National Institutes of Health/National Cancer Institute (NIH/NCI)
CA187561
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM58015
United States
National Institutes of Health/National Cancer Institute (NIH/NCI)
CA92267
United States
Citation
Journal: Nucleic Acids Res / Year: 2020 Title: DSS1 and ssDNA regulate oligomerization of BRCA2. Authors: Hang Phuong Le / Xiaoyan Ma / Jorge Vaquero / Megan Brinkmeyer / Fei Guo / Wolf-Dietrich Heyer / Jie Liu / Abstract: The tumor suppressor BRCA2 plays a key role in initiating homologous recombination by facilitating RAD51 filament formation on single-stranded DNA. The small acidic protein DSS1 is a crucial partner ...The tumor suppressor BRCA2 plays a key role in initiating homologous recombination by facilitating RAD51 filament formation on single-stranded DNA. The small acidic protein DSS1 is a crucial partner to BRCA2 in this process. In vitro and in cells (1,2), BRCA2 associates into oligomeric complexes besides also existing as monomers. A dimeric structure was further characterized by electron microscopic analysis (3), but the functional significance of the different BRCA2 assemblies remains to be determined. Here, we used biochemistry and electron microscopic imaging to demonstrate that the multimerization of BRCA2 is counteracted by DSS1 and ssDNA. When validating the findings, we identified three self-interacting regions and two types of self-association, the N-to-C terminal and the N-to-N terminal interactions. The N-to-C terminal self-interaction of BRCA2 is sensitive to DSS1 and ssDNA. The N-to-N terminal self-interaction is modulated by ssDNA. Our results define a novel role of DSS1 to regulate BRCA2 in an RPA-independent fashion. Since DSS1 is required for BRCA2 function in recombination, we speculate that the monomeric and oligomeric forms of BRCA2 might be active for different cellular events in recombinational DNA repair and replication fork stabilization.
History
Deposition
May 20, 2020
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Header (metadata) release
Jul 1, 2020
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Map release
Jul 1, 2020
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Update
Aug 26, 2020
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Current status
Aug 26, 2020
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Download / File: emd_21998.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation
Human BRCA2-DSS1-ssDNA complex with crosslinking
Voxel size
X=Y=Z: 1.48 Å
Density
Contour Level
By AUTHOR: 0.0063 / Movie #1: 0.0063
Minimum - Maximum
-0.009578008 - 0.06953412
Average (Standard dev.)
0.0001805619 (±0.0017589984)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
-128
-128
-128
Dimensions
256
256
256
Spacing
256
256
256
Cell
A=B=C: 378.88 Å α=β=γ: 90.0 °
CCP4 map header:
mode
Image stored as Reals
Å/pix. X/Y/Z
1.48
1.48
1.48
M x/y/z
256
256
256
origin x/y/z
0.000
0.000
0.000
length x/y/z
378.880
378.880
378.880
α/β/γ
90.000
90.000
90.000
start NX/NY/NZ
-64
-64
-64
NX/NY/NZ
128
128
128
MAP C/R/S
1
2
3
start NC/NR/NS
-128
-128
-128
NC/NR/NS
256
256
256
D min/max/mean
-0.010
0.070
0.000
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Supplemental data
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Sample components
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Entire : BRCA2-DSS1-ssDNA
Entire
Name: BRCA2-DSS1-ssDNA
Components
Complex: BRCA2-DSS1-ssDNA
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Supramolecule #1: BRCA2-DSS1-ssDNA
Supramolecule
Name: BRCA2-DSS1-ssDNA / type: complex / ID: 1 / Parent: 0 Details: Full-length human tumor suppressor BRCA2 protein in complex with DSS1 and dT100 oligo with crosslinking
Source (natural)
Organism: Homo sapiens (human)
Recombinant expression
Organism: Homo sapiens (human) / Recombinant cell: HEK293
Molecular weight
Experimental: 474 kDa/nm
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Experimental details
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Structure determination
Method
negative staining
Processing
single particle reconstruction
Aggregation state
particle
-
Sample preparation
Buffer
pH: 7.5
Staining
Type: NEGATIVE / Material: 2% uranyl acetate Details: BRCA2 complex was applied to glow-discharged continuous carbon copper grids and incubated for 2 min. Grids were washed briefly with 5 mM Mg(Acet)2 for three times to remove buffer, sucrose, ...Details: BRCA2 complex was applied to glow-discharged continuous carbon copper grids and incubated for 2 min. Grids were washed briefly with 5 mM Mg(Acet)2 for three times to remove buffer, sucrose, and glycerol. Samples were then stained with 2% uranyl acetate, blotted with filter paper, and air dried.
Grid
Model: Homemade / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE
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Electron microscopy
Microscope
JEOL 2100F
Image recording
Film or detector model: DIRECT ELECTRON DE-20 (5k x 3k) / Digitization - Dimensions - Width: 5120 pixel / Digitization - Dimensions - Height: 3840 pixel / Digitization - Sampling interval: 6.4 µm / Number grids imaged: 1 / Number real images: 1359 / Average exposure time: 1.0 sec. / Average electron dose: 37.5 e/Å2
Electron beam
Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
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