Journal: Mol Cell / Year: 2019 Title: Mechanism of DNA End Sensing and Processing by the Mre11-Rad50 Complex. Authors: Lisa Käshammer / Jan-Hinnerk Saathoff / Katja Lammens / Fabian Gut / Joseph Bartho / Aaron Alt / Brigitte Kessler / Karl-Peter Hopfner / Abstract: DNA double-strand breaks (DSBs) threaten genome stability throughout life and are linked to tumorigenesis in humans. To initiate DSB repair by end joining or homologous recombination, the Mre11- ...DNA double-strand breaks (DSBs) threaten genome stability throughout life and are linked to tumorigenesis in humans. To initiate DSB repair by end joining or homologous recombination, the Mre11-nuclease Rad50-ATPase complex detects and processes diverse and obstructed DNA ends, but a structural mechanism is still lacking. Here we report cryo-EM structures of the E. coli Mre11-Rad50 homolog SbcCD in resting and DNA-bound cutting states. In the resting state, Mre11's nuclease is blocked by ATP-Rad50, and the Rad50 coiled coils appear flexible. Upon DNA binding, the two coiled coils zip up into a rod and, together with the Rad50 nucleotide-binding domains, form a clamp around dsDNA. Mre11 moves to the side of Rad50, binds the DNA end, and assembles a DNA cutting channel for the nuclease reactions. The structures reveal how Mre11-Rad50 can detect and process diverse DNA ends and uncover a clamping and gating function for the coiled coils.
History
Deposition
Jul 8, 2019
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Header (metadata) release
Sep 4, 2019
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Map release
Sep 4, 2019
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Update
Nov 20, 2019
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Current status
Nov 20, 2019
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Download / File: emd_10114.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel size
X=Y=Z: 0.82 Å
Density
Contour Level
By AUTHOR: 0.0091 / Movie #1: 0.0091
Minimum - Maximum
-0.017892994 - 0.043078568
Average (Standard dev.)
0.00006128934 (±0.001980673)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
0
0
0
Dimensions
300
300
300
Spacing
300
300
300
Cell
A=B=C: 246.0 Å α=β=γ: 90.0 °
CCP4 map header:
mode
Image stored as Reals
Å/pix. X/Y/Z
0.82
0.82
0.82
M x/y/z
300
300
300
origin x/y/z
0.000
0.000
0.000
length x/y/z
246.000
246.000
246.000
α/β/γ
90.000
90.000
90.000
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
0
NC/NR/NS
300
300
300
D min/max/mean
-0.018
0.043
0.000
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Supplemental data
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Sample components
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Entire : Heterotetrameric complex of full-length Mre11-Rad50 (SbcCD) in co...
Entire
Name: Heterotetrameric complex of full-length Mre11-Rad50 (SbcCD) in complex with ADP and dsDNA
Components
Complex: Heterotetrameric complex of full-length Mre11-Rad50 (SbcCD) in complex with ADP and dsDNA
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Supramolecule #1: Heterotetrameric complex of full-length Mre11-Rad50 (SbcCD) in co...
Supramolecule
Name: Heterotetrameric complex of full-length Mre11-Rad50 (SbcCD) in complex with ADP and dsDNA type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)
Organism: Escherichia coli (E. coli)
Recombinant expression
Organism: Escherichia coli BL21(DE3) (bacteria)
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Buffer
pH: 7.5
Vitrification
Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 2 / Number real images: 12811 / Average electron dose: 73.6 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
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Image processing
Startup model
Type of model: NONE Details: The initial model was calculated de novo using cryoSPARC.
Final reconstruction
Resolution.type: BY AUTHOR / Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 152271
Initial angle assignment
Type: MAXIMUM LIKELIHOOD
Final angle assignment
Type: MAXIMUM LIKELIHOOD
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