15 research outputs found

    ARP2/3- and resection-coupled genome reorganization facilitates translocations [preprint]

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    DNA end-resection and nuclear actin-based movements orchestrate clustering of double-strand breaks (DSBs) into homology-directed repair (HDR) domains. Here, we analyze how actin nucleation by ARP2/3 affects damage-dependent and -independent 3D genome reorganization and facilitates pathologic repair. We observe that DNA damage, followed by ARP2/3-dependent establishment of repair domains enhances local chromatin insulation at a set of damage-proximal boundaries and affects compartment organization genome-wide. Nuclear actin polymerization also promotes interactions between DSBs, which in turn facilitates aberrant intra- and inter-chromosomal rearrangements. Notably, BRCA1 deficiency, which decreases end-resection, DSB mobility, and subsequent HDR, nearly abrogates recurrent translocations between AsiSI DSBs. In contrast, loss of functional BRCA1 yields unique translocations genome-wide, reflecting a critical role in preventing spontaneous genome instability and subsequent rearrangements. Our work establishes that the assembly of DSB repair domains is coordinated with multiscale alterations in genome architecture that enable HDR despite increased risk of translocations with pathologic potential

    The Tumor-Associated Variant RAD51 G151D Induces a Hyper-Recombination Phenotype

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    <div><p>The RAD51 protein plays a key role in the homology-directed repair of DNA double-strand breaks and is important for maintaining genome stability. Here we report on a novel human RAD51 variant found in an aggressive and therapy-refractive breast carcinoma. Expression of the RAD51 G151D variant in human breast epithelial cells increases the levels of homology-directed repair. Expression of RAD51 G151D in cells also promotes high levels of chromosomal aberrations and sister chromatid exchanges. <i>In vitro</i>, the purified RAD51 G151D protein directly and significantly enhances DNA strand exchange activity in the presence of RPA. In concordance with this result, co-incubation of G151D with BRCA2 resulted in a much higher level of strand-exchange activity compared to WT RAD51. Strikingly, the RAD51 G151D variant confers resistance to multiple DNA damaging agents, including ionizing radiation, mitomycin C, and doxorubicin. Our findings demonstrate that the RAD51 G151D somatic variant has a novel hyper-recombination phenotype and suggest that this property of the protein is important for the repair of DNA damage, leading to drug resistance.</p></div

    Increased replication fork tract length in MCF10A cells expressing RAD51 G151D.

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    <p><b>A.</b> Schematic of the experimental setup for the DNA fiber assay and a representative image of an elongating replication fork, which was the replication structure exclusively used to measure replication tract length. <b>B.</b> Replication tract length of elongating replication forks measured by DNA fiber assay in untreated, HU-treated (0.5mM for 2 hrs), or IR-treated (8GY) MCF10A cells expressing RAD51 WT or G151D. >100 replication tracts were measured for MCF10A RAD51 WT or G151D expressing cells for each treatment group. Data are representative of 3 independent experiments. Graphed as mean ± SD. <b>C,D.</b> Representative images of images of untreated MCF10A RAD51 WT or G151D expressing cells. Arrows indicate representative elongating replication fork structures used to measure replication tract length. Insets highlight representative replication tracts for RAD51 WT <b>(C)</b> and RAD51 G151D <b>(D)</b> cells.</p

    Single-molecule FRET assay for RAD51 WT- and RAD51 G151D- filament formation and RPA-RAD51 interaction.

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    <p><b>A.</b> Illustration of single-molecule RAD51-ssDNA filament assay with partial DNA duplex containing a 30—nucleotide tail. Upon RAD51 filament formation, there is a transition from high FRET (DNA-only) to low FRET (RAD51-bound). <b>B.</b> Histograms display a clear shift to low FRET upon addition of 400 nM RAD51 and 2 mM ATP with increased stability using the RAD51G151D mutant. Histograms were generated after subtracting the zero FRET values and truncating photobleached portions of FRET trajectories. A minimum of 75 smFRET trajectories was used to generate each histogram. <b>C.</b> Illustration of RPA disruption assay where 20 nM RPA is bound to DNA and then 400 nM RAD51 + 2 mM ATP is added in an attempt to create RAD51 filaments. <b>D</b>. RPA-bound DNA leads to a distinct medium FRET state as opposed to the low FRET state observed upon RAD51 filament formation (Fig 7B, Panel 2). There is a transition to low FRET upon addition of 400 nM RAD51 (or RAD51 G151D) and 2 mM ATP (Panels 3 and 4), indicating that both RAD51 WT and RAD51 G151D successfully disrupt bound RPA-DNA interactions and assemble filaments. However, RAD51 WT led to a tighter peak, indicating more efficient RPA removal as compared to the RAD G151D mutant.</p

    MCF10A cells expressing G151D repair IR-induced double strand breaks more rapidly than WT expressing cells.

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    <p><b>A-D.</b> MCF10A pools expressing WT or G151D were exposed to 8GY ionizing radiation, fixed at 0, 2, 4, 8 and 24 (γH2AX only) hours post IR exposure and immunofluorescence was performed. Cells were labeled with a γH2AX antibody (green) <b>(A,B)</b> or 53BP1 antibody (green) <b>(C,D)</b>. Labeled cells were visualized using confocal microscopy. <b>A,C.</b> The number of nuclei with >10 foci of γH2AX or 53BP1 was counted. The data are graphed as mean ± SEM (n>500 nuclei) **** p< 0.0001; *** p<0.001. <b>B,D.</b> Representative images of γH2AX foci <b>(B)</b> or 53BP1 <b>(D)</b> at 4 hours post-IR exposure in MCF10A RAD51 WT or RAD51 G151D expressing pools. <b>E, F.</b> MCF10A pools expressing RAD51 WT or G151D were exposed to 8GY ionizing radiation then allowed to recover for 0, 4 and 8 h. Cells were harvested and single cell electrophoresis was performed to quantitate DNA damage using the comet assay. <b>E.</b> Data are graphed as mean ± SEM (number of nuclei counted per group: WT 0hr; 76, G151D 0hr; 80; WT 4hr; 72, G151D 4hr; 97, WT 8hr; 91, G151D 8hr; 100). **** p< 0.0001. <b>F.</b> Representative images from each time point of recovery post IR-exposure.</p

    Increased genomic instability in MCF10A cells expressing RAD51 G151D.

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    <p>Metaphase spreads were prepared from undamaged, asynchronous RAD51 WT or G151D expressing MCF10A cells. <b>A.</b> Number of aberrations per metaphase. At least 50 metaphase spreads were scored for each cell line. ****p<0.0001, ***p<0.001, *p<0.05. <b>B.</b> Representative metaphase spread of MCF10A expressing RAD51 WT <b>(B)</b> or G151D <b>(C)</b>. <b>D.</b> Western blot demonstrates equivalent expression of exogenous RAD51 WT and G151D (I) in their respective MCF-7 pools, as well as the fold increase in expression over endogenous RAD51 expression (I/R). <b>E.</b> Cell invasion of MCF7 cells expressing either RAD51 WT or G151D was performed as described in Materials and Methods. Data are graphed as mean ± SD from 2 independent experiments. ** p<0.01.</p

    RAD51 G151D exhibits enhanced DNA strand exchange in the presence of RPA.

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    <p><b>A.</b> Schematic of the DNA strand exchange assay. RPA was incubated first with the 3’ tail DNA followed by addition of RAD51 (BRCA2 and RAD51 were added simultaneously in D & E) and finally the radiolabeled donor DNA was added to initiate the reaction. <b>B.</b> Autoradiograms of DNA strand exchange assays comparing RAD51 WT to G151D in the presence of increasing concentrations of RPA. Lanes 1 and 8 are no protein controls. Lanes 2 and 9 contain either RAD51 WT or G151D in the absence of any other protein. <b>C.</b> Quantification of the PAGE gels shown in <b>(B). D.</b> Autoradiograms of DNA strand exchange assays performed in the presence of increasing concentrations of BRCA2 utilizing a fixed concentration of RPA (100nM) and of RAD51 WT or G151D (300 nM). <b>E.</b> Quantification of the gel shown in <b>(D)</b>. Mean values from three independent experiments were plotted. Error bars represent S.D. <b>F.</b> Autoradiograms of DNA strand exchange assays utilizing a 3’ tail DNA substrate in the absence or presence of pre-incubation with RPA. No protein controls (lanes 1 & 8). WT or G151D RAD51 in the absence of RPA (lanes 2 & 9). WT or G151D RAD51 added after pre-incubation of 3’ tail DNA with 20 nM RPA (lanes 3 & 10). WT and G151D mixed together at the depicted ratios and added after 20nM RPA (lanes 4–6 & 11–13). WT or G151D RAD51 added after RPA as in lanes 3 & 10 (lanes 7 & 14). The total RAD51 (WT+G151D) concentration in each reaction was kept constant. <b>G.</b> Quantification of autoradiograms in (A). Black bars represent WT protein and red bars represent G151D protein. Proportion of black bar:red bar in graphs represent of WT:G151D ratios.</p
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