Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair
- Department of Molecular Genetics, Oncode Institute, Erasmus MC Cancer Institute, Erasmus University Medical Center, Netherlands
- Department of Radiation Oncology, Erasmus University Medical Center, Netherlands
Figures
Figure 1 with 3 supplements
Functional analysis of BRCA2 deletion variants in mouse embryonic stem cells, tagged at the endogenous locus with a HaloTag.
(A) Schematic overview of full-length mouse (top) and human (bottom) BRCA2 proteins, with key domains (DBD, CTD, NLS, BRC1-8: red bars; PALB2-binding: blue bar) and tags indicated. Deletion variants …
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Figure 1—source data 1
Excel file with the source data of the cell survival data in Figure 1C–F and HR assay in Figure 1G.
- https://cdn.elifesciences.org/articles/67926/elife-67926-fig1-data1-v2.zip
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Figure 1—source data 2
Full western blot images from Figure 1B.
- https://cdn.elifesciences.org/articles/67926/elife-67926-fig1-data2-v2.pdf
Figure 1—figure supplement 1
Generation of different variants of BRCA2-HaloTag knock-ins.
(A) Cartoon depicting the CRISPR/Cas9 targetting strategy, indicating the approximate position of the gRNA sequences (Supplementary file 1) and the targetting construct with the homology arm and …
Figure 1—figure supplement 2
Flow cytometry analysis of the CRISPR/Cas9 β-actin-P2A-mCherry targeting assay as shown in Figure 1G.
A plasmid expressing BFP2 was co-introduced for the selection of successfully transfected cells. The percentage of transfected cells (BFP+) that express mCherry targetted to the β-actin cells was …
Figure 1—figure supplement 3
Sequence alignment between human and mouse BRCA2 DBD and CTD.
Top panel indicates the conservation between human and mouse BRCA2 at the amino acid level. At the bottom, the aligned sequences between human and mouse DBD and CTD are plotted.
Figure 2 with 1 supplement
BRCA2 and RAD51 foci quantification.
(A) Representative confocal images (maximum intensity projections) of BRCA2 (red) and RAD51 (green) foci in mouse embryonic stem (mES) cells fixed 2 hr after mock or 2 Gy irradiation, without …
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Figure 2—source data 1
Excel file with exact n numbers and statistical tests of Figure 2B–G and the source data of the foci quantification.
- https://cdn.elifesciences.org/articles/67926/elife-67926-fig2-data1-v2.xlsx
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Figure 2—source data 2
Original uncropped images from Figure 2A.
Confocal z-projection images.
- https://cdn.elifesciences.org/articles/67926/elife-67926-fig2-data2-v2.zip
Figure 2—figure supplement 1
Additional images and plots for BRCA2 and RAD51 foci quantification.
(A) Confocal images (maximum intensity projection) of mouse embryonic stem (mES) cells producing the indicated variants of BRCA2 immunostained for RAD51 after pre-extraction and pulse-labeling with …
Figure 3 with 5 supplements
Single-particle tracking of BRCA2-HaloTag reveals immobilization of BRCA2 lacking either DBD or CTD upon DNA damage.
(A) Wide-field image of an S-phase cell visualized with iRFP720-PCNA and BRCA2-HaloTag::JF549. (B) Example of two tracks of BRCA2-Halo showing different diffusive behavior; see also Figure 3—videos 1…
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Figure 3—source data 1
Excel file with exact n numbers and statistical tests of Figure 3 and the source data of the single-molecule-tracking experiments.
- https://cdn.elifesciences.org/articles/67926/elife-67926-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Diffusion histograms of single-particle tracking experiments.
(A) Wide-field images of cells from the different BRCA2 variants (like in Figure 3A), showing iRFP720-PCNA in magenta and BRCA2-Halo::JF549 in green. (B) Histograms of the apparent diffusion …
Figure 3—video 1
Single-molecule recording of full-length BRCA2-Halo (JF549) in untreated mouse ES cells imaged at 30 ms interval.
Figure 3—video 2
Single-molecule recording of full-length BRCA2-Halo (JF549) in mouse ES cells treated with 2Gy of ionizing radiation imaged at 30 ms interval.
Figure 3—video 3
Single-molecule recording of BRCA2 ΔDBDΔCTD-Halo (JF549) in untreated mouse ES cells imaged at 30 ms interval.
Figure 3—video 4
Single-molecule recording of BRCA2 ΔDBDΔCTD-Halo (JF549) in mouse ES cells treated with 2Gy of ionizing radiation imaged at 30 ms interval.
Figure 4 with 4 supplements
C-terminal region of human BRCA2 contributes to the formation of BRCA2-BRCA2 oligomers.
(A) Representative scanning force microscopy (SFM) height images of full-length and ∆CTD BRCA2 in the presence and absence of RAD51. BRCA2 ΔCTD forms rod-shaped assemblies, like full-length BRCA2, …
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Figure 4—source data 1
Excel files with the source data of the data in Figure 4B and the replicate experiment.
- https://cdn.elifesciences.org/articles/67926/elife-67926-fig4-data1-v2.zip
Figure 4—figure supplement 1
Representative SFM height images of BRCA2 ΔDBD and BRCA2 ΔDBDΔCTD in the presence and absence of RAD51.
Both the constructs show reorganization of the protein into smaller globular assemblies on interaction with RAD51.
Figure 4—figure supplement 2
Plots showing distribution of full-length BRCA2 and c-terminal deletion constructs in the presence and absence of RAD51.
All the BRCA2 samples, full-length and the deletion constructs, show a similar rearrangement into globular and monomeric assemblies.
Figure 4—figure supplement 3
Control scanning force microscopy experiments of RAD51 and ssDNA alone.
(A) Representative scanning force microscopy (SFM) height images of RAD51 alone. (B) Representative SFM height image of single-stranded DNA (ssDNA) alone. (C) Distribution of volume of all the …
Figure 4—figure supplement 4
SDS-PAGE gels visualizing the purified protein preparations as used in this study (Figure 4 and Figure 5).
Coomassie staining and silver staining of protein preparations. Asterisks (*) indicate the expected molecular weight bands for the different proteins. Protein amounts differ in the lanes, as the …
Figure 5 with 3 supplements
C-terminal region of BRCA2 is essential for conformational rearrangement on interaction with ssDNA.
(A) Representative scanning force microscopy (SFM) height images of full-length BRCA2 and BRCA2 ΔDBD in the presence and absence of single-stranded DNA (ssDNA). Full-length BRCA2 rearranges into …
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Figure 5—source data 1
Excel files with the source data of the data in Figure 4B and the replicate experiment.
- https://cdn.elifesciences.org/articles/67926/elife-67926-fig5-data1-v2.zip
Figure 5—figure supplement 1
Representative SFM height images of BRCA2 ΔCTD and BRCA2 ΔDBDΔCTD in the presence and absence of ssDNA.
Neither of the constructs exhibited a change in conformation on incubation with single-stranded DNA (ssDNA). A noticeable amount of free ssDNA is visible in the background, showing lack of …
Figure 5—figure supplement 2
Distribution of full-length BRCA2 and the C-terminal deletion constructs with respect to their oligomerization and solidity.
Distribution of oligomerization and solidity of full-length BRCA2 and DNA-binding domain (DBD) and/or C-terminal domain (CTD) deletion variants. Full-length BRCA2 rearranges to form extended …
Figure 5—figure supplement 3
Control experiment showing representative scanning force microscopy (SFM) height images of full-length BRCA2 ± ssDNA in the absence of spermidine, showing that the conformational change observed on interaction with single-stranded DNA (ssDNA) is not an artifact due to presence of spermidine, which is used to facilitate adsorption of DNA on the mica surface for SFM imaging.
Tables
Table 1
Summary of results of the in vivo assays in this study.
| Ionizing radiation | DNA crosslinks | PARPi | HR | BRCA2 diffusion | Immobilization | RAD51 focus formation | ||
|---|---|---|---|---|---|---|---|---|
| Full-length | + | + | + | + | + | + | ++ | |
| ΔDBD | - | - | -- | - | + | + | + | |
| ΔCTD | +/- | + | - | +/- | + | + | + | |
| ΔDBDΔCTD | - | - | -- | - | + | +/- | + | |
Table 2
Summary of results of the in vitro assays in this study.
| Multimerization | Conformational change | ||
|---|---|---|---|
| + RAD51 | + ssDNA | ||
| Full-length | + | +++ | +++ |
| ΔDBD | +/- | ++ | — |
| ΔCTD | +/- | +++ | — |
| ΔDBDΔCTD | +/- | ++ | - |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (mouse) | Brca2 | Genbank | MGI:109337 | |
| Gene (human) | BRCA2 | Genbank | HGNC:1101 | |
| Cell line (mouse) | IB10, subclone of E14 129/Ola | Hooper et al., 1987 | IB10, mES | |
| Cell line (mouse) | Mouse ES cellsRad54 -/- | Tan et al., 1999 | ||
| Cell line (human) | HEK293T | Adapted to suspension culture | ||
| Antibody | Rabbit polyclonal anti-RAD51 | van Veelen et al., 2005 | 2307 | IF: 1:10 000 |
| Antibody | Rabbit polyclonal Anti-BRCA2 | Abcam | ab27976 | WB: mouse BRCA2 (1:1000) |
| Antibody | Mouse monoclonal Anti-HaloTag | Promega | G9211 | WB: 1:1000 |
| Antibody | Mouse monoclonal anti-PARP-1 | Enzo | BML-SA250-0050 | WB: 1:1000 |
| Antibody | Anti-rabbit IgG conjugated with CF568 | Biotium/Sigma | Cat# SAB4600310 | IF 1:1000 |
| Antibody | Donkey αRabbit IgG HR Peroxydase | Jackson Imm Res | Cat# 711-035-152 | WB: 1:2000 |
| Antibody | Sheep αMouse IgG HR Peroxydase | Jackson Imm Res | Cat# 515-035-003 | WB: 1:2000 |
| Antibody | Mouse monoclonal anti BRCA2 | Calbiochem | OP95 | WB: full-length, ΔDBD, ΔCTD, ΔDBDΔCTD proteins (1:500) |
| Recombinant DNA reagent | AAV_Actb HR donor plasmid | Yao et al., 2017; Addgene | Plasmid #97317 | |
| Recombinant DNA reagent | px459 | Ran et al., 2013 | ||
| Recombinant DNA reagent | px459 expressing two gRNAs | Zelensky et al., 2017 | Modified from Ran et al., 2013 | |
| Recombinant DNA reagent | BRCA2-HaloTag donor plasmid | This paper | Knock-in construct HaloTag -F2A-neomycin at mouse BRCA2 C-terminus; available on request from corresponding author | |
| Recombinant DNA reagent | BRCA2 ΔDBD-HaloTag | This paper | Knock-in construct HaloTag -F2A-neomycin at mouse BRCA2 C-terminus resulting in deletion of DBD; available on request from corresponding author | |
| Recombinant DNA reagent | BRCA2 ΔCTD-HaloTag | This paper | Knock-in construct HaloTag -F2A-neomycin at mouse BRCA2 C-terminus resulting in deletion of CTD; available on request from corresponding author | |
| Recombinant DNA reagent | BRCA2 ΔDBDΔCTD-HaloTag | This paper | Knock-in construct HaloTag -F2A-neomycin at mouse BRCA2 C-terminus resulting in deletion of DBD and CTD; available on request from corresponding author | |
| Recombinant DNA reagent | iRFP720-PCNA | This study | pMP37 pGb-iRFP720-I-PCNA | Expression construct flanked by piggyBac inverted terminal repeats; available on request from corresponding author |
| Recombinant DNA reagent | hyPBase | Yusa et al., 2011 | Expressing piggyBac transposase | |
| Recombinant DNA reagent | phCMV1-2MBP-TEV-fl BRCA2 | S Kowalczykoski lab | Expression clone for 293T HEK cells | |
| Recombinant DNA reagent | phCMV1-2MBP-TEV-BRCA2 ΔDBD | This study | Expression clone for 293T HEK cells; available on request from corresponding author | |
| Recombinant DNA reagent | phCMV1-2MBP-TEV-BRCA2 ΔCTD | This study | Expression clone for 293T HEK cells; available on request from corresponding author | |
| Recombinant DNA reagent | phCMV1-2MBP-TEV-BRCA2 ΔDBDΔCTD | This study | Expression clone for293T HEK cells; available on request from corresponding author | |
| DNA oligo | 90 nt ssDNA oligo | IDT | See sequence in 'Materials and methods' | |
| Commercial kit | Q5 site directed mutagenesis | NEB | Cat# E0554S | |
| Commercial assay or kit | MyTaq Red Mix | Bioline | BIO-25043 | |
| Chemical compound, drug | JF549 HaloTag-ligand | Grimm et al., 2015 | Gift from L Lavis | |
| Chemical compound, drug | JF646 HaloTag-ligand | Grimm et al., 2015 | Gift from L Lavis | |
| Chemical compound, drug | EdU (5-ethynyl-2’-deoxyuridine) | Cat# A10044 | ||
| Chemical compound, drug | Atto488-azide | ATTO-TEC GmbH | Cat# AD 488–101 | |
| Chemical compound, drug | Atto568 azide | ATTO-TEC GmbH | Cat# AD 594–101 | |
| Chemical compound, drug | MMC (mitomycin C) | Sigma-Aldrich | Cat# M503 | |
| Chemical compound, drug | Cisplatin | Sigma-Aldrich | Cat# P4394 | |
| Chemical compound, drug | Olaparib | Selleckchem | Cat# S1060 | |
| Reagent | FreeStyle 293 expression medium | Gibco | Cat# 10319322 | For growth of 293T HEK cells |
| Reagent | Serum-free hybridoma media | Gibco | Cat# 12045084 | For transfection of 293T HEK cells |
| Software, algorithm | DBD tracking | This paper | Software for analysis of single-moleucle tracking data Available at: https://github.com/maartenpaul/DBD_tracking (copy archived at swh:1:rev:19f3a47289830cf5dc139061a89627b6165da804, Paul, 2021) | |
| Software, algorithm | DBD foci | This paper | Scripts for analysis of foci data using CellProfiler Available at: https://github.com/maartenpaul/DBD_foci/ (copy archived at swh:1:rev:157c7953dbed176a65f2c55db7ad48ebfa7f3f5d, Pau, 2021) | |
| Software, algorithm | SOS Plugin | Reuter et al., 2014 | http://smal.ws/wp/software/sosplugin/ | |
| Software, algorithm | DL-MSS | Arts et al., 2019a Arts et al., 2019b | https://github.com/ismal/DL-MSS | |
| Software, algorithm | CellProfiler | Carpenter et al., 2006 | ||
| Software, algorithm | Fiji | Schindelin et al., 2012 | ||
| Software, algorithm | SFMetrics | Sánchez and Wyman, 2015 | http://cluster15.erasmusmc.nl/TIRF-SFM-scripts/ |
Additional files
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Supplementary file 1
Tables with gRNA sequences used for generating the BRCA2-Halo knock-in cell lines and primers used for genotyping the cell lines.
- https://cdn.elifesciences.org/articles/67926/elife-67926-supp1-v2.xlsx
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Supplementary file 2
Summary of average solidity of BRCA2 variants in the absence and presence of ssDNA (90-mer) oligomer.
- https://cdn.elifesciences.org/articles/67926/elife-67926-supp2-v2.xlsx
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Supplementary file 3
Percent rod-like assemblies in BRCA2-RAD51 interaction in various constructs.
- https://cdn.elifesciences.org/articles/67926/elife-67926-supp3-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/67926/elife-67926-transrepform-v2.docx
