Yra1 and the DNA damage response pathway

Background

Slx5 and Slx8 have been shown to become essential in the absence of the Sgs1 DNA helicase important for maintaining genome stability (Mullen et al., 2001; Prudden et al., 2007). Several studies have linked Slx5-Slx8 to DNA damage and the nuclear periphery proposing that the enhanced localization of irreparable DNA double strand breaks (DSBs) to nuclear pores may depend on physical interactions between Slx5-Slx8 and pore components (Cook et al., 2009; Nagai et al., 2008; Therizols et al., 2006).

SUMO ligases also localize at the site of damage where they modify targets involved in DNA repair (Chung and Zhao, 2015; Cremona et al., 2012). Moreover, the Slx5-Slx8 complex has been shown to affect sumoylation of DNA repair proteins and to negatively regulate recombination (Burgess et al., 2007). Finally, DNA Double Strand Breaks relocation to the nuclear periphery appears to depend on the level of sumoylation of targets regulated by the STUbL Slx5-Slx8 and by the SUMO-ligases Siz2 and Mms21 (Horigome et al., 2016). However, the targets important for DSB relocation are still unknown. Since Yra1 is ubiquitinated by Slx5-Slx8 and Sumoylated by Siz1/Siz2 (Project Background), one hypothesis is that it could be the target important for DSB relocation at the nuclear periphery. In this context we speculated that Yra1 overexpression could be toxic because it leads to genome instability through impairment of the DNA damage response (DDR) pathway. It is unclear whether transcription may have a role in DDR as studies in S. cerevisiae highlight the importance of transcription repression in the vicinity of DSBs (Manfrini et al., 2015).

However, this contrasts with data in human cells supporting the importance of transcription and active chromatin for activation of the DDR and repair by homologous recombination (Aymard et al., 2014; Manfrini et al., 2015).

Sensitivity of Yra1 mutants to DNA damage agents

Since it is well known that Yra1 overexpression (OE) is toxic for cell growth (Espinet et al., 1995; Gavalda et al., 2016; Preker and Guthrie, 2006; Preker et al., 2002;

Rodriguez-Navarro et al., 2002), and our data indicate that Yra1 is modified by Slx5-Slx8 (Project Background), a STUbL important for genome stability (Zhang et al., 2006), we decided to examine Yra1 overexpression mutants (Figure 3.1) for defects in genome integrity.

To be more confident on possible effects of yra1 mutants on genome stability, we compared the same yra1 mutants in three different related strain backgrounds: a) shuffled strains in which the wild-type YRA1 copy on a URA3 plasmid was replaced by a mutant copy on plasmid through counterselection on 5-FOA; b) strains in which the mutant copy was integrated directly into a W303 haploid replacing the wild-type gene;

c) strains in which the muant copy was integrated directly into a W303 diploid replacing one of the wild-type copies, followed by tetrad dissection and selection of mutant spores. (see below).

The mutants overexpressing Yra1 (Figure 2.5 C) are the yra1Δintron (Yra1 cDNA) that cannot be auto-regulated by splicing inhibition, the yra1(1-210) mutant lacking the 16 C-terminal amino acids also defective in splicing auto-regulation and the yra1(allKR) non ubiquitinated mutant in which all lysines are mutated to arginines and which is also overexpressed (Figure 3.1). We examined Yra1 protein levels by Western blot analysis in mutants that were constructed, as described above, either by shuffling (see Materials and Methods Figure 3.1 A); by integrating HA-YRA1 wild-type and mutant sequences directly into a W303 haploid strain (Figure 3.1 B); or by integrating HA-YRA1 wild-type and mutant sequences into a W303 diploid followed by tetrad dissection (Figure 3.1 C). Even though the Yra1 protein levels of specific mutants are not exactly the same in different strain constructions, their growth phenotype is comparable (Figure 3.1 D1, D2, D3). Overall, Yra1 overexpression affects growth, however there is no strong linear correlation between the level of overexpression and the growth defect.

Figure 3.1: Yra1 overexpression is toxic for cell growth  

A-B-C) Western Blot analysis of HA-Yra1 levels in HA-YRA1 WT, HA-yra1(1-210), HA-yra1Δintron, HA-yra1allKR was performed using αHA antibody and αPgk1 antibody was used as loading control.

The order of loading samples is shown from 0 to 4. One representative Western blot is shown for one out of at least three independent clones or spores. Blots A, B and C correspond to HA-Yra1 mutant strains obtained respectively by shuffling, direct integration into a W303 haploid strain, and integration

into a W303 diploid strain followed by dissection. The quantification is shown as the HA-Yra1/Pgk1 ratio below each blot. Western blot quantifications were performed using Lycor Software.

D) Spot test analysis of confluent cells at 25°C, 30°C, 34°C and 37°C of HA-yra1 mutants in shuffled strains (D1), integrated directly in W303 haploid (D2), integrated in W303 diploid and dissected (D3).

Spot test analyses in the presence of genotoxic agents causing different types of DNA lesions may allow to narrow down the condition in which Yra1 overexpression could be detrimental. We verified in all three types of strains obtained by shuffling (Figure 3.2 A), integration into haploid (Figure 3.2 B), integration into diploid and dissection (Figure 3.2 C), whether Yra1 mutants show sensitivity to i) the DSB agent Zeocin, ii) Hydroxyurea (HU) that depletes nucleotides causing replication fork arrest with consequent DNA damage, iii) Camptothecin (CMPT) that causes irreversible replication fork arrest by Topo-I block (DSB formation) and transcription block (R-loop), iv) MMS that causes DNA nicks. We cannot be precise on the degree of sensitivity to genotoxic drugs of the different yra1 mutants because they display different Yra1 protein levels and different growth defects in normal condition and passing from 25°C to 34°C. However, yra1 OE mutants tend to be more sensitive to HU (Figures 3.2 A-B-C *) and Zeocin (Figures 3.2 A-C *), which cause double strand breaks that are repaired by Non-Homologous End-Joining (NHEJ) if the damage occurs in G1 phase, and homologous recombination (HR) if the damage occurs in S-G2 phases.

We observed weak sensitivity of yra1 OE mutants to CMPT (Figures 3.2 A-B) that we did not investigate further since they were observed only with elevated doses of drug.

Interestingly, yra1 OE mutants were not sensitive to MMS (Figures 3.2 A-B-C) (Wyatt and Pittman, 2006), a drug inducing DNA nicks that are mainly repaired by the Base Excision Repair (BER) pathway, even though BER intermediates and unrepaired methyl base damage are repaired also by HR.

Overall the shuffled yra1 mutants are more strongly affected by HU and zeocyn (Figures 3.2 A) compared to the yra1 mutants integrated dyrectly into haploid cells, in which we observed only sensitivity to HU (Figures 3.2 B). The yra1 mutants integrated into diploid and dissected showed less pronunced sensitivity to HU and zeocin than the shuffled yra1 mutants as only the HA-yra1(1-210) and HA-yra1Δintron strains are slightly sensitive (Figures 3.2 C). The results are summarized in Table 1.

Figure 3.2 Yra1 overexpression mutants are sensitive to DSB agents. cultures of HA-yra1 mutants integrated directly into haploid W303 (one representative clone of at least 4 is shown for each yra1 mutant).

C) Spot test analysis in the presence of Zeocin 10 µg/ml, Zeocin 20 µg/ml, HU 50mM, HU 75mM and MMS 0,01% at 25°C of confluent cells of HA-yra1 mutants integrated into diploid W303 and dissected.

Sensitivity to drug is shown by *. Table 1: DNA damage sensitivity of HA-yra1 mutants.

The Table summarizes the DNA damage sensitivity observed for the HA-yra1 mutants in shuffled background, integrated in haploid or integrated in diploid and dissected strain backgrounds. (/)=not sensitive to the drug, (*)=sensitive to the drug. The growth defects present in YEPD are not shown.

To define whether the sensitivity to HU was in part dependent on the growth defect of yra1 OE mutants, exponentially growing cells were treated or not with 20mM HU for 2h30 at 25°C and three serial dilutions of 200/100/50 cells were plated to examine the recovery after damage. The graph in Figure 3.3 A shows the average of the percent of colonies of three dilutions for each yra1 integrated mutant (non treated and treated with HU) normalized to non treated WT (NT). The non treated conditions were set to 100% and corresponding values of yra1 OE mutants treated with HU were expressed as percentage of WT (HU) set to 100%, since nearly no differences were

observed between WT NT and WT HU. The yra1 OE mutants show a reduced number of colony forming units in comparison to the HA-YRA1 WT (which number of CFUs is not affected by HU), indicating that the HU sensitivity cannot be due to the difference in growth proficiency. The HA-yra1 (1-210) is the most affected with a level of recovery after damage of 50% comparable to Δrad52. The HA-yra1Δintron and the HA-yra1allKR show 80% of recovery after damage. The difference between yra1 mutants does not correlate with differences in Yra1 protein levels since they are all comparable in non treated and under damage condition (HU and Zeocin) (Figure 3.3 B). This observation may suggest that the highly conserved C-terminal region of Yra1 could be important for the recovery after damage.

The activation of the checkpoint under DNA damage condition was assessed by the appearance of a shifted Rad53 phosphorylated band on Western blot (Figure 3.3 B).

Figure 3.3: Yra1 OE mutants are defective in recovery after HU treatment.

A) Exponentially growing cells from integrated HA-yra1 mutants were treated with HU 20mM for 2h30 or not treated (NT), and serial dilutions of 200/100/50 cells were plated without drug. The percent of colonies was determined as the relative number of Colony Forming Unit (CFU) in each mutant compared to the HA-YRA1 WT NT and HU. The average of three dilutions from the same experiment is shown.

B) Western Blot analysis of Rad53 phosphorylation with αRad53 antibody as control for the treatment with HU and Zeocin (250ug/ml). The Rad53 band shifts up following treatment with these genotoxic drugs in all the strains. The % colonies recovering after Zeocin (250 µg/ml) treatment is not shown in A because this treatment was already detrimental in HA-YRA1 WT. The Western blot was also analyzed for

the levels of Pgk1 (αPgk1) as loading control and HA-Yra1 (αHA). The Yra1/Pgk1 ratio was quantified using Licor Software.

Since the sensitivity to HU could also be due to decreased RNR (ribonucleotide-diphophate reductase) gene expression (Mulder et al., 2005), we verified whether HA-yra1 mutants are defective in the expression of genes activated under DNA damage such us RNR3, DDI, HUG1 (Fu et al., 2008; Jaehnig et al., 2013). PHR1 was used as control since it is not activated under HU (Fu et al., 2008) (Figure 3.4). The results indicate that RNR3, DDI and HUG1 expression is induced in the mutants treated with HU, supporting that the sensitivity of HA-yra1 OE mutants to HU is specific and not due to indirect effects.

Figure 3.4: Yra1 OE mutants are not defective in the expression of genes activated under HU.

Analysis of RNR3, PHR1, DDI, HUG1 mRNA levels in HA-yra1 mutants treated with HU 20mM for 2h30. Total RNA was extracted from samples also examined in Figure 3.3. The indicated transcripts were quanitified by RT-qPCR normalized to SCR1.

Rad52 foci accumulation in Yra1 mutants

To define whether yra1 OE mutants accumulate DSBs in vivo, we monitored Rad52 foci accumulation under normal conditions without external genotoxic agent treatment. Rad52 foci are markers of homologous recombination (HR) centers that repair spontaneous DNA lesions in a conservative manner in S phase or G2/M using the replicated DNA or the homologous chromosome as template (Barlow and Rothstein,

2010; Lisby et al., 2001). The HA-yra1Δintron and HA-yra1allKR mutants show increased Rad52 foci in G1/S/G2M similar to the Δnup60 control, while the HA-yra1(1-210) mutant shows a mild increase in Rad52 foci specifically in G1 (Figure 3.5) (Alvaro et al., 2007; Palancade et al., 2007). G1 cells with Rad52 foci could arise from mitotic division before resolution of repair foci (Alvaro et al., 2007). The milder DSB accumulation in HA-yra1(1-210) compared to HA-yra1(Δintron) together with the comparable overexpression levels of these two mutant proteins (Figure 3.1), suggest that the Yra1 C-terminal box may be implicated in the detrimental effects of Yra1 overexpression.

Figure 3.5: yra1 OE mutants accumulate Rad52 foci, markers of HR centers.

Rad52 foci quantification in HA-YRA1 WT, HA-yra1(1-210), HA-yra1Δintron and HA-yra1allKR shuffled strains as well as in Δnup60 as control for Rad52 foci accumulation. Strains were transformed with a RAD52-YFP centromeric plasmid. Transformants were grown to exponential phase and fixed in Paraformaldehyde 4%. Z stack images were taken at 0.25-0.3 µm (25 slides) with a SP5 microscope.

The average of three independent experiments is shown. At least 300 cells were quantified for each strain. *: p <0.05; **: p <0.01 comparing the mutant values to HA-YRA1 WT. A) Quantification of cells with Rad52 foci in the different cell cycle stages (left) and the % of cells in each cell cycle phase (right).

B) Quantification of foci in the overall population distinguishing between the precence of 0, 1 or more foci. C) One representative Z stack for each strain. The Rad52-foci form yellow dots. The percent of cells forming Rad52-foci in the overall population is indicated below each picture.

Experiment performed by Audrey Zihlmann and supervised by Valentina Infantino.

Yra1 genetic interactions with DDR components  

To assess whether yra1 OE mutants indeed impair the DNA damage response pathway activated in response to Double Strand Breaks and specific to HR, we tested genetic interactions between yra1 mutants and i) Mre11, a component of MRX, one of the first complexes recruited at DSBs; ii) Sae2, important together with Mre11 for the first resection event; iii) Exo1, which mediates extensive resection generating long ssDNA bound by RPA complexes; and iv) Rad52 important for replacing RPA complexes (Barlow and Rothstein, 2010; Gobbini et al., 2013). DDR deletion mutants were generated in the YRA1 shuffle background deleted for the endogenous YRA1 gene (Δyra1::HIS) and complemented with a YRA1 WT gene on a URA3 centromeric plasmid. The effect of the mutations can be monitored by transformation with yra1 mutant genes on TRP1 centromeric plasmids and spotting on 5-FOA plates to select for loss of the YRA1 WT URA3 plasmid (Figure 3.6). The yra1(Δintron) mutant is synthetic sick with Δsae2, Δmre11, Δrad52 but not with Δexo1, indicating impairment in the initial resection events and early steps of HR. Interestingly, the yra1(1-210) mutant appears to suppress the synthetic sickness of Yra1 overexpression, suggesting that the C-terminal box may be implicated in the phenotype. Finally, the yra1allKR mutant is sick already in the WT YRA1 shuffle background and it is therefore difficult to interpret the phenotypes of the double mutants.

Figure 3.6: The yra1Δintron mutant is synthetic sick with Δsae2, Δmre11 and Δrad52

A) Spot test of WT, Δsae2, Δmre11, Δrad52, Δexo1 in YRA1 shuffle background (Δyra1::HIS, YRA1 WT, URA3 CEN) transformed with plasmids (TRP1, CEN) expressing HA-Yra1 WT (1), HA-yra1(1-210) (2), HA-yra1allKR (3), HA-yra1Δintron (4), on TRP(-) or 5-FOA plates.

Synthetic sickness is indicated by *.

We also addressed the possible synthetic lethality of yra1 OE mutants with Δsae2, Δmre11, Δrad52 and Δexo1 in W303 background by cross with strains generated by integration of HA-YRA1 wild-type or mutant sequences in a diploid W303 followed by dissection (Figure 3.7). Looking at the size of the spores, HA-yra1 OE mutants are not synthetic sick with Δsae2, Δmre11, Δexo1. The HA-yra1 OE mutants seem to be sicker in Δrad52 background although the difference in size between the single and the double mutants is not so strong (Figure 3.7). These data were confirmed by crossing the DDR deletion mutants with HA-yra1 mutants generated by integration of HA-YRA1 wild-type or mutant sequences directly into haploids (data not shown).

Overall we showed in different approaches that HA-yra1 OE mutants are synthetic sich with Δrad52.

Figure 3.7: Yra1 OE mutants are synthetic sick with Δrad52  

Tetrad analyses following the cross of HA-yra1 OE mutants with Δsae2, Δexo1, Δrad52 and Δmre11 deletion mutants. One representative tetrad is shown for each cross. The HA-yra1 mutants used in the cross have been generated by integration of HA-YRA1 wild-type or mutant sequences into a diploid strain followed by dissection. Black triangles: spores with the deletion only; turquoise rectangles: spores with the HA-yra1 mutation only; Red circles: double mutant spores.

To define whether HA-yra1 OE may enhance the sensitivity of these HR mutants to genotoxic agents, we performed additional spot tests in the presence of HU, Zeocin, MMS and CMPT with the strains generated by cross. Spot test analysis of HA-yra1 OE mutants in Δsae2, Δmre11, Δrad52 and Δexo1 background reveals that HA-yra1(1-210) and HA-yra1(Δintron) mutations increase the sensitivity of Δexo1 (Figure 3.8 A and C) and Δsae2 (Figures 3.8 A-B-C) to DNA damaging agents. We noticed also strong sickness of the double mutant HA-yra1(Δintron), Δrad52 (Figures 3.8 A-B-C) and HA-yra1(Δintron), Δmre11 (Figure 3.8 C) withouth any drug. The HA-yra1(1-210) is also sicker in the Δrad52 and Δmre11 backgrounds but not to the extent of the HA-yra1(Δintron) (Figure 3.8 C). Overall we can conclude that the HA-yra1(1-210) and the HA-yra1(Δintron) enhance the sensitivity to genotoxic drugs when components of the DNA damage repair pathway by Homologous Recombination are limiting. The weaker phenotypes of the HA-yra1 mutants obtained by direct integration into a haploid instead of a diploid strain could be due to suppressor effects that cannot be excluded in this type of strain construction (compare Figures 3.8 A-B with C).

Table 2 summarizes these observations.

Figure 3.8: Yra1 overexpressing mutants increase the sensitivity of Δsae2 and Δexo1 to DSB agents.

A) Spot test analysis under YEPD 2% Glu, HU 50mM, HU 75mM, Zeocin 10 µg/ml, Zeocin 20 µg/ml, MMS 0,01%, MMS 0,02% at 25°C of confluent cells of HA-yra1 mutants integrated directly into W303 haploid, followed by cross with Δsae2, Δrad52 and Δexo1 mutants to generate the indicated double mutants.

B) Spot test analysis under DMSO 2% (as no drug control), CMPT 15 µg/ml, CMPT 30 µg/ml, HU 50mM, HU 75mM, Zeocin 10 µg/ml, Zeocin 20 µg/ml, MMS 0,01%, MMS 0,02% at 25°C, 30°C, 34°C of confluent cells of HA-yra1 mutants integrated directly into W303 haploid followed by cross with Δsae2, Δrad52 and Δexo1 mutants to generate the indicated double mutants.

C) Spot test analysis under YEPD 2% Glu, HU 50mM, HU 75mM, Zeocin 10 µg/ml, Zeocin 20 µg/ml, MMS 0,01%, at 25°C of confluent cells of HA-yra1 mutants integrated into diploid and dissected in W303 followed by cross with the Δsae2, Δrad52, Δexo1, Δmre11 mutants to generate the indicated double mutants.

In all 3 panels, sensitivity and sickness are highlighted by rectangular black squares.

Integrated in haploid YEPD   Zeocin     HU     MMS   CMPT  

HA-YRA1 WT /   /   /   /   /  

HA-yra1(1-210) /   /   *   /   /  

HA-yra1allKR /   /   *   /   /  

HA-yra1∆intron /   /   /   /   /  

Table 2: Summary of the additive effect of HA-yra1 mutants on the sensitivity of DDR mutants to DNA damage agents.

The Table summarizes the DNA damage sensitivity observed for the HA-yra1 mutants integrated in haploid WT or Δsae2, Δrad52, Δexo1, Δmre11 backgrounds, diploid and dissected, in Δsae2, Δrad52, Δexo1, Δmre11 strain backgrounds. (/)=not sensitive to the drug, (*)=sensitive to the drug, (**)=additive effect on Drug sensitivity, (+)=lethality. The growth defects in YEPD are not shown.

To exclude that the increased sensitivity to the drugs observed in the double mutants of HA-yra1 and DDR components could be due to mRNA export defects, we

transformed the integrated strains with 2µ plasmids overexpressing Nab2 or Mex67.

The growth defect of the HA-yra1(1-210) can be marginally rescued overexpressing Nab2 and Mex67 in WT as well as in Δsae2 and Δexo1 background (Figure 3.9 A).

Under damage condition, Nab2 and Mex67 overexpression partially rescues the sensitivity of HA-yra1(1-210), as well as HA-yra1(1-210), Δsae2 and HA-yra1(1-210), Δexo1 double mutants (Figure 3.9 D). The growth defect of HA-yra1Δintron can be rescued by Mex67 overexpression in the WT and Δsae2 background, as well as by Nab2 overexpression in Δrad52 (Figure 3.9 B). Mex67 overexpression can also partially rescue the sensitivity of HA-yra1Δintron to DNA damage agents (Figure 3.9 E). Thus, the sensitivity to DNA damage observed in HA-yra1(1-210) and HA-yra1Δintron in WT background or in Δsae2 and Δexo1 could be partially due to mRNA export defects. As control the temperature sensitive GFP-yra1-8 shuffled strain is fully

Under damage condition, Nab2 and Mex67 overexpression partially rescues the sensitivity of HA-yra1(1-210), as well as HA-yra1(1-210), Δsae2 and HA-yra1(1-210), Δexo1 double mutants (Figure 3.9 D). The growth defect of HA-yra1Δintron can be rescued by Mex67 overexpression in the WT and Δsae2 background, as well as by Nab2 overexpression in Δrad52 (Figure 3.9 B). Mex67 overexpression can also partially rescue the sensitivity of HA-yra1Δintron to DNA damage agents (Figure 3.9 E). Thus, the sensitivity to DNA damage observed in HA-yra1(1-210) and HA-yra1Δintron in WT background or in Δsae2 and Δexo1 could be partially due to mRNA export defects. As control the temperature sensitive GFP-yra1-8 shuffled strain is fully