Using budding yeast, S cerevisiae, we show that Arp2/3 actin branching complex has an evolutionarily conserved role in promoting chromosome mobility of double-strand breaks (DSBs). Disrupting Arp2/3 activity after DSB induction, by drug treatment with CK-666 or auxin-induced degron depletion of nucleation promoting factors Las17WASP or the type-1 myosins (Myo3 and Myo5), markedly reduced the radius of confinement of DSBs. Arp2/3 was found to be required to initiate and maintain 5’to 3’ resection of DSB ends. Conversely, inhibiting Exo1 and Dna2- dependent long-range resection or deleting FUN30 impaired the mobility of the DSB, while overexpression of Exo1 suppressed resection inhibition by CK-666. Inactivation of Arp2/3 before DSB induction triggered a shortened checkpoint arrest through the Tel1ATM/Mre11 (TM) checkpoint, while inactivation of Arp2/3 after DSB induction caused cells to escape arrest prematurely. Shortened checkpoint arrest correlated with a reduction in repair by interchromosomal homologous recombination. These results suggest that Arp2/3 regulation plays an unanticipated role in the regulation of processing of DSB ends that is correlated with an increase in DSB mobility and DSB repair.
Article
Arp2/3 and Type-I myosins control chromosome mobility and end-resection at double-strand breaks in S. cerevisiae
https://doi.org/10.21203/rs.3.rs-5125308/v1
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double-strand break (DSB) mobility
DSB repair
Arp2/3
actin
myosin
Las17WASP
homology search
DSB end resection
Repair of double-strand breaks (DSBs) is required to maintain genome integrity. Homologous recombination (HR) requires that the ends of the DSB find a homologous donor sequence to use as a template for repair (reviewed in [1]). In budding yeast, a DSB causes an increase in chromatin mobility near a DSB break site [2, 3], which is thought to facilitate the Rad51-mediated search for homology by increasing the volume that the DSB ends explore within the nucleus, defined as the radius of confinement (Rc) [4]. While processes such as the activation of the Mec1ATR-dependent DNA damage checkpoint, the phosphorylation of histone H2A, and recruitment of repair proteins to the break have been shown to be required for DSB-induced chromatin mobility [2, 3], there is still no mechanism that explains how a DSB causes increased chromatin mobility or how changes in mobility affect DSB repair (reviewed in [4, 5]).
Several studies have suggested that actin branching is directly involved in repairing double-strand breaks. Recently it has been shown in mammalian cells and Drosophila that inactivation of the actin nucleator Arp2/3 by the drug CK-666 lowers DSB mobility [6, 7]. Treatment of Drosophila cells with CK-666 or RNAi treatment of nuclear myosin proteins impaired the directed movement of damaged DNA in heterochromatin to the nuclear periphery [6]. In addition, CK-666 caused an apparent reduction in the amount of single-stranded DNA (ssDNA) formed at DSB ends [7]. A proteomics study in Xenopus egg extracts has shown that all seven-subunits of Arp2/3 and actin are enriched at DSBs [7].
The branching of polymerized actin is dependent on the Arp2/3 complex and a set of nucleation promoting factors (NPF) (reviewed by [8]. The Arp2/3 actin nucleator Wiskott-Aldrich syndrome protein (WASP) [9, 10] has been shown to affect the loading of the single-strand binding protein RPA onto single-stranded DNA in mammalian cells [11] and mutations in Las17, the budding yeast homologue for WASP, was shown to have hypersensitivity to the DNA damaging agents hydroxyurea (HU) and methyl methanesulfonate (MMS) [11]. Las17 mutants accumulated more Rad52 foci when exposed to HU or MMS, suggesting a deficiency in repair by HR [11].
Here we have investigated the role of Arp2/3 and its associated NPFs in S. cerevisiae in more detail. In budding yeast, Arp2/3 and the NPFs Las17 and the type-I myosins (Myo3 and Myo5) (reviewed in [12]) are primarily found in cortical actin patches and are required for clathrin-mediated endocytosis (CME) [8]. These factors are well-studied in endocytosis [12], but their roles in other cellular processes remain poorly defined.
Here we have used Ddc2-GFP to directly label DSBs [13] and mean-square displacement (MSD) analysis (reviewed in [14]) to track and measure changes in mobility of an irreparable site-specific DSB at the MAT locus on chromosome III. We found that blocking Arp2/3 activity, either through the drug CK-666 or through a deletion/mutation of NPFs, lowered DSB mobility. Moreover, 5’ to 3’ resection was reduced when Arp2/3 was inactivated after DSB formation. When Arp2/3 activity is blocked before DSB induction, the initiation of resection was severely impaired. Additionally, we show that altering the rate of resection, through deleting the chromatin remodeler Fun30 or inactivating the Exo1 and Dna2 nucleases responsible for long-range resection, lowered DSB mobility. Altogether our study shows that Arp2/3 has an evolutionarily conserved role in regulating both DSB mobility and DSB end-resection, and that directly impairing the rate of resection prevents the increased motion of DSB ends.
Labeling and tracking the mobility of double-stranded breaks
To generate, track, and quantify the mobility of a single-irreparable DSB we modified the well-characterized strain JKM179, in which a single site-specific cut in the MAT locus on chromosome III is induced by a galactose-regulated HO-endonuclease gene (Gal-HO) [15]. To visualize and track the DSB, we monitored the DNA damage checkpoint protein Ddc2 (Ddc2-GFP) which we have previously shown forms a damage-dependent GFP focus in ~ 80% of cells 3 h after adding galactose [13]. An mCherry-tagged spindle pole body protein Spc42 (Spc42-mCherry) was used as a fiducial marker to account for random nuclear motion. Cutting by Gal-HO is highly efficient with nearly 90% of cells being cleaved 1 h after galactose addition [15–17]. In these strains the homologous HML and HMR donors are deleted, thus preventing repair of MAT by HR. When HO is continually expressed, repair of the DSB by altering the cleavage site via nonhomologous end-joining occurs in only 0.2% of cells [18]. We measured DSB mobility 3 h after adding galactose and quantified the mobility of the DSB using mean-squared displacement (MSD) analysis (see Methods for details) (Fig. 1A-B).
Blocking Arp2/3 activity lowers DSB mobility
To test whether Arp2/3 activity has a conserved role in DSB mobility in budding yeast, we added 10µM CK-666 to block Arp2/3 activity 20 min before imaging (160 min after adding galactose) (Fig. 1C). Addition of CK-666 significantly lowered the radius of confinement (Rc) of the DSB from 0.92 µm to 0.69 µm (Fig. 1B and Table S1). Addition of CK-666 did not affect the mobility of the spindle pole bodies relative to the bud neck (Figure S1A-B, Table S1), suggesting that the changes seen in DSB mobility when cells are treated with CK-666 were due to the changes in the behavior of the DSB.
Since Arp2/3 is primarily known as a component of endocytosis in budding yeast, we asked whether the change in DSB mobility was due to impaired endocytosis. Deletion of SLA2, a key component of endocytosis [19], did not affect the behavior of the DSB (Fig. 1D-E and Table S1). This result suggests that endocytosis is not required for DSB mobility and that the role of Arp2/3 in DSB mobility is independent of its role in endocytosis.
Arp2/3 affects changes in local and global chromatin mobility in response to a DSB
While the direct labeling of DSBs with Ddc2-GFP is suitable for tracking the mobility of DSBs, it cannot be used to establish a basal level of chromatin mobility in the absence of a DSB. To test whether Arp2/3 is required for increasing chromatin mobility near a DSB, we induced an irreparable DSB at MAT in a derivative of strain JKM179 that expresses the GFP-LacI protein that binds to a lacO array inserted 4.4 kb away from the MAT locus [20] (Fig. 2A). As previously reported [20], we found that inducing a DSB caused an increase in local chromatin mobility near the DSB site: RC 0.63 µm (uncut) and 1.1 µm (cut) (Fig. 1B, G and Table S1). CK-666 did not significantly affect basal levels of chromatin mobility: Rc uncut (0.63 µm) and uncut + CK-666 (0.54 µm) (Fig. 1B, G and Table S1); however, treatment with CK-666 either 20 min before (Fig. 2C) or 160 min after (Fig. 2E) DSB induction resulted in no increase in DSB mobility compared with the uncut condition, monitored at 180 min (Fig. 2D, F-G and Table S1). Thus, CK-666 impairs DSB-induced chromatin mobility.
Nucleation promoting factors are required DSB-induced increase in mobility
In humans, Arp2/3 requires the NPF WASP to nucleate new branches of actin [9, 10]; Las17 is the budding yeast homologue of WASP (Fig. 3A) [21]. To test whether Las17 was required for DSB mobility, we added an auxin-inducible degron (AID) to the C-terminus of Las17 (Las17-AID) [22, 23]. Adding 1mM auxin (IAA) caused degradation of Lad17-AID within 1h (Fig. 3B). The AID-tag did not affect the MSD curve of Las17-AID, but when IAA was added 2 h after Gal-HO induction, the mobility of the DSB measured at 3 h dropped: Rc WT (0.83 µm), Las17-AID (0.77 µm), and Las17-AID + IAA (0.49 µm) (Fig. 3C-D, Table S1).
To further characterize the role of Las17, we deleted functional domains that are responsible for Arp2/3 activation. Deletion of the acidic patch (CA) domain (las17-CA∆), which directly binds to Arp2/3 to activate Arp2/3 [24], did not result in a significant change in the mobility of the DSB: Rc WT (0.91 µm) and las17-CA∆ (0.90 µm) (Fig. 3E-F, Table S1). However, additional deletion of the WH2 domain, which brings monomeric actin to Arp2/3, along with the CA domain (las17-WH2-CAΔ) lowered the mobility of the DSBs: RC las17-WH2-CAΔ (0.53 µm) (Fig. 3E-F, Table S1). This suggests that Las17 in its role as an Arp2/3 actin nucleator is required for DSB mobility.
The type-I myosins Myo3 and Myo5 play a role in DSB mobility
Type-I myosins Myo3 and Myo5 promote actin nucleation by Arp2/3 during endocytosis in budding yeast [25] and depletion of Myo1a and Myo1b in Drosophilia increases sensitivity to ionizing radiation [6]. We asked if Myo3 or Myo5 have a role in DSB mobility in budding yeast. We found that a single deletion of MYO3 or MYO5 was sufficient to lower the mobility of the DSB: Rc WT (0.83 µm), myo3∆ (0.60 µm), and myo5∆ (0.58 µm) (Fig. 4A, C and Table S1). The effect of deleting either type-I myosin was different from what is seen in endocytosis, where Myo3 and Myo5 are functionally redundant [26]. We then asked whether a second copy of one type-I myosin, added to a deletion of the other, would restore DSB mobility. We integrated a second copy of MYO5 at URA3 on chromosome V, under control of its own promoter, in a myo3Δ background and found that there was no significant difference in the Rc between the WT (0.83 µm) and the myo3Δ Myo5 (+ Myo5) (0.80 µm) strains. (Fig. 4B-C, Table S1).
To determine which functional domains of Myo5 are required for DSB mobility, we integrated a series of MYO5 domain deletion plasmids with their endogenous promotors [25] into a myo5D strain. In the presence of Myo3, we found that there was no significant difference in the RC between the wildtype strain and a deletion of the Myo5 motor-domain, TH1, or TH2 domains: WT (0.89 µm), myo5-motorΔ (0.86µm), myo5-TH1Δ (0.85 µm), and myo5-TH2Δ (0.96 µm) (Figure S2A-C, E and Table S1). However, a deletion of the SH3 domain (myo5-SH3Δ) did not restore the mobility of the DSBs back to wildtype levels; RC WT (0.89 µm) and myo5-SH3Δ (0.62 µm) (Figure S2D-E, Table S1).
Because a double deletion of MYO3 and MYO5 is synthetically lethal in most backgrounds [27, 28], we added an AID tag to Myo5 (Myo5-AID) in a myo3D strain. In a MYO3 strain, the addition of the AID tag to Myo5 did not affect the mobility of the DSB and adding IAA 2 h after DSB induction reduced mobility, similar to the MYO5 deletion. (Figure S3, Table S1). Unexpectedly, in the myo3∆ Myo5-AID strain, treatment with IAA increased the mobility of the DSB to near WT levels: Rc WT (0.81 µm), myo3∆ Myo5-AID (0.6 µm), and myo3∆ Myo5-AID + IAA (0. 81 µm) (Fig. 4D-E, Table S1).
Since Las17 has a WH2 domain and acidic (CA) patch, it is possible that the increase in DSB mobility when both Myo3 and Myo5 are absent could be due to the Arp2/3 actin nucleation by Las17. To test this, we deleted the WH2 and CA domains of Las17 (las17-WH2-CAΔ) in a myo3Δ Myo5-AID background and measured the mobility of the DSB ± IAA as described above. We found that the las17 mutant prevented the restoration of Rc to WT levels that was seen when both Myo3 and Myo5 are inactivated: Rc WT (0.99 µm), myo3Δ Myo5-AID las17-WH2-CAΔ (0.57 µm), and myo3Δ Myo5-AID las17-WH2-CAΔ + IAA (0.58 µm) (Figure S4, Table S1). Together these results suggest that Myo3 and Myo5 in their role as Arp2/3 actin nucleators play a role in DSB mobility and Las17 can compensate for the loss of both Myo3 and Myo5 to maintain near WT levels of DSB mobility.
Arp2/3 and myosins are required for damage-dependent focus formation with Ddc2-GFP or Rad51-GFP
Ddc2-GFP and Rad51-GFP form damage-dependent foci in ≥ 80% of cells 3 h after DSB induction [13]. However, when Arp2/3 activity was blocked by CK-666 before DSB induction, we observed that Ddc2-GFP and Rad51-GFP formed foci in fewer than 15% of cells (Figure S5A-B). Similar inhibition of the DSB-induced foci was found in a deletion of VRP1, or when IAA was added to Las17-AID or myo3Δ Myo5-AID cultures. Deletion of KU70 did not restore damage-dependent Ddc2-GFP foci formation in CK-666 treated cells (Figure S5C). This finding raised the possibility that blocking Arp2/3 might interfere with either Gal-HO cutting or with long-range resection. By using a pair of PCR primers that flank the HO-cut site in the MAT locus (Figure S5A) we showed that Gal-HO cutting was unaffected in Las17-AID or myo3∆ Myo5-AID mutants treated 1 h before DSB induction with IAA (Figure S5B-D).
Arp2/3 and type-I myosins are required for the initiation and maintenance of resection
Since Gal-HO cutting is normal, blocking Arp2/3 activity might interfere with 5’ to 3’ resection of the DSB. We first monitored the disappearance of GFP-LacI/lacO foci in a strain where the lacO array was inserted 4.4 kb away from the HO cleavage site at MAT (Fig. 2A). As resection erodes the lacO array, GFP-LacI will lose its binding sites and the GFP-LacI/lacO focus will vanish. Within 2 h after DSB induction there was a steady drop in the percentage of cells with a GFP-LacI focus (Fig. 5A). However, addition of CK-666 1 h before HO induction prevented the loss of the GFP-LacI focus. Resection in cells treated with CK-666 after 1 h after DSB induction was also significantly impaired.
We used a restriction enzyme-based qPCR resection assay to measure the generation of ssDNA around the HO-cut site itself [29, 30]. qPCR analysis was done using a series of primers flanking STY1 cut sites at different distances from the HO-cut site in the MAT locus (0.7 kb, 5 kb, and 10 kb) (Fig. 5B). As resection converts dsDNA to ssDNA, StyI cleavage sites are lost and qPCR threshold signal is detected.
We measured resection in Las17-AID, myo3∆, myo5∆, and myo3Δ Myo5-AID mutants. IAA was added either 1 h before or 2 h after adding galactose. Las17-AID without IAA had a similar resection profile as the wildtype; however, when IAA was added either before or after galactose, long range resection was severely impaired (Fig. 5C) suggesting that Las17 is required for the initiation and maintenance of resection.
Single deletions of MYO3 or MYO5 did not greatly affect resection (Fig. 5D). Resection in the myo3∆ Myo5-AID mutant without IAA was comparable to resection in the WT, as expected from the myo3∆ resection data (Fig. 5D-E); but when IAA was added to myo3∆ Myo5-AID 1 h before DSB induction, there was little to no resection up to 6 h after DSB induction (Fig. 5E). Finally, when IAA was added 2 h after DSB induction, resection in myo3D Myo5-AID appeared to be greatly inhibited within an hour of degrading Myo5 (Fig. 5E). Together these data suggests that Arp2/3 and type-I myosins have a role both in the initiation of resection and in its maintenance.
Changes in resection lower the mobility of DSBs
Since resection was affected by changes in Arp2/3 activity, we asked whether there was a correlation between the rate of resection and DSB mobility. Previously we and others have shown that a deletion of the chromatin remodeler FUN30 greatly reduced Exo1 and Sgs1/Dna2-dependent long-range resection [31–33]. Although resection through the 0.7 kb site was not affected in fun30∆, long-range resection past the 5 and 10 kb sites was markedly reduced (Fig. 6A). MSD analysis of a DSB showed the RC of fun30D (0.5 µm) was significantly lower than the WT (0.8 µm) (Fig. 6B-C, Table S1).
Long-range resection requires both the exonuclease Exo1 and the helicase/endonuclease DNA2-Top3-Rmi1-Sgs1 complex [34, 35]. Since the rate of resection is not affected by deleting either EXO1 or SGS1 alone [33], we blocked long range resection by deleting EXO1 and degrading Dna2-AID. There was no difference in DSB mobility between the wildtype and exo1D Dna2-AID, but when IAA was added 2 h after DSB induction the mobility of the DSB measured at 3 h was reduced: Rc WT (0.8 µm), exo1Δ DNA2-AID (0.8 µm), and exo1Δ DNA2-AID + IAA (0.5 µm). (Fig. 6D-E, Table S1).
To test whether increasing resection affected DSB mobility, we measured mobility in a strain with a second copy of EXO1 expressed under control of a galactose promoter (pGal:EXO1) at the LEU2 locus [36]. We found that there was no difference between the Rc in WT and when Exo1 was overexpressed (Fig. 6F, H and Table S1) even though the rate of resection was increased (Fig. 6I). However, pGal::EXO1 suppressed the reduction in Rc from CK-666 (Fig. 6F-H, Table S1), but did not rescue Ddc2-GFP focus formation in cells treated with CK-666 before DSB induction (Figure S5D). Taken together, continuous resection is required for the high mobility of DSBs and overexpression of Exo1 can overcome the reduced rate of resection caused by CK-666 but could not prevent the block to the initiation of resection.
Activation of the TM checkpoint in response to blocking Arp2/3 activity
When a DSB is detected, the DNA damage checkpoint (DDC) is activated to halt cell cycle progression and allow cells a chance to repair the DSB before proceeding through mitosis [37, 38]. In budding yeast, activation of the DDC is mainly controlled through the effector kinase Mec1ATR with a minor contribution by the Tel1ATM kinase [39–42]. Mec1 is recruited to DSBs by its binding partner Ddc2ATRIP, which binds to RPA loaded on ssDNA [43, 44]. Arrest by the DDC is typically monitored by cell morphology, as cells shift towards a large-budded G2/M state, and by Western blot analysis of phosphorylation of the Mec1/Tel1 target, Rad53 [45].
We hypothesized that preventing resection - by blocking Arp2/3 activity prior to HO induction - should interfere with cell cycle arrest through the DDC. Typically, a cell with a single irreparable DSB will arrest for 12–15 h before escaping arrest through a process known as adaptation [46]. Here we find that when Arp2/3 activity was blocked before DSB induction, either by drug treatment with CK-666 or degradation of Las17-AID with IAA (Fig. 7A), G2/M arrest was shortened to 4 h (Fig. 7D-G). Deletion of either Myo3 or Myo5 did not shorten checkpoint arrest (Fig. 7D-E).
While Mec1 is primarily responsible for checkpoint arrest by the DNA damage checkpoint, the Tel1-Mre11 (TM) checkpoint is an alternative checkpoint response in response to a DSB, notably in mec1D cells in which initial resection by the Mre11 complex is impaired [47]. We confirmed that the short checkpoint response we observed was attributable to the TM checkpoint, as tel1D and mre11D strains treated with CK-666 before DSB induction did not exhibit a checkpoint response (Fig. 7B-C). A mre11Δ Las17-AID double mutant treated with auxin also showed little or no G2/M arrest and western blot analysis showed that Rad53 was not phosphorylated (Fig. 7H-I).
Since degrading Las17 after DSB induction impaired further resection, we assayed whether Arp2/3 is required to maintain checkpoint arrest by adding CK-666 to our wildtype strain or IAA to Las17-AID 2 h after adding galactose and monitored for changes in cell morphology (Fig. 7A). We found that treatment with CK-666 or degrading Las17-AID resulted in cells escaping G2/M arrest early despite Rad53 remaining hyperphosphorylated (Fig. 7J-K). These results show that the shortened DSB-induced checkpoint arrest was dependent on the TM checkpoint which was activated when resection was impaired by inhibiting Arp2/3 or its associated factors.
DSB Mobility and Repair by Gene Conversion
If DSB-induced chromatin mobility facilitates homology search, then lowering the mobility of DSBs might impact the repair efficiency carried out by gene conversion (GC). To study how changes in chromatin mobility affect the efficiency of DSB repair, we modified the well-characterized strain YJK17 which has an ectopic, mutated copy of MAT (MATa-inc) that cannot be cut by the HO-endonuclease and which serves as a donor to repair the cleavage at MATα by interchromosomal gene conversion (Fig. 8A) [33, 48]. With this strain we could generate and monitor the repair of a single DSB event.
Deletion of MYO3, MYO5, or MRE11 did not impact repair; however Las17-AID showed a significant decrease in viability from YP-Gal (79%) and YP-Gal + IAA (46%) plates (Fig. 8B). An mre11Δ Las17-AID double mutant showed an even more significant drop in viability (65% on YP-Gal and 10% on YP-Gal + IAA) (Fig. 8B). Las17-AID survivors from the YP-Gal and YP-Gal + IAA plates grew normally on YPD and YP-Gal plates (Figure S7) as expected.
To further test the effects of mobility-deficient mutants on GC, we deleted MYO5 and modified Las17 in a series of strains we had previously characterized with repair efficiencies ranging from 46–9% [49]. The differences in repair efficiencies reflect the contact probability between the DSB site and the LEU2 donor sequence. As with YJK17, we found that myo5D did not affect repair efficiency (Fig. 8D) but Las17-AID + IAA showed a significant decrease in the percentage of survivors in all 4 strains (Fig. 8E).
Since degradation of Las17 showed reduced resection, we wanted to know how this would affect repair by single-strand annealing (SSA) using the previously characterized strain YMV2, which has an HO cut-site inserted in LEU2 on chromosome 3 (leu2-cs) and a 1.3 kb fragment of the 3’ end of LEU2 (U2) inserted 30 kb upstream of leu2-cs (Fig. 8F) [50]. The DSB can be repaired both by Rad51-dependent break-induced replication and by Rad51-independent SSA [33]. Degradation of Las17 reduced the survival rate from 86–63% (Fig. 8G). Deletion of RAD51 did not affect the repair efficiency, 85% (Fig. 8G). Yet when Las17 was degraded in the rad51Δ background, repair efficiency dropped to 36% (Fig. 8G). This suggests that Las17 and Arp2/3 have a role in repair by SSA and have much less effect on BIR, where much less resection is required.
When a cell commits to repair a DSB through HR, there is an increase in chromatin mobility near the DSB [2, 3, 20]. While it is thought that increased chromatin mobility near a DSB facilitates homology search, it is unclear what role DSB-induced chromatin mobility has in DSB repair by HR. In this study we investigated the role of the Arp2/3 actin nucleator in DSB mobility and repair by HR.
Here we show that Arp2/3, previously shown to be required for DSB mobility in Drosophila and mammalian cells [6, 7], has a conserved role in DSB mobility in budding yeast and that this process is separate from Arp2/3’s previously well-characterized function in endocytosis [51]. It appears that Arp2/3 is required at 2 different steps during homology-based repair: first, for the initiation, and then for the maintenance of long-range resection and, perhaps consequently, for the increased mobility of the DSB during homology searching.
Long-range resection was not seen when Arp2/3 activity was blocked before DSB-induction, either through treatment with the Arp2/3 inhibitor CK-666, deletion of VRP1, degradation of Las17WASP, or when both type-I myosins, Myo3 and Myo5, were not present. Unlike a SAE2 deletion mutant, which has been reported to have delayed DSB-induced chromatin mobility [3] and delayed resection [52], we saw little or no resection up to 6 h after inducing an HO break when Arp2/3 was blocked prior to DSB induction.
In previous studies, deletion of YKU70/80 allowed Exo1-dependent resection of DSB ends in G1-arrested cells [17] and overexpression of YKU70/80 interfered with Mre11 association with DSB ends and acted as a barrier for resection [53]. Here we show that a deleting YKU70 did not overcome the CK-666 block to forming Ddc2-GFP foci, suggesting that Arp2/3 does not affect the residence of yKu70/80 at DSB ends.
The connection between increased chromatin mobility and end-resection is complex. As in mammalian cells, slowing down resection reduces the mobility of DSB ends, though whether the same factors are involved remains unclear. In mammals, inhibition of the Mre11 by mirin reduced mobility [7], while in budding yeast inhibition of the Mre11 complex by deletion of SAE2 only delays resection and the increase in Rc [3, 52]. Here we found that Rc was reduced by impairing long-range resection, either by deleting the chromatin remodeler FUN30 or by inactivation of both Exo1 and Dna2. Thus, a defect in long-range end-resection is sufficient to impair damage-induced chromatin mobility. We also found that EXO1 overexpression suppressed the reduction in Rc caused by adding CK-666, but overexpression of EXO1 was not able to bypass the block in initiating resection when Arp2/3 was inhibited prior to DSB induction. We were unable to test DSB mobility in a triple deletion mutant of the primary resection proteins because an mre11-nd (nuclease dead) exo1 sgs1Δ deletion is inviable [35]; but it would appear that inactivating Arp2/3 in an mre11D background eliminates both long- and short-range resection and thus recombination. These findings confirm and extend the analysis of the role of Arp2/3 in mammalian cells, where treatment with CK-666 impaired both mobility and end-resection in human cells [7].
Detection of a DSB triggers cell cycle arrest through the DNA damage checkpoint (DDC) for 12–15 h [46]. We found that when Apr2/3 was blocked, cells experienced a 4 h checkpoint which was dependent on the Tel1-Mre11 (TM) checkpoint which arrests cells in response to unresected DSB ends [54, 55]. Disruption of both the TM checkpoint and Arp2/3 activity prevented cells from entering DSB-induced checkpoint arrest as evidenced by the lack of cells accumulating in G2/M arrest and the lack of phosphorylation of Rad53. Arp2/3 was also required to maintain checkpoint arrest as cells began to escape arrest, despite continued Rad53 phosphorylation. Impaired Mec1-mediated checkpoint arrest in budding yeast is consistent with the lower ATR signaling reported in WASP-deficient human cells [11].
Of the known Arp2/3 actin nucleation promoting factors in budding yeast, we focused on Las17WASP and the type-I myosins Myo3 and Myo5. In humans WASP has been shown to bind to RPA and is implicated in loading RPA onto ssDNA in response to a DSB as mutations in WASP were shown to have less RPA-bound DNA when exposed to the topoisomerase I inhibitor camptothecin (CPT), which causes replication fork collapse [7]. We show that the highly conserved C-terminal region across WASP orthologs (WH2 and CA domains) of Las17 is required for DSB mobility. Human WASP binds to RPA, through this highly conserved region and mediates RPA binding to ssDNA [11]. Impaired RPA binding to ssDNA would prevent Ddc2ATRIP-mediated Mec1 activation of the DDC.
Budding yeast Myo3 and Myo5 are functionally redundant in endocytosis and deletion of either Myo3 or Myo5 did not affect resection, checkpoint arrest, or repair by gene conversion. However, a deletion of either myosin protein lowered DSB mobility and the insertion of a second copy of Myo5 with its endogenous promotor into a myo3 deletion mutant restored DSB mobility to wildtype levels. Through domain deletion mutants of MYO5, we found that the SH3 domain of Myo5, which mediates interactions with Vrp1 [56, 57], is required for wildtype levels of DSB mobility. Since SH3 domains mediate a wide range of protein-protein interactions, the loss of either type-I myosin might result in an imbalance of interactions required for DSB mobility and the deletion of MYO3 and MYO5 might restore that balance. The loss of both type-I myosins after DSB induction brought DSB mobility back to wildtype levels in a Las17-dependent manner. Deletion of VRP1 impaired the initiation of resection. Since Vrp1 brings Myo3 and Myo5 to Arp2/3 [56], the deletion of VRP1 would likely be similar to when both Myo3 and Myo5 are degraded.
Changes in DSB mobility did not necessarily affect overall DSB repair efficiency through HR. While the degradation of Las17 negatively affected the repair efficiency of DSBs through GC and SSA, deletion of either MYO3 or MYO5 did not affect the repair efficiency of DSBs, even when the donor location was moved to unfavorable locations. This difference in repair efficiencies among these mobility deficient mutants might be due to the length of G2/M arrest imposed by the DDC. Since myo3Δ and myo5Δ mutants did not affect the length of checkpoint arrest, these mutants had the same amount of time to repair the break as the wildtype. The shortened checkpoint arrest when Las17 was degraded might have triggered an early adaptation response.
By using single particle tracking and MSD analysis of a single DSB in the MAT locus on chromosome III in budding yeast, we were able to show that Arp2/3 has a conserved role the DNA damage response and plays an important part in the initiation and facilitation of the resection of DSB ends. The lower DSB mobility that we observe when Arp2/3 activity is blocked, either through drug treatment or degradation of Las17WASP, is likely linked to a lower rate of resection, as DSBs in other resection mutants, such as a FUN30 deletion or an exo1 dna2 double deletion, are less mobile than their wildtype counterparts. The rate of resection is not the only determinant of DSB mobility as a deletion of either Myo3 or Myo5 is sufficient to lower DSB mobility but did not affect the rate of resection. Preventing resection triggered a shortened checkpoint arrest through the TM checkpoint and stopping continued resection allowed cells to escape checkpoint arrest early.
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Methods Details
Strain and plasmid construction
All strains are derived from JKM179 [15] which is 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 [13]. Ddc2-GGSGGSRIPGLIN-eGFP and Rad51-GGSGGSRIPGLIN-eGFP are referred to as Ddc2-GFP and Rad51-GFP respectively in this study. All AID-tagged mutants strains were derived from a modified version of JKM179 expressing osTIR1 at URA3 created by cutting pNHK53 [23] with StuI and integrating into the genome. For degron tagged derivatives, PCR products were generated with mixed oligoes with homology to the C-terminal end and plasmid pJH2892 or pJH2899 [22] to create an 9xMyc-AID (AID) inserts with the KAN or NAT marker. Myo5-domain deletion plasmids [25] 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 [58]. Las17 domain deletion mutants were created by CRISPR/Cas9 as previously described [59] with material listed in supplemental tables. Transformants were verified by PCR and western blot. Strains are listed in Table S3.
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 5ml YP-lactate for ~15 hours overnight at 30° C with agitation. The following day, 500-100ml 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 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 1mM 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 timepoint by centrifugation at 3000x for 3 minutes and prepared for microscopy, resection assays, and western blot analysis. Cells prepared for microscopy were washed 3 times with a Leu- media.
Plating assays
The efficiency of DSB repair by homologous recombination or single-strand annealing was determined as previously described for YJK17 [48]. Briefly, cells were selected from a single colony on YPD plates and grown overnight in 5ml 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 (1mM) and YPD in triplicate and incubated at 30OC until visible colonies formed. 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.
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 objectives, a Yokogawa CSU-W1 spinning-disk head, and an Andor iXon 897U EMCCD camera. Fluorophores were excited at 488nm (GFP) and 561nm (mCherry). Time-lapse series were acquired with 15 optical slices of 0.3µm thickness every 30 seconds for 20 minutes. Time-lapse image stacks were analyzed using a custom MATLAB program designed by the Bloom lab as previously described [60]. Coordinates of DSB and the SPB were tracked with Speckle Tracker, a custom MATLAB program [61, 62]. 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 (Rc) to an Excel spreadsheet [60]. In MATLAB spot positions were fitted to [µx, σx] = normfit (x – xmean) and [µy, σy] = normfit (y – ymean). The variance of the distribution of spot position was then calculated as σ2 = mean (σx2, σy2). The average squared deviation from the mean position is (∆r02) = (∆x02) + (∆y02). Using σ2 and (∆r02), we calculated Rc as 
Due to variations in day-to-day collection, wildtype data was only compared to data collected on the same day.
Statistical analysis
Statistical analysis for Rc comparison was done in PRISM using a t-test or one-way ANOVA analysis. For one-way ANOVA analysis, Dunnett test was used to correct for multiple comparisons. Wildtype comparisons were conducted with data collected from the same day.
Resection and cutting assays
Resection was measured by quantitative PCR (qPCR) analysis using a restriction enzyme digest as previously described [29]. Briefly, cells are grown in YEP-Lac as described above. 50ml of culture was harvested and DNA extracted using a DNA extraction kit (Masterpure Yeast DNA Purification Kit Cat# MPY80200). DNA was diluted to 10ng/µl. Sty1-HF (digest) or an equivalent amount of water (mock) in Cutsmart buffer at 37CO for 4 h. qPCR samples were run in triplicate on a Biorad 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 Table S4 for primers. Resection was calculated measuring the fraction of cells that had passed the Sty1 restriction site (RS).
f is the fraction of cells where HO has been cleaved. ERS and EADH1 are the primer efficiencies for the primer pairs 0.7kb, 5kb, 10kb away from the HO-cut site and the ADH1 primers. ∆Cq (digest–mock) is the difference between quantification cycles between the mock and digested samples.
For cutting assays, DNA was collected as described above. DNA was diluted to 10ng/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.
TCA protein extraction
Protein extracts were prepared for western blot analysis by the standard TCA protocol described in [63]. Briefly, 15-10ml of harvested cells were incubated on ice in 1.5ml microcentrifuge tubes with 20% TCA for 20 minutes. Cells were washed with acetone and the pellet was air dried. 200ul of MURBs buffer (50mM sodium phosphate, 25mM MES, 3M urea, 0.5% 2-mercaptoethanol, 1 mM sodium azide, and 1% SDS) was added to each sample allow with acid washed glass beads. Cells were lysed by mechanical shearing with glass beads for 2 minutes. Supernatant was collected by poking a hole in the bottom of the 1.5ml microcentrifuge tube and spun in a 15ml conical tube. Samples were boiled at 95C for 10 minutes.
Western blotting
Denatured protein samples prepared by TCA extraction were centrifuged at max speed for 1 minute and 8 – 20 µl of samples were loaded into a 10% or 8% SDS page gel. Proteins were separated by applying 90 V constant 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 1x 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 1x TBS-T and incubated with anti-mouse or anti-rabbit HRP secondary antibody for 1 h at room temperature. After washing 3 times with 1x 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 BioRad ChemiDoc XR+ imager and prepared for publication using Image Lab software (BioRad) and Adobe Photoshop CC 2017.
Competing Interests
The authors declare no competing interests.
Acknowledgments
We are grateful to Kerry Bloom for training in MSD measurements, Bruce Goode for donation of CK-666 and expertise, Helle Ulrich for donations of the AID and TIR1 plasmids, David Drubin for donations of the Myo5 plasmids and Susan Gasser for donation of strains. Research was supported by NIH grant R35 GM127029. F.Y.Z, M.A., N.A., K.C., and K.B.F. were supported by NIH Genetics Training Grant TM32GM007122.
Author Contributions
Conceptualization and initial data collection was conducted by Felix Y. Zhou and James E. Haber. Data collection, imaging, primer design, data analysis, and strain making was conducted by Felix Y. Zhou, Marissa Ashton, Yiyang Jiang, Neha Arora, Kevin Clark, and Kate B. Fitzpatrick. The manuscript was authored and edited by Felix Y. Zhou and James E. Haber.
LEAD CONTACT AND MATERIALS AVAILABILITY
Further information and requests for reagents may be directed to, and will be fulfilled by the corresponding author, Dr. James E. Haber ([email protected]).
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