Using budding yeast, we show that the Arp2/3 actin branching complex has an evolutionarily conserved role in promoting chromosome mobility of double-strand breaks (DSBs). The radius of confinement of a broken chromosome is reduced by inhibiting Arp2/3 or by auxin-induced degron depletion of the nucleation-promoting factor Las17WASP or type-1 myosins. Arp2/3 and Las17 are required both to initiate and maintain 5 ' to 3' resection of DSB ends, whereas depleting Myo3 or Myo5 impairs broken chromosome motion without affecting resection. Conversely, inhibiting Exo1- and Dna2-dependent long-range resection reduces DSB mobility. Inactivating Arp2/3 before DSB induction leads to shortened checkpoint arrest, activating the Tel1ATM/Mre11 (TM) checkpoint. Shortened checkpoint arrest, but not reduced broken chromosome mobility per se, results in reduced interchromosomal homologous recombination. These results suggest that regulating the Arp2/3 complex plays a key role in the processing of DSB ends, which is correlated with an increase in DSB mobility and DSB repair.

Strain and plasmid construction

All strains are derived from JKM179 (17), a well-characterized strain with the HML and HMR donor domains deleted. Ddc2 and Rad51 were GFP-tagged with a 13 amino acid linker GGSGGSRIPGLIN-eGFP as previously described (15). Ddc2-GGSGGSRIPGLIN-eGFP and Rad51-GGSGGSRIPGLIN-eGFP are referred to as Ddc2-GFP and Rad51-GFP, respectively, in this study. All AID-tagged mutant strains were derived from a modified version of JKM179 expressing osTIR1 at URA3 created by cutting pNHK53 (28) with StuI and integrating it into the genome. For degron-tagged derivatives, PCR products were generated with mixed oligos with homology to the C-terminal ^nd and plasmid pJH2892 or pJH2899 (27) to create a 9xMyc-AID (AID) insert with the KAN or NAT marker. Myo5-domain deletion plasmids (30) were digested with BamHI/SacI to excise a linear fragment for genome integration. Deletion of ORFs, myo5 mutants, and AID tags were introduced with the one-step PCR homology cassette amplification and the standard yeast transformation method (70). The Las17 domain deletion mutants were created by CRISPR/Cas9 as previously described (71) with the material listed in the supplemental tables. Transformants were verified by PCR and Western blot. Strains are listed in Supplementary Data (5). Primers are listed in Supplementary Data 6. Plasmids are listed in Supplementary Data (7). Key Resources are listed in Supplementary Data (8).

Growth conditions

Strains containing degron fusions and galactose-inducible HO were cultured using standard procedures. Briefly, a single colony grown on a YPD plate was inoculated in 5 ml YP-lactate for 15 h overnight at 30 °C with agitation. The following day, 500–100 ml of YP-lactate was inoculated with the starter culture so that the cell density reached OD600~ = 0.5 the following day after overnight growth at 30 °C with agitation. Once cultures reached the appropriate density, HO expression was induced by the addition of 20% galactose to 2% vol/vol final concentration. For auxin treatment, indole-3-acetic acid (IAA) was resuspended in ethanol to a working concentration of 500 mM. For AID degradation, cultures were split at the indicated timepoints to add auxin. IAA was added to one culture to a final concentration of 1 mM either 1 h before or 2 h after adding galactose. The equivalent volume of 100% ethanol was added to the second culture. Cells were harvested at the indicated time point by centrifugation at 3000× for 3 min and prepared for microscopy, resection assays, and western blot analysis. Cells prepared for microscopy were washed 3 times with Leu-media.

Plating assays

The efficiency of DSB repair by homologous recombination or single-strand annealing was determined as previously described for YJK17(55). Briefly, cells were selected from a single colony on YPD plates and grown overnight in 5 ml of YEP-lactate. Cells were diluted to OD₆₀₀ = 0.2 and allowed to grow until OD₆₀₀ = 0.5–1.0. Approximately 100 cells from each culture were then placed on YEP-Gal (2% gal per volume) or YEP-Gal+IAA (1 mM) and YPD in triplicate and incubated at 30 °C until visible colonies formed. For plating experiments with CK-666, overnight YEP-lactate cultures were treated with CK-666 (100 µM) 20 min prior to being plated on YPD and YEP-Gal plates in triplicate, as previously described.

For plating assays with IAA and/or nocodazole, YP-Lac cultures were split and given 100% ethanol (control), IAA (1 mM), nocodazole (10 µg/ml)(72), or both IAA and nocodazole 1 h prior to galactose. Ethanol-treated cultures were directly plated on YPD and YP-Gal plates. Galactose (2%) was added to cultures treated with IAA and/or nocodazole and allowed to grow at 30 °C for 6 h before plating on YEP-Gal or YEP-Gal+IAA plates. Viability was calculated by dividing the number of colonies on YEP-Gal or YEP-Gal+IAA plates by the number of colonies on YPD plates. Viability in nocodazole-treated cells was calculated by dividing the percentage of survivors in nocodazole-treated cells by the percentage of survivors of wild-type cells treated with nocodazole.

Live cell microscopy and mean-squared displacement analysis

Live cell microscopy was performed using a spinning disc confocal microscope using Nikon Elements AR software on a Nikon Ni-E upright microscope equipped with a 100× (NA, 1.45) oil immersion objective, a Yokogawa CSU-W1 spinning-disk head, and an Andor iXon 897U EMCCD camera. Fluorophores were excited at 488 nm (GFP) and 561 nm (mCherry). Time-lapse series were acquired with 15 optical slices of 0.3 µm thickness every 30 s for 20 min. Time-lapse image stacks were analyzed using a custom MATLAB program designed by the Bloom lab as previously described (73). Coordinates of DSB and the SPB were tracked with Speckle Tracker, a custom MATLAB program (74), (75). A custom PERL script was used to convert the pixels to nanometers and subtract the distance of the SPB from the Ddc2-GFP coordinates to eliminate cell and nuclear motion, subtract the mean position of the corrected GFP coordinates, calculate the MSD of each time lapse, and export MSD coordinates and radius of confinement (R_c) to an Excel spreadsheet (73). In MATLAB, spot positions were fitted to [µ_x, σ_x] = normfit (x – x_mean) and [µ_y, σ_y] = normfit (y – y_mean). The variance of the distribution of spot position was then calculated as σ² = mean (σ_x², σ_y²). The average squared deviation from the mean position is (∆r₀²) = (∆x₀²) + (∆y₀²). Using σ² and (∆r₀²), we calculated R_c as 𝑅𝐶 =√54⁢(2⁢σ2+Δ⁢r20). Due to variations in day-to-day collection, wild-type data were only compared to data collected on the same day.

Statistics and reproducibility

Statistical analysis for Rc comparison was done in PRISM using a two-tailed t-test or one-way ANOVA analysis. For one-way ANOVA analysis, the Dunnett test was used to correct for multiple comparisons. Wild-type comparisons were conducted with data collected from the same day. MSD and R_C analyses were done from at least 10 cells (n ≥ 10) collected across at least 2 days to account for variations in microscopy conditions. Resection analysis was done from 3 biological replicates collected in triplicate. Changes in cell morphology were measured across 3 days, with >100 cells counted at each time point per day. Western blots to show degradation of AID-tagged proteins were run at least twice: first to validate the creation of the strain and second to measure degradation of AID-tagged proteins under experimental conditions.

Resection and cutting assays

Resection was measured by quantitative PCR (qPCR) analysis using a restriction enzyme digest as previously described (36). Briefly, cells are grown in YEP-Lac as described above. 50 ml of culture was harvested and DNA extracted using a DNA extraction kit (Masterpure Yeast DNA Purification Kit, Cat# MPY80200). DNA was diluted to 10 ng/µl. Sty1-HF (digest) or an equivalent amount of water (mock) in Cutsmart buffer at 37 °C for 4 h. qPCR samples were run in triplicate on a Bio-Rad CFX384 Real-Time System C1000 Touch Thermal Cycler qPCR machine using Bio-Rad CFX Maestro 1.1 Version 4.1.2433.1219. ADH1 was used as a control gene. See Supplementary Data (5) for primers. Resection was calculated by measuring the fraction of cells that had passed the Sty1 restriction site (RS).

𝑥=2(𝐸𝑅⁢𝑆Δ⁢𝐶𝑞⁢(𝑑⁢𝑖⁢𝑔⁡𝑒⁢𝑠⁢𝑡−𝑚⁢𝑜⁢𝑐⁢𝑘)𝐸𝐴⁢𝐷⁢𝐻⁡1Δ⁢𝐶𝑞⁢(𝑑⁢𝑖⁢𝑔⁡𝑒⁢𝑠⁢𝑡−𝑚⁢𝑜⁢𝑐⁢𝑘)+1)*𝑓

1

f is the fraction of cells where HO has been cleaved. E_RS and E_ADH1 are the primer efficiencies for the primer pairs 0.7 kb, 5 kb, and 10 kb away from the HO-cut site and the ADH1 primers. ∆C_q (digest–mock) is the difference between the quantification cycles between the mock and digested samples.

For cutting assays, DNA was collected as described above. DNA was diluted to 10 ng/µl and run using primers flanking the HO-cut site in the MAT locus on chromosome III. ADH1 was used as a control. Gal-HO cutting was measured by the fold increase with the following equation, using the 0 h timepoint as a control.

𝑓⁡𝑜⁢𝑙⁢𝑑𝑖⁢𝑛⁢𝑐⁢𝑟⁢𝑒⁢𝑎⁢𝑠⁢𝑒𝑡=2−(Δ⁢𝐶𝑡−Δ⁢𝐶𝑐⁢𝑜⁢𝑛⁢𝑡⁢𝑟⁢𝑜⁢𝑙⁢𝑎⁢𝑣⁢𝑒⁢𝑟⁢𝑎⁢𝑔⁡𝑒).

2

TCA protein extraction

Protein extracts were prepared for western blot analysis by the standard TCA protocol described in ref. (76). Briefly, 15–10 ml of harvested cells were incubated on ice in 1.5 ml microcentrifuge tubes with 20% TCA for 20 min. Cells were washed with acetone, and the pellet was air-dried. 200 µl of MURBs buffer (50 mM sodium phosphate, 25 mM MES, 3 M urea, 0.5% 2-mercaptoethanol, 1 mM sodium azide, and 1% SDS) was added to each sample, allowed to incubate with acid-washed glass beads. Cells were lysed by mechanical shearing with glass beads for 2 min. Supernatant was collected by poking a hole in the bottom of the 1.5 ml microcentrifuge tube and spun in a 15 ml conical tube. Samples were boiled at 95 °C for 10 min.

Western blotting

Denatured protein samples prepared by TCA extraction were centrifuged at max speed for 1 min, and 8–20 µl of the samples were loaded into a 10% or 8% SDS page gel. Proteins were separated by applying a constant 90 V until the 37 kDa marker reached the bottom of the gel. Gels were then transferred to an Immun-Blot PVDF using a wet transfer apparatus set to 100 V constant voltage for 1 h. The resulting membranes were blocked in 5% nonfat dry milk or OneBlock buffer (Genesee Scientific, 20-313) for 1 h at room temperature or overnight at 4 °C with gentle agitation. After washing 3 times with 1× TBS-T, blots were incubated with either mouse anti-Myc [9E11] (Abcam, ab56) to detect Tir1 and AID fusions, rabbit anti-Rad53 (Abcam, ab104232), or mouse anti-Pgk1 antibody (Abcam Cat# ab113687, RRID: AB_10861977) for 1 h at room temperature. Blots were then washed 3 times with 1× TBS-T and incubated with anti-mouse or anti-rabbit HRP secondary antibody for 1 h at room temperature. After washing 3 times with 1× TBS-T, ECL Prime was added to fully coat the blots and left to incubate for 5 min at room temperature with gentle agitation. Blots were imaged using a Bio-Rad ChemiDoc XR+ imager and prepared for publication using Image Lab software (Bio-Rad) and Adobe Photoshop CC 2017. Uncropped and unprocessed Western blots are provided in the Source Data file.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.