PMC:6145447 / 19185-46803 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/6145447","sourcedb":"PMC","sourceid":"6145447","source_url":"https://www.ncbi.nlm.nih.gov/pmc/6145447","text":"METHODS\n\nChromatin binding assay and western blot\nPreparation of Low-Speed Supernatant (LSS) Xenopus egg extracts and isolation of demembranated sperm nuclei (chromatin) were performed as previously described 34. For chromatin binding experiments, 15 μl of LSS extracts were supplemented with demembranated sperm nuclei (2,500 sperm/μl) and incubated for 10 min at 21ºC before addition of DMSO or one of the following compounds for 10 min: CK-666 (160 μM), CK-548 (160 μM) KU5593 (100 μM), VE821 (50 μM). Subsequently, the PflMI restriction endonuclease was added to each sample (0.05U/μl) before incubation for an additional 60 min at 21ºC. Extracts were diluted in chromatin isolation buffer (50 mM Hepes-KOH, pH 7.8, 100 mM KCl, 2.5 mM MgCl2) supplemented with 0.125% Triton X-100 and overlaid on top of sucrose cushions (chromatin isolation buffer plus 30% sucrose) in 1.5 ml low-retention tubes (Thermo Fisher Scientific). Samples were spun at 7180 RPM for 30 min at 4ºC in a swing-bucket Sorval rotor (HB-6). Chromatin pellets were resuspended in 10 μl Laemmli buffer, boiled, and fractionated on a 4 – 22% Tris-Glycine gel (Invitrogen) according to standard procedures, followed by transfer of resolved proteins onto PVDF membranes. Following a 1 hr block with 5% milk, membranes were incubated overnight at 4ºC with one of the following primary antibodies against: β-actin (Sigma Aldrich: A5316, 1/1000), Arpc4 (Novus Biologicals: NBP1-69003, 1/500), Capping protein β-2 subunit (Developmental Studies Hybridoma Bank: E00007, 1/10000), Histone H3 (Cell Signaling Technology, 9715, 1/2000) and Xenopus Mre11 34. HRP-conjugated secondary antibodies were used (Anti-Rabbit IgG HRP, Anti-Mouse IgG HRP, Fisher Scientific) and chemiluminescence (Supersignal West Pico Chemiluminescent Substrate, 34077) was used.\n\nMass spectrometric analysis of DSB-containing chromatin\nChromatin samples were isolated as described above with the following modifications: 100μl of LSS extracts were supplemented with sperm nuclei (5,000 sperm/μl) and incubated for 10 min at 21ºC before addition of PflMI restriction endonuclease (0.05U/μl) and incubated for an additional 60 min. Chromatin was isolated through sucrose cushions as described above and extensively digested with 100U of micrococcal nuclease for 30 min at 37ºC (NEB). Chromatin proteins were reduced with DTT (5mM, 30 min 60ºC) and alkylated with iodoacetamide (15mM, 30 min RT) before fractionation on SDS-PAGE. “in-gel” digestion was performed with proteomic-grade trypsin (Promega) at 5 ng/μl for 16 hrs at 37ºC. The resulting digestion peptides were extracted from the gel pieces with 1:2 (vol/vol) 5% formic acid: acetonitrile solution. The resulting peptides were labeled with isobaric mass tags (ITRAQ 4-plex, Sciex), combined, purified with in-house made STAGE tips 35, resuspended in 0.5% acetic acid and loaded onto a home-packed reverse phase C18 column (75 μm I.D.). The peptides were separated using a linear gradient (0 % to 42 % acetonitrile, 0.5 % acetic acid, 120 min, 150 nL/min) and directly sprayed into an LTQ-Orbitrap-Velos mass spectrometer for analysis (Thermo). The repetitive analytical cycle incorporated a high-resolution mass scan in the Orbitrap (resolution = 30,000) followed by tandem MS scans of the five most intense peaks observed in each Orbitrap mass spectrum. Peptides were identified and quantified using Proteome Discoverer software (Thermo, Version 1.4).\n\nCell culture and drug treatment\nU2OS and mouse-tail fibroblast cell lines were cultured in high-glucose Dulbecco’s modification of Eagles media supplemented with L-Glutamine, 10% fetal bovine serum, and 1% penicillin-streptomycin. For Neocarzinostatin-dependent DSB induction, cells were cultured on 8-well chamber slides (Thermo Fisher Scientific) and were treated with 0.5 μg/ml NCS for 60 min at 37 °C. Cells were washed twice with PBS before adding media containing DMSO or one of the following compounds: CK-666 (Sigma Aldrich: SML-006, 100 μM), CK-548 (Sigma Aldrich: C7499, 50 μM), CK-689 (Sigma Aldrich: 18251750, 100 μM), and Wiskostatin (Sigma Aldrich: W2270, 3 μM). After drug treatment, cells were then incubated at 37 °C for the indicated times. For mirin experiments, cells were pre-treated with mirin (Sigma Aldrich: M9948, 50 μM) for 1 hour, prior to addition of NCS,\nER-AsiSI U2OS cells stably expressing the AsiSI restriction endonuclease tethered to the estrogen receptor were generously provided by Dr. G. Legube, Centre de Biologie Integrative – Toulouse. For cell synchronization, cells were treated with 2 mM thymidine for two 18 hr intervals separated by an 11 hr release in fresh media. Cells underwent double-thymidine block and were released into fresh media for 7 hrs (G2) or 15 hrs (G1) prior to AsiSI-dependent DSB induction with media supplemented with 300 nM 4OHT (Sigma Aldrich, H7904) plus CK-666, CK-548, wiskostatin, or DMSO. Cells were subsequently incubated at 37 °C for 4 h.\nArpc2−/− fibroblast cell lines (generously provided by Dr. J.E. Bear, University of North Carolina) stably express the Cre-recombinase tagged to the estrogen receptor. Upon 4-OHT treatment, the Arpc2 locus is floxed by Cre and cells are depleted of Arpc2 following 4 days of 4-OHT treatment.\nCB33 and RD lymphoblastoid B-cell lines were a kind gift of Dr. Riccardo Dalla-Favera (Columbia University). Two B-lymphocyte cell lines bearing distinct mutations in the gene WAS were obtained from the NIGMS Human Genetic Cell Repository (Coriell: GM1267, GM1268). GM1267 harbors a G\u003eA transition at position 4 in intron 6 of the WAS gene (IVS6+5G\u003eA). This mutation activates a cryptic splice site leading to splicing of a 38-nucleotide sequence from intron 6 onto exon 7. GM1268 harbors a G\u003eA transition at nucleotide 257 in exon 2 on the WAS gene. This mutation results in a methionine for valine substitution at codon 75 (V75M). All B-cell lines were grown in Rosewell Park Memorial Institute Medium with 20% heat-inactivated bovine serum with 2mM L-glutamine added.\n\nImmunohistochemistry and quantification of DSB repair protein foci\nU2OS cells were cultured on 8-well chamber slides (Thermo Fisher Scientific) and subjected to drug treatments as described above. Cells were then washed once with PBS before fixation with freshly-prepared 4% PFA (pH 7.4) for 10 min. Cells were subsequently washed twice with PBS for 10 min prior to permeabilization with 0.1% PBS-Triton X-100 for 10 min. Cells were then washed once with PBS for 5 min before incubation with blocking buffer (3% BSA in 0.2% PBS-Tween) for 1 h. Cells were stained with primary antibodies diluted in blocking buffer under a Hybrislip (Invitrogen) overnight in a humidified chamber at 4°C. Primary antibodies include γH2AX (EMD Millipore: 05-636, 1/500 or Abcam: ab81299, 1/500), WASP (Santa Cruz: sc-5300, 1/50), Rad51 (Santa Cruz: sc-8349, 1/50), 53BP1 (Novus Biologicals: NC100-304, 1/250) and FLAG (Sigma Aldrich: F3165, 1/250). Cells were then washed three times in PBS for 15 min and incubated with a fluorescent-conjugated secondary antibody in PBS with DAPI (Invitrogen, 1/10000) for 1 hr at room temperature. Secondary antibodies were Alexa 488 conjugated goat anti-mouse Ig (Abcam: ab150113, 1/1000), Alexa 488 conjugated goat anti-rabbit Ig (Thermo Fisher Scientific, A-11034, 1/10000), and Alexa 594 conjugated goat anti-mouse Ig (Thermo Fisher Scientific, A-11005, 1/10000). Cells were then washed three times with PBS for 15 min. Vectashield was applied to each slide and slides were coverslipped. Slides were analyzed under 40X magnification using a Zeiss Axio Imager Z2 microscope, equipped with a CoolCube1 camera (Carl Zeiss, Thornwood, NY, USA). Images were processed for contrast enhancement and background reduction using ImageJ.\nMetaCyte software (Metasystems, Newton, MA, USA, Version 3.10.6) was used to detect U2OS nuclei based on DAPI staining and perform automated quantification of γH2AX, Rad51, and WASP foci within each nucleus. For foci size and clustering analyses, images were converted to Grayscale and processed with Icy Software (Version 1.8.6.0; Institut Pasteur, Quantitative Image Analysis Unit; http://www.icy.bioimageanalysis.org). Regions of interest were drawn around individual nuclei and foci were detected using the Spot Detector plug-in (Channel 0) 36. For clustering analyses, spots detected using Scale 2 (3 px) were exported to the spatial analysis plug-in, which utilizes Ripley’s K-function to assess the deviance of pairs of points from total randomness 24,36. The software reports statistically significant clustering (p\u003c0.05) when the K-function crosses the clustering threshold. The software further corrects for the number of points in the computations of the quantiles of the Ripley’s K function. Thus, there is no loss of statistical power for samples with lower numbers of foci. For foci size analysis, foci with minimum sizes of 1 px and 3 px were counted using the Spot Detector plug-in. Scale 1 was used to detect foci that were ≥ 1 px in size, whereas Scale 2 was used to detect foci that were ≥ 3 px. Foci that met criteria for Scale 1 (≥ 1 px) and Scale 2 (≥ 3 px) were excluded from the Scale 1 group to avoid duplicate detections. Foci counts were subsequently averaged to determine the mean size of foci per nucleus (100 nuclei were detected per condition or as indicated). For co-localization studies, images were imported into Image J (NIH) and separated into green (γH2AX) and red (WASP) channels. These files were subsequently imported into Icy and regions of interest were drawn around individual nuclei. Pearson r was calculated using the Co-localization Studio plug-in, which compares the coincidence of spots inside a region of interest between green and red channels.\n\nChromatin immunoprecipitation\nChIP experiments followed previously published protocols with the following modifications 20: 50 million ER-AsiSI U2OS cells were cultured on 150mm2 plates in high-glucose Dulbecco’s modification of Eagles media supplemented with L-Glutamine, 10% fetal bovine serum, and 1% penicillin-streptomycin. Following double-thymidine block, cells were released into fresh media for 7 hr (G2) or 15 hr (G1) before incubation with 300 nM 4-OHT for 4 h. Cells were then trypsinized, washed with PBS, cross-linked in 1% methanol-free formaldehyde fixing buffer for 10 min, quenched with 0.125M glycine for 5 min, and snap frozen at −80 ºC. Cell lysis and nuclear isolation were performed using NP40 lysis buffer and SDS shearing buffer (Covaris). Nuclei were sonicated using the S220 Ultrasonicator (Covaris) to obtain chromatin fragments of 500 – 1000 bp in length. Sheared chromatin was incubated with 10 μg antibodies to Rad51 (Santa Cruz: sc-8349), DNA-PKcs (Abcam: ab1832), WASP (Santa Cruz: sc-5300), Arpc2 (Santa Cruz: sc-32195) or IgG (Jackson ImmunoResearch Laboratories) overnight. These antibodies were validated for ChIP by previous studies 12,20. Protein A/G magnetic beads were added overnight, followed by sequential washes at increasing stringency and reverse cross-linking at 65 ºC. Immunoprecipitated DNA and input DNA were analyzed in triplicate by qPCR. ChIP efficiencies (measured as percent of input immunoprecipitated) were measured by qPCR at sites 80 bp downstream of DSBs. Graphs show ChIP efficiencies in G1, G2, or No 4-OHT cells, pooled from three experiments, and normalized against input material. Sequences for primers are listed below: DSB I: 5′-GTCCCTCGAAGGGAGCAC-3′, 5′-CCGACTTTGCTGTGTGACC-3′; DSB II: 5′-CCGCCAGAAAGTTTCCTAGA-3′, 5′-CTCACCCTTGCAGCACTTG-3′; DSB III: 5′-TCCCCTGTTTCTCAGCACTT-3′, 5′-CTTCTGCTGTTCTGCGTCCT-3′; DSB IV: 5′-ATCGGGCCAATCTCAGAGG-3′, 5′-GCGACGCTAACGTTAAAGCA-3′. Primer pairs used to measure Rad51, DNA-PK(cs), WASP, and Arpc2 were located 80 bp from DSB sites or at an undamaged genomic locus.\n\nI-SceI-induced DSB repair assays\nU2OS cell lines which harbor chromosomally-integrated I-SceI-based GFP reporter substrates were used to monitor the efficiency of HDR, SSA, MMEJ, and c-NHEJ repair processes. The DR-GFP, SA-GFP, EJ2-GFP, and EJ5-GFP cell lines (generous gifts from Dr. J. Stark, City of Hope Cancer Center) were transfected with 1 μg of I-SceI expressing vector (FuGENE 6 transfection reagent (Promega), Opti-MEM (Gibco)) in the media supplemented with DMSO or one of the following compounds: CK-666 (Sigma Aldrich: SML-006, 50 μM), CK-548 (Sigma Aldrich: C7499, 25 μM), CK-689 (Sigma Aldrich: 18251750 μM), SMIFH2 (Sigma Aldrich: S4826, 5 μM), or wiskostatin (Sigma Aldrich: W2270, 3 μM). The I-SceI expressing vector was a gift of Dr. Richard Baer. For the FLAG actin construct overexpression experiments, DR-GFP cells were transfected with FLAG-WTactin-NLS or FLAG-R62Dactin-NLS for 48 hr prior to transfection with I-SceI in media supplemented with DMSO or CK-666. For the mCherry actin construct overexpression experiments, DR-GFP cells were co-transfected with I-SceI and mCherry-WTactin, mCherry-WTactin-NLS, mCherry-R62Dactin, or mCherry-R62Dactin-NLS. Cells were harvested 48 hr post I-SceI transfection and % GFP+ events were counted by FACS (10,000 cells per biological replicate). For all conditions, parallel transfection with 1 μg of pEGFP-N3 vector (Clontech) was used to determine transfection efficiency. Percentage refers to the number of GFP+ cells divided by the number of pEGFP+ cells.\n\nResection assay\nA detailed protocol for the resection assay has been recently described 37. Briefly, ER-AsiSI U2OS cells were treated with 300 nM 4-OHT in media supplemented with DMSO, CK-548 (50 μM), or CK-666 (100 μM). For mirin experiments, cells were pre-treated with mirin (Sigma Aldrich: M9948, 50 μM) for 1 hour prior to addition of 4-OHT, and subsequently washed and incubated with mirin for indicated time points. After 4 hours, cells were trypsinized and washed with cold PBS before resuspension in lysis buffer (100 mM NaCl, 10 mM TrisCl pH 8, 25 mM EDTA, pH 8, 0.5% SDS, 0.1 mg/mL proteinase K). Cells were agitated at 37°C overnight. After phenol/chloroform purification and salt/ethanol extraction, precipitated DNA underwent restriction enzyme digest by BamHI-HF, BsrGI, or HindIII-HF (1 unit of enzyme per 7 ng of DNA) at 37 °C overnight. DNA digests were subsequently used as templates in a 20 uL qPCR reaction containing 10.0 μL 2× TaqMan Universal PCR Master mix (Thermo), 0.5 μM of each primer, and 0.2 μM of probe. The extent of ssDNA generation at sites downstream of various DSBs was determined by calculating the ΔCt value for each site (mock-digested Ct value - digest Ct value) and employing the following equation: ssDNA = 1/(2^(ΔCt-1)+0.5)*100. Sequences for primers and probes are listed below: DSB V (335 bp site): 5′-GAATCGGATGTATGCGACTGATC-3′, 5′-TTCCAAAGTTATTCCAACCCGAT-3′; 6FAM-CACAGCTTGCCCATCCTTGCAAACC-TAMRA; DSB V (1618 bp site): 5′-TGAGGAGGTGACATTAGAACTCAGA-3′, 5′-AGGACTCACTTACACGGCCTTT-3′; 6FAM-TTGCAAGGCTGCTTCCTTACCATTCAA-TAMRA; DSB V (3500 bp site): 5′-TCCTAGCCAGATAATAATAGCTATACAAACA-3′, 5′-TGAATAGACAGACAACAGATAAATGAGACA-3′; 6FAM-ACCCTGATCAGCCTTTCCATGGGTTAAG-TAMRA; DSB VI (364 bp site): 5′-CCAGCAGTAAAGGGGAGACAGA-3′, 5′-CTGTTCAATCGTCTGCCCTTC-3′; 6FAM-CCAGGCCCTCAAAATCCCTCCACTG-TAMRA; DSB VI (1754 bp site): 5′-GAAGCCATCCTACTCTTCTCACCT-3′, 5′-GCTGGAGATGATGAAGCCCA-3′; 6FAM-CACTCCCTGTTCTTCTTCTGCTCCCGA-TAMRA; DSB VI (3564 bp site): 5′-GCCCAGCTAAGATCTTCCTTCA-3′, 5′-CTCCTTTGCCCTGAGAAGTGA-3′; 6FAM-CTGCAGCCCTCAAGCCCGGAT-TAMRA. No DSB site: 5′-ATTGGGTATCTGCGTCTAGTGAGG-3′, 5′-GACTCAATTACATCCCTGCAGCT-3′; 6FAM-TCTCTGCACAGACCGGCTTCCCTTC-TAMRA.\n\nAsiSI-AID repair assay\nThe repair assay followed recently published protocols with the following modifications 20,24. 5 million ER-AsiSI-AID U2OS cells were cultured on 100mm2 plates in high-glucose Dulbecco’s modification of Eagles media supplemented with L-Glutamine, 10% fetal bovine serum, and 1% penicillin-streptomycin. Following double-thymidine block, cells were released into fresh media for 7 hr (G2) or 15 hr (G1) before incubation with 300 nM 4-OHT and DMSO or CK-666 (100 μM) for 4 h. Cells were subsequently washed 3 times with PBS and then incubated in media containing auxin (Sigma: I5148, 500 μg/mL) and DMSO or CK-666. Cells were harvested 0, 2, or 8 hr after auxin exposure. Phenol/chloroform purification followed by salt/ethanol extraction was performed to precipitate DNA. DNA were subsequently used as templates in a 20 μL qPCR reaction containing 10.0 μL 2× TaqMan Universal PCR Master mix (Thermo), 0.5 μM of each primer, and 0.2 μM of probe. The level of unrepaired DSBs was determined by normalizing the Ct value for each DSB site with a control, undamaged site. Sequences for primers and probes are listed below. DSB V (Across DSB): 5′-GATGTGGCCAGGGATTGG-3′, 5′-CACTCAAGCCCAACCCGT-3′; DSB VI (Across DSB): 5′-GAGGAGCCTCTCCTGCAGC-3′, 5′-GAACCAGACCTACCTCCAGGG-3′.\n\nLive cell imaging\nU2OS cells stably expressing Rad52-mCherry, 53BP1-YFP, and Geminin-CFP constructs 21 were cultured on 35 mm glass bottom microwell dishes (MatTek, P35GC-1.5–10-C). Cells were treated with 0.5 μg/ml NCS to induce DSBs for 60 min at 37 °C. Subsequently, cells were washed twice with PBS before adding media containing DMSO or CK-666 (100 μM). For mirin experiments, cells were pre-treated with mirin (Sigma Aldrich: M9948, 50 μM) for 1 hour prior to addition of NCS, and subsequently washed and incubated with mirin for indicated time points. S-phase cells were selected for Rad52 foci analysis by screening for Geminin-CFP positivity. G1 cells were selected for 53BP1 foci analysis by screening for Geminin-CFP/Rad52-mCherry negative cells. After 20 hours, images were acquired on an A1RMP confocal microscope (Nikon Instruments, Melville, NY), on a TiE Eclipse stand equipped with a 60×/1.49 Apo-TIRF oil-immersion objective lens, an automated XY stage, stage-mounted piezoelectric focus drive, and a heated, humidified stagetop chamber with 5% CO2 atmosphere. Cells expressing Rad52-mCherry and 53BP1-YFP foci were imaged in GaAsP detectors using 561 nm and 488 nm excitation, respectively, and standard RFP and GFP emission filters. The confocal pinhole was set to 1 Airy unit for the red channel. Foci movements were examined by collecting z series at 0.4 μm intervals throughout the entire nucleus every 5 min for 2.5 h. Focus was maintained by the Perfect Focus System (Nikon).\n\nNuclear actin-chromobody-tag-GFP imaging and analysis\nFor live cell imaging experiments, U2OS cells were cultured on 35 mm glass bottom microwell dishes at 90% confluency (MatTek, P35GC-1.5–10-C) and transfected with 0.3 μg of nuclear actin-chromobody-tag-GFP expressing vector (Chromotek, acg-n; EMD Millipore, FuGENE 6 transfection reagent; Promega) After six hours, cells were treated with 1 μg/ml NCS for 60 min at 37 °C. Cells were washed twice with PBS before adding media containing DMSO. After 3 hours, images were acquired on an A1RMP confocal microscope using equipment and settings described above. Cells with actin-cb foci were classified using the following criteria: actin-cb foci exhibited movement and frequent turnover, were round in morphology and often appeared to have fuzzy “tails”. Cells with actin-cb rods were classified using the following criteria: actin-cb rods exhibited limited movement, no turnover, were very bright, and were filamentous in morphology. Foci movements were examined by collecting z series at 0.4 μm intervals throughout the entire nucleus every 30 seconds for 10 min. Following initial image acquisition, cells were subsequently treated with 100 μM CK-666. Images of actin-cb foci were subsequently acquired 1 hour post CK-666 treatment. Actin-cb foci were scored as the number of discrete punctate structures appearing per nucleus, as detected by the Icy Spot Detector plug-in. For experiments assessing localization of actin cb foci with RPA, U2OS cells were co-transfected with actin-chromobody-tag-GFP and RPA32-NLS-mCherry (a kind gift of Jiri Lukas) for 16 hr prior to NCS treatment. After 12 hours, foci movements were examined by collecting z series at 0.4 μm intervals throughout the entire nucleus every 5 min for 2.5 h.\nFor experiments in fixed cells, U2OS cells were cultured on 8-well chamber slides (Thermo Fisher Scientific), subjected to transfection and drug treatments as described above, and processed for IHC as described above. Cells were stained with primary antibodies against Rad51 (Santa Cruz: sc-8349, 1/50) and GFP (Abcam: ab13970, 1/100) overnight. Secondary antibodies were Alexa 488 conjugated goat anti-chicken Ig (Invitrogen: A11039, 1/1000) and Alexa 594 conjugated goat anti-mouse Ig (Thermo Fisher Scientific, A-11005, 1/10000). Actin chromobody-expressing cells were selected for analysis by screening for actin-cb foci. Images were acquired on an A1RMP confocal microscope equipped with a 60×/1.49 Apo-TIRF oil-immersion objective lens. Z series at 0.4 μm intervals throughout the entire nucleus were acquired. For co-localization analysis, actin-cb and Rad51 foci were detected in Z using the spot detector plug-in available on Icy Bioimaging Software platform. The co-localization of Rad51 and actin-cb spots was assessed using the Co-localization Studio plug-in, object-based methodology. Here, co-localization was defined as the number of Rad51 spots that directly overlapped with or touched actin-cb foci (within a 0.7 μm radius).\n\nDSB foci movement analysis\nDSB movement analysis followed the approach recently described 9. Nikon NIS Elements data files were processed in ImageJ 38. Green and red channels were split and a maximum-intensity projection of each z stack was generated. T stacks were subsequently aligned using the StackReg plugin 25 to correct for cell movements over the duration of the experiment. Cells that underwent large-scale nuclear deformations or expansions were discarded. The TrackMate plug in for ImageJ 39 was used to perform single-particle tracking of DSB foci over a 100-min interval and monitor foci intensity. Foci trajectories were subsequently exported to Matlab and analyzed using the class @msdanalyzer 44. The mean-square displacement (MSD) of DNA damage foci plots the average squared distance foci travel at increasing time intervals, whereas the diffusion coefficient D(t) is approximated through the linear-weighted fit of the initial mean MSD curve 40. Mean-Square Displacement curves for DSB foci in a given cell were computed using the formula MSD = \u003c(x(t+Δt)-x(t))2\u003e, where × reflects focus position and t is the time in minutes. For Rad52 foci, the weighted mean of all the MSD curves acquired from \u003e 2000 tracks in 3 independent experiments is shown. The error bars for each point on the weighted mean of the MSD curve represent the weighted standard error of the mean over all MSD curves. The diffusion coefficient D(t) was estimated from the linear fit of the first 20% of each MSD curve. Calculation of the time-dependence coefficient α was determined using Matlab via log-log fitting of the power law MSD(t) = Γ × tα. Cumulative distance (CD) traveled by foci over 100 min was calculated relative to their starting position using the formula: D(i)=sqrt(((X(i)-X(i-1))2 + ((Y(i)-Y(i-1))2))) where X and Y refer to the x and y coordinates of the focus at time i.\n\nDSB foci clustering analysis\nFor analysis of DSB foci clustering during live cell imaging, a clustering event was defined as complete co-localization of ≥ 2 foci over ≥ 3 consecutive frames (15 min) over a 100 min interval. For actin-cb clustering during live cell imaging, a clustering event was defined as complete co-localization of ≥ 2 foci over ≥ 3 consecutive frames (3 min) over a 20 min interval.\n\nQuantification of RPA association to chromatin by flow cytometry\nDetection of chromatin-bound RPA in S-phase cells followed recently published protocols with the following modifications: 2 × 106 cells were treated with DMSO or 1 μg/ml camptothecin (CPT) for 24 hours. Cells were subsequently washed once with cold PBS, then incubated in cold CSK buffer + 1% Triton-X for 5 min. Cells were then washed with PBS and fixed in 0.5% PFA for 15 min. Cells were subsequently washed twice in BD Perm/Wash buffer (Becton-Dickinson: BD554723) and stained with RPA2/RPA32 antibody (Abcam: ab2175, 1:50) overnight. Cells were then washed with PBS and incubated in secondary antibody (Abcam: ab150113, 1/50 or Alexa 647 conjugated donkey anti-mouse Ig, Invitrogen: A31571, 1/50) for 1 h. Cells were then washed and stained with propidium iodide in the presence of RNAse A overnight. The percentage of S-phase cells as identified by PI staining was quantified by flow cytometry. The gate for RPA-positive cells was established using a negative control sample that was stained with mouse IgG following extraction. RPA-positive cells following DMSO and CPT treatment were subsequently quantified by flow cytometry. A minimum of 10000 cells were counted per biological replicate.\n\nClonogenic and cell survival assays\nU2OS cells were seeded onto 10 cm dishes (1000 cells per dish) overnight. Cells were subsequently treated with 0, 100, 200, or 300 μM CK-666 in the presence of DMSO, camptothecin (CPT, 10 nM), or aphidocolin (APH, 400 nM) for 12 hours. Cells were then washed with PBS and incubated in fresh media 10–14 days. Cells were subsequently washed with PBS, fixed for 10 min with 100% methanol, and stained with 0.1% crystal violet for 10 min. For olaparib studies, cells were treated with 0, 10, or 20 μM CK-666 in the presence of DMSO or 50 nM Olaparib for 14 days, and fixed and stained was above. Each experiment was performed in triplicate.\nFor colony quantification, images of dishes were converted to Grayscale and processed with Icy Software (Version 1.8.6.0; Institut Pasteur, Quantitative Image Analysis Unit; http://www.icy.bioimageanalysis.org). Regions of interest were drawn around individual dishes and colonies were counted using the Spot Detector plug-in (Channel 0). Counts from two biological replicates were normalized for plating efficiency.\nFor Annexin V/propidium iodide survival assay, 1–5 × 105 cells were incubated in DMSO or 1 μg/ml CPT for 0, 12, or 24 hours. Cells were then washed in 1X binding buffer (Thermo Fisher Scientific: 00-0055) and stained with APC-conjugated Annexin V (Thermo Fisher Scientific: 17-8007) for 10 min. Cells were then washed in 1X binding buffer and stained in propidium iodide (Thermo Fisher Scientific: 00-6990). Percent viability following CPT treatment was assessed by measuring the percentage of APC-Annexin V-negative and propidium iodide-negative cells by flow cytometry. A minimum of 10,000 cells counted per condition.\n\nQuantification and Statistical Analysis\nStatistical parameters are reported in the Figures and the Figure Legends. Two-tailed t-tests and Ordinary one-way ANOVAs were used for comparisons of means of normally distributed data and two-tailed Mann-Whitney U tests were used for comparison of non-normally distributed data. Statistical analyses were performed using Prism 7.0.\n\nResearch Animals\nThe use of Xenopus laevis (African clawed frog) in this research proposal is approved (protocol AAAK0551) by the Institutional Animal Care and Use Committee (IACUC) of Columbia University Medical School. All procedures comply with Public Health Service’s policies for the humane care and use of laboratory animals.\n\nData Availability\nThe data that support the findings of this study are available from the corresponding author upon reasonable request. The authors declare that the data supporting the findings of this study are available within the paper [and its Supplementary Information 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