 0.44±0.01 (DNA+CMG; Fig. 6b and Supplementary Fig. 12) in mean FRET (x0), due to both a reduction in molecular flexibility relative to naked single-stranded DNA and CMG-mediated single-stranded DNA stretching. Similar results were obtained with ATP (x0=0.96±0.01 for naked DNA and x0=0.48±0.01 for CMG), although binding/stretching events occurred with lower frequency (Supplementary Fig. 12). These data agree with the observation that use of a slowly hydrolysable ATP analogue is required to stabilize the interaction between the MCM AAA+ domain and single-stranded DNA612, and support the notion that CMG binding stabilizes and stretches single-stranded DNA. We note that the lower frequency of DNA binding/stretching observed when CMG is ATP incubated correlates with the predominance of the relaxed ATPase form observed in the ATP–CMG cryo-EM data set.

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