Fabiola Garcia Fernandez

Brenda Lemos

Yasmine Khalil

Renaud Batrin

Emmanuelle Fabre
email: emmanuelle-g.fabre@inserm.fr

F Lazzaro

V Sapountzi

M Granata

A Pellicioli

M Vaze

J E Haber

P Plevani

Chromosome structure due to phospho-mimetic H2A modulates DDR through increased chromatin mobility

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Eukaryotic cells have developed sophisticated machineries to respond to the multiple 29 stresses they are constantly confronted with. In the presence of DNA Double-Strand Breaks

The DDR activation by Mec1 ATM and Tel1 ATR is a critical first step in the response (Seeber et 117 al., 2013;Smith et al., 2018;Dion et al., 2012;Miné-Hattab and Rothstein, 2012). Mec1 ATM 118 and Tel1 ATR activation have diverse consequences, such as centromeric relaxation 119 (Strecker:2016jj;Lawrimore et al., 2017) and chromatin modifications (Herbert et al., 2017; 120 Hauer et al., 2017;Miné-Hattab et al., 2017). It has been proposed that the repulsive forces 121 of the negative charges due to H2A phosphorylation could render the chromosome stiffer 122 (Herbert et al., 2017), or that histone depletion could be the cause of a more expanded 123 chromatin (Hauer et al., 2017;[START_REF] Cheblal | DNA Damage-Induced Nucleosome Depletion Enhances Homology 790 Search Independently of Local Break Movement[END_REF], both chromatin alterations resulting in 124 the observed increase of chromosome mobility. It is remarkable that increased chromosome 125 mobility, as a response to genomic insults, is a conserved feature in metazoan genomes, 126 with regulations involving repair proteins 53BP1 or γ-H2AX, like in yeast (Ryu et al., 2015; 127 Clouaire et al., 2018;Lottersberger et al., 2015;Dimitrova et al., 2008;Schrank et al., 2018). To better understand how γ-H2A(X) affects different aspects of chromosome structure and 130 movement, we have created yeast strains in which both copies of the H2A encoding genes 131 have been mutated to S129E, which appears to be phosphomimetic (Eapen et al., 2012).

132

We find that H2A-S129E fully recapitulates the increased apparent stiffness of chromatin 133 observed after damage, resulting in increased intra-chromosomal distances and 134 chromosome dynamics. An increased repair by NHEJ and a decreased rate of translocation correlate with this change in chromatin. Surprisingly, we find that H2A-S129E rescues ∆rad9 136 survival deficiency after DNA damage, further supporting the notion that changes in 137 chromatin structure are a key contributor to DNA damage response. To study the consequences of mimicking H2A-S129 phosphorylation on chromosome 143 mobility, we created isogenic yeast strains in which both HTA1 and HTA2 were mutated to 144 H2A-S129E by CRISPR/Cas9-mediated gene editing (see Materials and Methods). We used 145 four strains (P1-P4) that carry fluorescently labeled green loci (LacI-GFP bound to an array 146 of LacO sites) at different regions of the right arm of chromosome IV (Figure 1A). As control, 147 we replaced S129 by an alanine to impede phosphorylation, using the same strategy (H2A-148 S129A). We first compared the growth of the H2A-S129 mutants to their wild-type 149 counterpart, in the presence or in the absence of DNA damage generated by the radio-150 mimetic drug Zeocin (Dion et al., 2012;Seeber et al., 2013;Herbert et al., 2017). In the 151 absence of Zeocin, growth rates of P1-P4 WT, H2A-S129E and H2A-S129A strains were 152 indistinguishable, suggesting no particular endogenous damage caused by these mutations.

153

WT strains exhibited a sensitivity to Zeocin (Figure 1A, Supplementary Figure 1A), as 154 previously shown (Seeber et al., 2013;Strecker et al., 2016;Krol et al., 2015). In the 155 presence of Zeocin, growth of H2A-S129E was similar to that of WT, whereas H2A-S129A 156 mutants showed increased sensitivity (Figure 1A, Supplementary Figure 1A). To precisely 157 measure the sensitivity to Zeocin of the mutant strains, we performed colony forming unit 158 (CFU) assays by calculating the ratio between colonies grown on media with and without 159 Zeocin. CFU ratios were close to 70% in P1-P4 WT cells (Figure 1B). Likewise, CFU ratios 160 in H2A-S129E mutant strains ranged between 81% ± 3.1 and 96 ± 3.2%, with no significant 161 difference from WT, except for the P4 strain, which showed a slightly higher survival (Downs 162 et al., 2000;Moore et al., 2007;Redon et al., 2003). In H2A-S129A strains, CFU ranged 163 from 47.5% ± 4.0 to 64.5% ± 7.2 confirming the enhanced sensitivity of these mutants to 164 Zeocin compared to WT, as previously shown (Downs et al., 2000;Moore et al., 2007; 165 Redon et al., 2003). The simplest interpretation for a similar survival to DNA damage 166 between H2A-S129E mutant and WT is that glutamic acid can replace the function normally 167 provided by S129 phosphorylation, a conclusion further supported by the analysis of the 168 H2A-S129A mutants.

169

We then explored chromosome mobility in H2A-S129E mutant cells by tracking the 170 fluorescently labeled green loci (Herbert et al., 2017). Green labels are located at 180 kb and the four Gr1-Gr4 loci by tracking hundreds of cells in each P1-P4 strain, by high-speed 6h. Under these conditions, in which ~ 80% of the cell population showed damage, as seen 177 by Rad52-GFP foci formation (Supplementary Figure 1) (Herbert et al., 2017), WT cells 178 exhibited a global increase in chromosome mobility (Figure 1C). Strikingly, in the absence of 179 Zeocin treatment, the MSDs in the H2A-S129E mutant strains, were as high as those 180 observed in wild type strains after induction of damage (Figure 1C and Supplementary 181 Figure 2A). In these mutant cells, addition of Zeocin for 6h did not significantly increase 182 MSDs, suggesting that H2A-S129E mutation maximizes mobility that Zeocin cannot increase 183 any further (Figure 1C and Supplementary Figure 2A). After Zeocin exposure, the lower 184 increase in mobility of the four tagged Gr1-Gr4 loci in H2A-S129A mutated cells 185 (Supplementary Figure 2B and2C), confirmed that γ-H2A(X) is required for full global 186 mobility upon DNA damage (Herbert et al., 2017). Together, these results suggest that the (Hauer et al., 2017;Herbert et al., 2017;Miné-Hattab et al., 2017).

229

Based on polymer modeling and multi-scale tracking of chromatin after damage, a global 230 stiffening of the chromatin fiber is consistent with a simultaneous increase in chromosomal 231 mobility and spatial distances between loci on the same chromosome (Herbert et al., 2017; 232 Miné-Hattab et al., 2017). H2A phosphorylation was proposed as a potential molecular 233 mechanism, since the negative repulsive charges due to H2A phosphorylation could 234 increase the stiffness of the chromatin fiber, as seen in vitro and by modeling (Cui and 235 Bustamante, 2000;Qian et al., 2013;Herbert et al., 2017). In addition, based on similar 236 experiments, but using different modeling assumptions, chromatin decompaction has also 237 been proposed to play a role (Hauer et al., 2017;Amitai et al., 2017). We therefore 238 hypothesized that the increase in H2A-S129E chromosome motion could be linked to a change in chromosomal structure. To address this question, we measured red-green 240 pairwise distances in P1-P4 WT and H2A-S129E mutated strains (Figure 2D). In these 3C). Thus, the failure of the ∆rad9 mutant to survive and activate cell cycle arrest in 4). To check whether changes in 286 chromosome dynamics also translate into chromatin structural changes in the ∆rad9 H2A-287 S129E double mutant, we also measured intra-chromosomal 2D distances (Figure 3E). We 288 confirmed that in the absence of Zeocin treatment, the distances in ∆rad9 mutant were not 289 significantly different to those of the wild-type strain and that the distances in the ∆rad9 H2A-290 S129E double mutant were comparable to the single H2A-S129E mutant. As expected from 291 the dynamic behavior, in the presence of Zeocin, ∆rad9 showed a modest increase in intra-292 chromosomal distances as compared to the WT but increase as much as H2A-S129E in the 293 double ∆rad9 H2A-S129E mutant (Figure 3E). Thus, the structural changes induced by the [START_REF] Zhou | The DNA damage response: putting checkpoints in 1010 perspective[END_REF]Moore et al., 2007;Eapen et al., 2012). In contrast, the number of His + 320 colonies was ~ 1.3 times higher in the H2A-S129E mutant than in WT, indicating that 321 mimicking H2A phosphorylation allowed more effective repair by NHEJ than in WT (Figure 322 4B). In the ∆rad9 mutant, the number of His + cells was 4 to 8 times lower than in the WT

323

(Figure 4B), an expected result given the role of Rad9 in limiting 5' DNA end resection 324 (Ferrari et al., 2015). Remarkably, the number of His , 2015;Dimitrova et al., 2008).

330

We next explored whether large-scale chromatin mobility could also promote the joining of

331

DSBs that are at long range distance, leading to translocation mis-repair, as observed in 332 mammals (Lottersberger et al., 2015). To test for translocation rates, we used a genetic

)) and significantly reduced in the double mutant ∆rad9 H2A-S129E (Figure 4D). In mammals, regulation of ATM response by chromatin structure was also proposed to 399 contribute to the extremely rapid and sensitive response to damage, across the nucleus, as

400
shown by ATM activation in the absence of detectable damage, during hypotonic shock or 401 during histone deacetylation [START_REF] Bakkenist | DNA damage activates ATM through intermolecular 757 autophosphorylation and dimer dissociation[END_REF]Kumar et al., 2014). The changes 402 in chromatin mobility that we report here, where no damage was induced, were et al., 2000). The fact that in the H2A-S129E mutant, the protection 410 pattern or the length of nucleosomal repeats was conserved (Downs et al., 2000), is also 411 compatible with a stiffer chromatin, but the number of nucleosomes per length unit (i.e. Image analyses were performed using Fiji plugins (Herbert et al., 2017). Briefly, images were 670 corrected for chromatic aberrations using the plugin "Descriptor-based registration (2d/3d)".

671

Once correction applied to all acquired images, the "Detect ROI" script allowed automatically 672 selecting non-dividing cells and computing the (x, y) coordinates of the red and green loci

187Figure 3 ).

 3 Figure3). These results reject the hypothesis that enhanced chromatin dynamics observed

  S129 phosphorylation increases intra-chromosomal distances without DNA 226 damage. 227 It has been established that an increase in genomic mobility could be explained by a change 228 in chromatin structure

275

  response to Zeocin, is rescued by H2A-S129E mutation independently of Rad53 276 phosphorylation. These results suggest that an early DDR response -the phosphorylation 277 of histone H2A -enables cells to delay cell division in the absence of Rad9 and thus provide 278 more time for DNA repair (see Discussion). 279 We then measured global chromatin mobility in these mutants in the absence and the 280 presence of Zeocin (Figure 3D). Whereas the ∆rad9 mutant behaved similarly to WT in 281 undamaged conditions, the absence of RAD9 partially impeded the enhancement of global 282 chromatin mobility observed in WT strains upon DNA damage, in agreement with (Seeber et 283 al., 2013). Interestingly, in both undamaged and damaged conditions, the ∆rad9 H2A-S129E 284 double mutant induced a massive increase of global dynamics, comparable to the single 285 H2A-S129E mutant (Figure 3D, Supplementary Figure

294H2A-

  S129E mutant in the ∆rad9 background corroborate the observations on the 295 chromosomal dynamics. Our results highlight the capacity of H2A-S129E mutant to suppress 296 the specific contribution of Rad9 to cell survival, DNA damage checkpoint and global 297 chromosome dynamics when genome is damaged, possibly through chromatin structure mammalian ortholog 53BP1, are known to facilitate NHEJ (Ferrari et al., 2015; 303 Zimmermann et al., 2013). Since the Zeocin sensitivity of ∆rad9 is suppressed by the H2A-304 S19E mutant that increases chromosome mobility, and since chromosome mobility could be 305 a means for promoting the joining of double-stranded extremities by NHEJ (Lottersberger et 306 al., 2015; Dimitrova et al., 2008), we investigated the effect of both ∆rad9 and H2AS129E 307 mutants on the NHEJ repair pathway. We used a plasmid repair assay, in which repair of a 308 linear plasmid can only be mediated by NHEJ. We generated linear plasmids with cohesive ends, by cutting a HIS3-containing centromeric plasmid by EcoRI enzyme. In this assay, 310 only circularized plasmids confer histidine prototrophy to the transformed cells. A ∆yku70 311 mutant, which is impaired in NHEJ, was used as control for plasmid linearization. After 312 transformation by the digested plasmids, His + colonies were obtained in the ∆yku70 mutant 313 10-fold less than in WT cells. These data indicate that most transforming molecules were 314 efficiently re-circularized in WT (Figure 4A). The number of His + colonies recovered in the 315 H2A-S129A mutant was almost as low as in ∆yku70 mutant, revealing that plasmid 316 circularization by NHEJ is compromised in the absence of ƴ-H2A(X). This result is consistent 317 with previous studies showing that truncated forms of H2A lacking S129 are deficient for 318 NHEJ and is consistent with an increased resection rate observed in H2A-S129A mutant 319 (Downs

  + colonies was similar to WT in the ∆rad9, H2A-S129E double mutant. Thus the restoration of cell survival to DNA damage 326 observed in ∆rad9 H2A-S129E double mutant could be linked to an increase in NHEJ 327 efficiency. In turn, increased chromosome motion observed in H2A-S129E could promote 328 NHEJ, as it was observed for dysfunctional telomeres in mammalian cells (Lottersberger et 329 al.

333

  system in which recovery of Ura + MATα colonies is mediated by a reciprocal translocation 334 between two DSBs generated in truncated URA3 on two distinct chromosomes, 335 chromosomes III and V (Lee et al., 2008) (Figure 4C). DSBs were induced by expressing 336 the HO endonuclease from the galactose inducible GAL1-10 promoter. We first checked by 337 qPCR that the cutting efficiency was similar after 1h of galactose induction at the two HO 338 cutting sites in WT and H2A-S129E mutant cells (Supplementary Figure 5). Then, 339 translocation rates were measured after permanent induction of HO on galactose-containing 340 plates. The number of Ura + colonies growing on GAL-URA plates was divided by the total 341 number of cells grown in non-selective YPGAL plates for each strain. While phospho-(Figure 4D and (Lee et al., 2008)), H2A-S129E decreased translocation rates by 345approximately 13-fold (1.1% ± 0.6%) (Figure4D). As expected, the efficiency of 346 translocations in the mutant ∆rad9 is comparable to that ofWT (Figure 4D and (Lee et al., 347

403

  systematically accompanied by an increase in distances between the arrays along the 404 chromosome, a parameter which can be explained by a change in chromatin structure 405(Hauer et al., 2017; Amitai et al., 2017; Herbert et al., 2017). The higher order chromatin 406 structure modification due to H2A-S19E was previously proposed to correspond to a 407 decreased compaction(Downs et al., 2000), in agreement with a more relaxed profile of 2µ 408 plasmid from H2A-S12E containing cells and a faster digestion of the H2A-S129E chromatin 409 by MNase (Downs

  412compaction) remains to be determined. Similarly, treatment with Zeocin has been shown to 2017). Histone degradation could explain the additional modest effect on mobility and intra-415 chromosomal distances observed in H2A-S129E mutant when treated with Zeocin since the 416 global level of H4 was not affected in undamaged H2A-S19E cells, but substantial 417 degradation was observed in both, WT and H2A-S19E cells in the presence of Zeocin. Thus 418 chromatin stiffening due to H2A phosphorylation and chromatin de-compaction due to 419 histone loss could exist together in Zeocin treated cells. Only close examination of the 420 chromatin structure by super resolution will help to determine the nature of these chromatin 421 changes. 422 Our live-cell chromosome dynamics observations combined with the genetic evidence 423 presented here suggest a novel mode of DDR regulation via an extremely efficient 424 modification of the chromosome structure. The activation of DDR is evident after DNA 425 damages, which restores cell cycle arrest in the double mutant ∆rad9, H2A-S129E. Which 426 effectors could be responsible for the reactivation of the checkpoint? It was tempting to test 427 whether the phosphorylation of Rad53 was restored under these conditions. The lack of 428 detectable phosphorylated form of Rad53 indicates that downstream checkpoint activation 429 occurs through other effectors that have yet to be identified. Another hypothesis is linked to 430 the Mad2-mediated Spindle Assembly Checkpoint (SAC) that we previously show to sustain 431 arrest (Dotiwala et al., 2010). Activation of this extended G2/M arrest is dependent on 432 formation of γ-H2AX (Dotiwala et al., 2010). A possible explanation to the bypass of ∆rad9 433 by H2A-S129E could be an extended arrest through the Mad2 SAC. Regardless the nature 434 of the effectors activated by H2A-S129E, our results indicate that H2A post-translational 435 modification of the chromatin that can be achieved in the absence of any protein synthesis, 436 results in a very proficient DDR, probably faster than any transcriptional regulation. of H2A-S129E, Rad9 is no longer essential for arresting cells in G2/M after 440 DNA damage or for facilitating damage-associated chromosome mobility. Based on the 441 evidence presented here, we propose that chromosome mobility induced by chromatin 442 structural changes, is a manner to efficiently signal damage and help repair. The chromatin 443 changes and the enhanced dynamics may favor accessibility of the NHEJ repair machinery 444 to the extremities of the break. In agreement with this, Tel1 ATM that phosphorylates H2A-445 S129, facilitates NHEJ repair protein recruitment and prevents dissociation of broken DNA 446 ends (Lee et al., 2008

Figure 1 .Figure 2 .Figure

 12 Figure 1. H2A-S129E increases global dynamics in absence of DNA damage.

673

  using a custom-made Fiji plugin implementing Gaussian fitting. For tracking in time-lapse 674 microscopy, we used the same custom-written Fiji plugin, which allowed extracting locus 675 positions over the entire time course for each nucleus. A custom-made MATLAB script that 676 corrected global displacements and computed MSD curves for each trajectory using non-677 overlapping time further analyzed these trajectories. Finally, other MATLAB scripts were 678 used to fit power laws to individual MSD curves or population-averaged MSDs over time grown overnight in 3 ml of YPD. The day after each culture was 682 appropriately diluted and ~ 200 colonies were plated onto YPD plates and YPD plates 683 supplemented with Zeocin at a concentration of 12.5 µg/ml in order to induce random DSBs 684 (as described in Seeber et al., 2013). Colonies were counted after 2-3 days of incubation at 30°C. For each strain, three independent experiments were performed with the 686 (HIS3) was linearized with either EcoRI, which generates cohesive 689 ends. Equal numbers of competent cells are transformed with 100ng of either linear or 690 circular plasmids. Following transformation with linear plasmid, the cell must repair the HIS3-691 containing plasmid to survive subsequent plating on Dropouts plates lacking histidine. Each 692 strain's repair efficiency was quantified, relative to the isogenic wild type, by assessing the 693 number of colonies obtained with the cut plasmid relative to the circular plasmid. Each 694 mutant was assayed a minimum of three times in triplicate, the averaged results, standard 695 errors and unpaired t tests are reported.

  Figure1

  Figure4

the regulation of DDR by changes in chromatin structure 382

  Chromatin structure is one of the key factors in the DDR since it is the first in being altered

	380
	381
	348 A model for 383
	349 384	Altogether, our results indicate that H2A phosphorylation mimicry induces an increase in both by the damage itself that generates DSBs and by the accumulation of histone
	350 385	chromosomal dynamics favorable to a local NHEJ repair concomitant with a sharp decrease modifications and repair proteins at the damaged site. DSB might alter topological
	351 386	in the inter-chromosomal translocation rate. constraints on the chromatin fiber, although little is known about these constraints
	352 387	associated with higher order chromosome organization (Canela et al., 2017). Histone post-
	353 388	Discussion translational modifications on the other hand, such as phosphorylation of H2A, which can
	354 389	Here we show that in the absence of DNA damage induction, the H2A-S129E mutation fully extend from the DSB over areas as large as 50-kb on either side of the DSB (Shroff et al.,
		recapitulates global chromosome/chromatin dynamics previously observed upon Zeocin 2004; Lee et al., 2014), also change the chromatin structure, the repulsive negative charges
	372
	373	DNA damage sensitivity and checkpoint defects are rescued by H2A-S129E suggests that
	374	H2A-S129E may have a positive effect on repair. We tested this proposal by examining the
		repair by NHEJ of a linearized plasmid and found that it was favored in H2A-S129E cells.

355

treatment

(Hauer et al., 2017; Herbert et al., 2017)

. The specificity of this H2A-S129E mutant 356 is to mimic H2A phosphorylation by the criterion of being recognized by a γ-H2AX-specific 357 antibody

(Eapen et al., 2012)

. One intriguing consequence of H2A-S129E mutation is to 358 allow bypassing an essential function of the DNA damage checkpoint protein, RAD9 53BP1 , 359 when Zeocin is present. Locus tracking in living cells reveals that H2A-S129E mutant 360 restores the high chromatin mobility induced by DNA damage, which is defective in ∆rad9. 361 Enhanced global dynamics was not related to tethering of the centromeres, histone depletion 362 or checkpoint activation, these mechanisms being proposed to explain the increase in 363 overall chromosome mobility observed when the genome is damaged (Strecker et al., 2016; 364 Smith et al., 2018; Lawrimore et al., 2017; Seeber et al., 2013). Rather, we found that the 365 negative charges of glutamic acid (but not the uncharged alanine) enhanced global 366 dynamics together with increased intra-chromosomal distances. These two characteristics 367 indicate a structural modification of the chromatin compatible with its stiffening (Herbert et al., 368 2017). If transcription of some key regulators involved in the DDR would be modified in H2A-369 S129E mutated cells, a similar phenotype could be expected. However, no such changes in 370 transcription could be detected by RNAseq analyses of H2A-S129E mutant compared to WT 371 (S. Bohn, B. Lemos, J.E. Haber and N. Krogan, unpublished result). The fact that ∆rad9 375 These observations are consistent with the idea that chromatin mobility of DNA extremities 376 may favor their rejoining as proposed in mammalian cells (Lottersberger et al., 2015). 377 However, the direct measurement of DSB translocations revealed that the level of joining in 378 conformation due to H2A phosphorylation are a means to efficiently modulate the DDR. 390 due to phosphorylation being compatible with a stiffer chromatin (Reina-San-Martin et al., 391 2003;

[START_REF] Celeste | Histone H2AX phosphorylation is 786 dispensable for the initial recognition of DNA breaks[END_REF] Lee et al., 2008; Miné-Hattab et al., 2017; Herbert et al., 2017)

. 392 Normally, γ-H2AX modifications occur around the site of a DSB, which exhibits enhanced 393 local motion, while inducing global mobility changes in undamaged regions. Here we see 394 that H2A-S129E-containing chromatin in undamaged regions display enhanced mobility. 395 Whether this represents global motion or is a manifestation of a local motion, we yet cannot 396 determine.

397 398

Saccharomyces Rad53 Checkpoint Kinase during Adaptation from DNA Damage-

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	697	Translocation assay
	698	Strains were grown overnight in 20mL of synthetic minimum medium SC (2% raffinose) at
	699	30°C, diluted to a density corresponding to an OD of 0.2 at 600nm, and grown for 3 h in the
		same synthetic minimum medium in order to reach exponential growth phase (OD = 1).
	702
	703	Colonies were counted after 2-3 days of incubation at 30°C. Translocation efficiency (%) is
	704	done by calculating the ratio between the number of colonies in -URA SC plates containing
	705	Galactose and the number of colonies in non selective SC plates containing Galactose. For
	706	each strain, three independent experiments were performed.
	707	FACS analyses
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kb from the centromere IV or at 476kb and 180kb from the right telomere IV (Gr1, Gr3,

Gr2 and Gr4 respectively, Figure1A). We analyzed Mean Square Displacements (MSDs) of

Rad53 phosphorylation was not restored in the ∆rad9, H2A-S129E double mutant (Figures

± 2.6 and 42.4% ± 4.8 for WT and H2A-S129A, respectively), as previously documented

doi:10.1016/j.celrep.2017.01.018.

28:1701-1711. doi:10.1091/mbc.E16-12-0846.

Acknowledgments 740

We thank S. Eun Lee and K. Dubrana for strains and advice, S. Duchez for her help with the 741 FACS experiments and A. Carré Simons for drop tests. We acknowledge P. Lesage, A.

Funding 729 This work was supported by Labex "Who am I?" (ANR-11-LABX-0071, Idex ANR-11-IDEX-730 0005-02). EF has support from Agence Nationale de la Recherche (ANR-13-BSV8-0013-01), 731 IDEX SLI (DXCAIUHSLI-EF14) and Cancéropôle Ile de France (ORFOCRISE PME-2015).

100ms time-lapse microscopy, for a time period of 5 minutes (Spichal et al., 2016;Herbert et 175 al., 2017). To induce DNA damage, we used a final concentration of Zeocin of 250 µg/ml for Supplementary Figure 1. arrays inserted at specific locations on the chromosome IV (Robinett et al., 1996). These