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Current Genetics (2018) 64:697–712 https://doi.org/10.1007/s00294-017-0786-4

ORIGINAL ARTICLE

Fumarase is involved in DNA double-strand break resection through a functional interaction with Sae2

Michael Leshets1 · Dharanidharan Ramamurthy2 · Michael Lisby3 · Norbert Lehming2 · Ophry Pines1,2

Received: 9 October 2017 / Revised: 19 November 2017 / Accepted: 22 November 2017 / Published online: 4 December 2017 © Springer-Verlag GmbH Germany, part of Springer Nature 2017

AbstractOne of the most severe forms of DNA damage is the double-strand break (DSB). Failure to properly repair the damage can cause mutation, gross chromosomal rearrangements and lead to the development of cancer. In eukaryotes, homologous recombination (HR) and non-homologous end joining (NHEJ) are the main DSB repair pathways. Fumarase is a mitochon-drial enzyme which functions in the tricarboxylic acid cycle. Intriguingly, the enzyme can be readily detected in the cyto-solic compartment of all organisms examined, and we have shown that cytosolic fumarase participates in the DNA damage response towards DSBs. In human cells, fumarase was shown to be involved in NHEJ, but it is still unclear whether fumarase is also important for the HR pathway. Here we show that the depletion of cytosolic fumarase in yeast prolongs the presence of Mre11 at the DSBs, and decreases the kinetics of repair by the HR pathway. Overexpression of Sae2 endonuclease reduced the DSB sensitivity of the cytosolic fumarase depleted yeast, suggesting that Sae2 and fumarase functionally interact. Our results also suggest that Sae2 and cytosolic fumarase physically interact in vivo. Sae2 has been shown to be important for the DSB resection process, which is essential for the repair of DSBs by the HR pathway. Depletion of cytosolic fumarase inhibited DSB resection, while the overexpression of cytosolic fumarase or Sae2 restored resection. Together with our finding that cytosolic fumarase depletion reduces Sae2 cellular amounts, our results suggest that cytosolic fumarase is important for the DSB resection process by regulating Sae2 levels.

Keywords Fumarase · DNA damage response · Homologous recombination · HR repair pathway · Double-strand break resection · Sae2

Introduction

Preservation of genomic integrity is one of the most impor-tant challenges of all organisms. An average cell in our body suffers approximately 100,000 DNA lesions each day (Alberts et al. 2004; Jackson and Bartek 2009; Lindahl and Barnes 2000). Stochastic or chronic failure to properly repair the damage can lead to multiple pathological conditions, the most prominent of which is cancer (Hanahan and Weinberg 2011; O’Driscoll 2012). Therefore, identification of previ-ously unknown factors as key players in the cellular response to DNA damage, and the characterization of their particular function, is extremely important.

One of the most dangerous forms of DNA damage is the double-strand break (DSB). Accordingly, different cellular mechanisms that repair DSBs have evolved (Shiloh and Lehmann 2004; van Gent et al. 2001). In yeast and human cells there are two main DSB repair pathways; the first, non-homologous end joining (NHEJ), directly re-joins the DNA

Communicated by M. Kupiec.

Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00294-017-0786-4) contains supplementary material, which is available to authorized users.

* Ophry Pines ophryp@ekmd.huji.ac.il

1 Department of Microbiology and Molecular Genetics, IMRIC, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem, Israel

2 CREATE-NUS-HUJ Program and the Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

3 Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen N, Denmark

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broken ends and is mainly orchestrated in the yeast Saccha-romyces cerevisiae by the yKu70/80 complex, Dnl4 and Lif1 (Durdikova and Chovanec 2017; Lewis and Resnick 2000). The repair of DSBs using NHEJ has been shown to be more error-prone and infrequently used in yeast (Kramer et al. 1994; Lewis and Resnick 2000; Moore and Haber 1996). The second, and more dominant, DSB repair mechanism in yeast is the homologous recombination (HR) pathway, in which an intact homologous DNA sequence is used to accurately repair the DSB (Aylon and Kupiec 2004; Shiloh and Lehmann 2004; van Gent et al. 2001).

In yeast, the DNA damage response (DDR) to DSBs is initiated by the recruitment of the highly conserved Mre11/Rad50/Xrs2 (MRX) complex (Game and Mortimer 1974; Hopfner et al. 2001; Lisby et al. 2004; Shroff et al. 2004; Trujillo et al. 2003). Following the association with the DSB, this complex initiates two central processes; (1) Acti-vation of the checkpoint machinery by the recruitment of the Tel1 kinase (Nakada et al. 2003; Usui et al. 2001), and (2) Initiation of the DSB resection process (Cannavo and Cejka 2014; Mimitou and Symington 2008; Zhu et al. 2008).

During DSB resection, the DNA flanking the DSB under-goes nucleolytic cleavage to form a 3′ overhang structure (White and Haber 1990). This process is composed of two sequential steps. In the initial step, MRX and Sae2 induce the resection of a short DNA region flanking the DSB. Next, the DNA is extensively resected by two parallel pathways involving the Exo1 exonuclease and Dna2-Sgs1/Top3/Rmi1 (STR) complex (Bermudez-Lopez and Aragon 2017; Mimi-tou and Symington 2008; Zhu et al. 2008). The length of single-stranded DNA (ssDNA) revealed by the resection process can extend up to several kbp (Chung et al. 2010). Importantly, this ssDNA is essential for the repair of the DSB by the HR pathway. Furthermore, initiation of resec-tion is proposed to serve as decision point which determines whether the DSB will be repaired by HR or NHEJ (Daley and Wilson 2005; Frank-Vaillant and Marcand 2002; Ira et al. 2004; Zhang et al. 2009).

The initiation of resection is concomitant with the dis-sociation of MRX and the recruitment of replication pro-tein A (RPA) to the resulting ssDNA (Lisby et al. 2004; Wang and Haber 2004). Following its recruitment, RPA has been shown to participate in two central processes; (1) The recruitment of multiple checkpoint complexes includ-ing Mec1/Ddc2 and Rad24/RFC (Lisby et al. 2004; Majka et al. 2006), and (2) The activation of the HR pathway by the recruitment of Rad52 (Hays et al. 1995; Lisby et al. 2004). Following its association, Rad52 recruits Rad59 and facili-tates the replacement of RPA with the Rad51 recombinase. Rad51 mediates strand invasion to pair the ssDNA with a homologous sequence on an intact DNA molecule. Next, Rad54 ATPase facilitates the dissociation of Rad51 and the initiation of the homologue-dependent DNA synthesis,

thus starting a sequence of additional events leading to the repair of the DSB by HR (Davis and Symington 2003; Li and Heyer 2009; Lisby et al. 2004; Milne and Weaver 1993; Miyazaki et al. 2004; Song and Sung 2000; Sung 1997).

The enzyme fumarase is a member of the class II fuma-rase family which is highly conserved from bacteria to humans. In the budding yeast Saccharomyces cerevisiae fumarase is encoded by the FUM1 gene located on chromo-some 16. In vivo, the enzyme functions as a homotetramer with a molecular weight of 200 kDa (Burak et al. 2013; Woods et al. 1988; Wu and Tzagoloff 1987). Canonically, fumarase is localized to mitochondria where it functions in the tricarboxylic acid (TCA) cycle. The enzyme catalyzes the hydration of fumarate to L-malate and the reverse dehy-dration reaction (Mann and Woolf 1930; Woods et al. 1988).

In addition to the mitochondrial population of fumarase, the enzyme can be readily detected in the cytosolic com-partment. This cytosolic localization is highly conserved as fumarase can be found in the cytosol of all eukaryotic organ-isms from yeast to human (Akiba et al. 1984; Edwards and Hopkinson 1979; Kobayashi and Tuboi 1983; O’Hare and Doonan 1985; Tolley and Craig 1975; Wu and Tzagoloff 1987). In budding yeast, both mitochondrial and cytosolic fumarase populations are encoded by the FUM1 gene (Wu and Tzagoloff 1987). During translation, a subset of FUM1 translation products, although partially translocated, are prevented from full mitochondrial import by a mechanism termed reverse translocation. Following translation, these translation products remain in the cytosol forming the cyto-solic fumarase population (Sass et al. 2001, 2003; Stein et al. 1994).

To investigate the function of the cytosolic fumarase population in yeast, we previously constructed a S. cerevi-siae strain termed FumM. In this strain, the FUM1 gene was transferred from its original location on chromosome 16 to the mitochondrial DNA. This genetic manipulation resulted in the depletion of cytosolic fumarase, thus presenting an opportunity to determine its extra-mitochondrial function (Yogev et al. 2010).

The FumM strain exhibited significant sensitivity to DSB inducing chemicals, γ-irradiation and HO-induced DSBs. In addition, following DSB induction, fumarase cellular lev-els increased and it has been shown to localize to the cell nucleus. The DSB sensitivity of the FumM strain was sup-pressed by the expression of cytosolic fumarase or exposure of the cells to fumarate (Yogev et al. 2010). These results suggest that the enzymatic activity of cytosolic fumarase is important for the DDR to DSBs. However, the exact role of fumarase in the yeast DDR is still unknown.

The protein level of the human homolog of fumarase, termed fumarate hydratase (FH), was shown to increase following induction of DSBs, and its nuclear localization could be observed. In addition, FH knockdown rendered

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cells more sensitive to DNA DSBs and impaired the acti-vation of the DNA damage checkpoint machinery (Yogev et al. 2010). These results suggested that the importance of fumarase to the DSB DDR is not specific to yeast and can also be observed in human cells. This observation is particularly interesting since biallelic loss of FH can lead to the development of hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome (Kiuru et al. 2001; Launonen et al. 2001; Tomlinson et al. 2002).

A recent study by Jiang et al., suggested that in human cells, FH is important for the NHEJ repair pathway (Jiang et al. 2015). The phosphorylation of FH on Thr 236 was shown to be essential for its function by this mechanism. Nevertheless, cells harboring Thr 236-mutated FH did not exhibit the full phenotype observed upon FH knockdown (Jiang et al. 2015; Yogev et al. 2010). This result suggested that FH may be involved in additional mechanisms of the DSB DDR. This possibility motivated us to investigate whether fumarase can also be important for the HR repair.

Here we show that the cytosolic fumarase population is important for the DNA DSB resection process through a functional interaction with the Sae2 endonuclease. Cytosolic fumarase is also required for the timely repair of DSBs by HR. These results indicate for the first time, that in yeast, fumarase is important for the HR repair pathway.

Results

To study the role of cytosolic fumarase in the DDR to DSBs, we examined the effect of cytosolic fumarase deple-tion on recruitment and dissociation of DDR regulators at DSB sites. For this purpose, five yeast strain sets were con-structed; each set contained a wild type (WT) and a FumM strain, in which a particular DDR regulator was fused to yellow florescent protein (YFP) (Lisby et al. 2004). DDR regulators are recruited to the DSB in a particular order to form structures termed nuclear foci. These structures can be readily detected by fluorescence microscopy (Supplementary Fig S1A) (Lisby et al. 2004). We used this phenomenon to follow the DSB recruitment kinetics and the DSB dissocia-tion kinetics of Mre11, Rad9, Ddc2, Rad24 and Rad54.

First, we determined the fraction of cells, in a logarithmic culture, which presented at least one nuclear focus of the dif-ferent DDR proteins (Fig. 1a). Interestingly, depletion of the cytosolic fumarase population significantly increased (about threefold) the percentage of cells harboring Mre11 foci. The increase of the other DDR regulators was minor and insig-nificant. The exclusivity of the Mre11 increase suggests that the depletion of cytosolic fumarase prolongs the presence of Mre11 at endogenous DSBs. Next, the DSB dissocia-tion kinetics of Mre11 following an exogenous induction of DSBs, was examined (Fig. 1b). Logarithmic cultures of

WT and FumM strains expressing YFP-tagged Mre11 were treated with methyl methanesulfonate (MMS) to induce replication-associated DSBs (Ensminger et al. 2014). Fol-lowing DSB induction the cells were washed and grown in the same medium lacking MMS. With time, aliquots of the cultures were analyzed to determine the percentage of cells containing at least one Mre11 focus. Following the induction of DSBs, WT cells were shown to readily recruit Mre11 to form nuclear foci (time points: 0–120). Notably, this process was less evident for the FumM strain probably due to the higher basal percentage of Mre11 foci presenting cells in this strain. Two hours from the beginning of the experiment, the percentage of WT cells with Mre11 foci started to decline, indicating the dissociation of Mre11 from the sites of dam-age (time points: 120–240). The Mre11 dissociation kinetics were significantly delayed in the FumM strain. These results suggest that the depletion of cytosolic fumarase can prolong the presence of Mre11 at the sites of both endogenous and exogenously induced DSBs.

There are a number of DDR regulators whose impair-ment has been similarly shown to prolong the presence of Mre11 at the site of the DSB. One group of such proteins includes the Ddc2-Mec1 and Rad24-RFC complexes (Lisby et al. 2004). To determine whether the effect of cytosolic fumarase depletion on Mre11 is mediated by these com-plexes, the recruitment kinetics of Rad24 and Ddc2 were examined (Supplementary Fig S1B, C). The experiment was conducted using WT and FumM strains expressing YFP-tagged Rad24 and Ddc2. Following the induction of DSBs by MMS, WT and FumM cells were shown to recruit both Rad24 and Ddc2 at similar rates to the damage sites. Thus, the Mre11 phenotype, observed in the absence of cytosolic fumarase, is probably not mediated by the recruitment of Ddc2 and Rad24 proteins.

The second group of DDR proteins, impairment of which can lead to a similar Mre11 phenotype, includes the Sae2 endonuclease and Mre11 itself (Clerici et al. 2006; Kim et al. 2008; Lisby et al. 2004; Usui et al. 2001). Notably, defects in these factors have been shown also to delay the DSB recruitment and dissociation of the HR machinery (Lisby et al. 2004). To gain support for the notion that the cytosolic fumarase effect on Mre11 is mediated by one of these proteins, we examined whether depletion of cytosolic fumarase can also influence the process of DSB repair by the HR pathway. For this, the kinetics of Rad54 nuclear foci for-mation and disassembly in the FumM strain were determined (Fig. 1c) (Sugawara et al. 2003). WT and FumM strains expressing Rad54, fused to YFP, were examined following exposure to MMS. WT cells are shown to recruit Rad54 to form nuclear foci (Fig. 1c, time points: 0–180) and 5 h later Rad54 dissociation from the sites of damage can be observed (time points: 180–300). In contrast, cytosolic fumarase depletion delays the recruitment and dissociation of Rad54

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following DSBs induction, as can be observed by the differ-ences at time points 120 and 300, respectively. These find-ings may suggest that cytosolic fumarase is important for DSB repair by HR.

We also examined the DSB dissociation kinetics of the Rad9 checkpoint adaptor. Rad9 is recruited to the DSB and facilitates the checkpoint activation process while its dis-sociation can be detected following the conclusion of repair (Lisby et al. 2004; Toh et al. 2006). Therefore, delayed Rad9 dissociation can provide indirect evidence for a delay or an

impairment of DSB repair. WT and FumM strains, express-ing YFP-tagged Rad9, were induced for DSBs with MMS (Fig. 1d). In both the WT and FumM strains, Rad9 is rapidly recruited to the sites of damage and subsequently starts its dissociation processes. The depletion of cytosolic fumarase delayed the dissociation of Rad9 as seen by the differences at time points 40–180. This result is consistent with fumarase being important for DSB repair.

To directly assess whether the depletion of cytosolic fumarase can alter the repair of DSBs by HR, we induced a

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Fig. 1 The depletion of cytosolic fumarase prolongs the presence of Mre11 at DSBs, and delays DSB repair by the HR pathway. a WT (BY4741) and FumM strains in which DDR regulators Ddc2, Mre11, Rad9, Rad24 and Rad54 were C-terminally fused to YFP within the genome (Lisby et  al. 2004). Logarithmic cultures of these strains were examined using fluorescence microscopy to determine the percentage of cells containing at least one nuclear focus. Statistical significance was determined using exact binomial confidence inter-vals (Lisby et  al. 2004). Cells examined n = 2595, *p = 6.99 × 10−8. b–d Logarithmic cultures of the strains described in a were exposed to 0.03%(v/v) methyl methanesulfonate (MMS) for one hour and washed (Wash). Samples were collected at indicated time points and analyzed using fluorescence microscopy to determine the percent-age of cells containing at least one nuclear focus. Statistical signifi-

cance was determined as in a. Cells examined: b n = 2736, c n = 2775, d n = 1646. *p ≤ 0.03. e Schematic representation of the PCR-based method used to determine the kinetics of DSB repair by HR (Haber et  al. 1993). The approximate location of the primers is designated by blue arrows. f HO nuclease was induced by 2% (w/v) galactose in logarithmic cultures of WT (BY4742) and FumM strains, grown in YP-raffinose. Thirty minutes later, HO expression was suppressed by the addition of 2% (w/v) glucose. At indicated time points the repair product was detected by semi-quantitative PCR. Amplification of a region in the ARG80 open reading frame was used as a control (Con-trol). g The rate of DSB repair by HR was calculated using densito-metric analysis of the gels described in F (Schneider et al. 2012). The results were normalized to the size of the MATα population and the HO cleavage efficiency. *p = 4.38 × 10−3

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single DSB at the MAT locus on chromosome III by expres-sion of the HO endonuclease (Fig. 1e). The repair of this DSB by the HR pathway leads to gene conversion; “copy” of sequences from a distant locus, termed HMR, to the MAT locus (Hicks et al. 1979). Using this phenomenon, the repair kinetics of the DSB were determined by following the accumulation of a specific PCR product, generation of which is possible only after HR repair (Fig. 1f) (Haber et al. 1993). The WT strain exhibited fast HR repair following the induction of HO, while the depletion of cytosolic fumarase strongly inhibited the HR kinetics. Densitometric quantifica-tion of the results allowed us to calculate the relative DSB repair rate by HR, which was reduced approximately five-fold in cells depleted of cytosolic fumarase (Fig. 1g). Taken together, these results indicate that the cytosolic fumarase population is important not only for the timely dissociation of Mre11, but also for DSB repair by the HR pathway.

Prolonged presence of Mre11 at the site of damage and delayed kinetics of HR were previously observed in strains lacking Sae2 (Δsae2) or in Mre11 nuclease-dead (mre11-nd) mutants (Clerici et al. 2006; Lisby et al. 2004). The similar phenotypes of the cytosolic fumarase depletion and mutations of Mre11 or Sae2, prompted the assumption that cytosolic fumarase may affect the function of these pro-teins. So next we looked into the known DDR functions of Mre11 and Sae2. Both Mre11 and Sae2 have been shown to be important for the initial step of the DSB resection, and

therefore we examined whether fumarase may be important for the resection process (Cannavo and Cejka 2014; Clerici et al. 2005; Mimitou and Symington 2008; Rattray et al. 2001; Zhu et al. 2008). Our first approach was indirect and took advantage of the fact that the Ku family of proteins has been shown to negatively regulate the DSB resection process (Clerici et al. 2008; Lee et al. 1998). In this regard, the depletion of yKu70 suppressed the DSB sensitivity of the Δsae2 and mre11-nd mutants (Mimitou and Symington 2010). To determine whether cytosolic fumarase is impor-tant for DSB resection we deleted yKu70 in the FumM strain. Our rationale was that if the FumM strain suffers from a defect in the resection process, the depletion of a negative regulator of this process may improve resection, thus reduc-ing the sensitivity of FumM to DSBs. The DSB sensitivity of the cells was determined by examining their survival on plates containing hydroxyurea (HU), which induces repli-cation-associated DSBs (Fig. 2a, Supplementary Fig S1D) (Adamczyk et al. 2016; Saintigny et al. 2001). The knock-out of yKu70 partially inhibited the DSB sensitivity of the cytosolic fumarase depleted yeast. This result provided the first indication for the importance of the cytosolic fuma-rase to the DSB resection process. To rule out the possi-bility that the depletion of cytosolic fumarase may simply increase yKu70 expression levels, thereby inhibiting DSB resection, we determined the yKu70 transcript levels in a strain depleted for cytosolic fumarase (Supplementary Fig

Fig. 2 Cytosolic fumarase is important for the DSB resection. a Overnight cultures of the indicated yeast strains were diluted to OD600 = 0.5 and incubated for 90  min at 30  °C. The cultures were then serially diluted and plated on SC-Dex with or without 200 mM hydroxyurea (HU). b Logarithmic cultures of the indicated strains

were treated with 350  mM HU. At the indicated time points sam-ples were analyzed by Western blot using anti-Rad53 antibody. c Densitometric quantification of the experiments described in b. *p = 4.58 × 10−2 and **p = 4.01 × 10−2. Error bars SEM

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S1E). No significant differences in the yKu70 transcript lev-els were detected under cytosolic fumarase depletion.

The Rad53 checkpoint kinase is activated by multi-site phosphorylation following DNA damage induction (Lee et al. 2003; Sanchez et al. 1996; Sun et al. 1996). Efficient phosphorylation of Rad53 appears to be dependent on DSB resection, and accordingly, we determined the capacity of the FumM strain to induce Rad53 phosphorylation upon DSB induction (Ira et al. 2004; Pellicioli et al. 1999; Vaze et al. 2002). Logarithmic cultures of WT and FumM yeast were treated with HU, and the phosphorylation state of Rad53 determined by the reduced electrophoretic mobility of the phospho-Rad53 species (Fig. 2b, detected as an up-shift area above the non-phosphorylated form). The deple-tion of cytosolic fumarase reduced Rad53 phosphorylation compared to the WT strain. Following DSB induction, the FumM strain exhibited significantly reduced phospho-Rad53 to nonphospho-Rad53 ratios compared to the WT (Fig. 2c, see supplementary Fig S1F for details regarding the quanti-fication). Interestingly, our preliminary data suggested that FumM cells also exhibit a reduction in the total Rad53 pro-tein level, therefore, we cannot rule out the possibility that the depletion of cytosolic fumarase can also influence Rad53 abundance. The phosphorylation and activation of Rad53 have been shown to require Tel1/Mec1 activity (Sanchez et al. 1996; Sun et al. 1996). Worth mentioning is the fact that we find no significant changes in Tel1 and Mec1 tran-script levels due to the depletion of cytosolic fumarase (Sup-plementary Fig S2A). Together, these results support the suggestion that cytosolic fumarase may be important for the resection process.

To further investigate the importance of cytosolic fuma-rase in the resection process, we used mre11-nd (nuclease-dead) yeast mutants (Moreau et al. 1999). The nuclease activity of Mre11 has been shown to be important for the initial step of the resection process both in yeast and human (Anand et al. 2016; Cannavo and Cejka 2014). Accord-ingly, we overexpressed the cytosolic form of fumarase (ΔMTS-Fum) in the mre11-nd mutant strains (Fig. 3a). We assumed that if cytosolic fumarase is important for DSB resection, then its overexpression may improve the resec-tion ability of the mre11-nd yeast, thereby rendering them less sensitive to DNA DSBs. Overexpression of cytosolic fumarase partially suppressed the sensitivity of the mre11-nd cells to DNA DSBs (Fig. 3a, see plating efficiency in the presence and absence of HU, compare row 2 to row 4 and row 3 to row 5). Worth motioning is that the differ-ence in the HU sensitivity between the mre11(D56N)-nd and the mre11(H125N)-nd mutants, seen following cyto-solic fumarase overexpression, is probably due to different inherent properties of these strains (Hamilton and Maizels 2010). Next, we examined if this positive effect can also be achieved by exposure of the mre11-nd yeast to fumarate,

a product of fumarase enzymatic reaction (Fig. 3b, Sup-plementary Fig S2B). WT and mre11 (H125N)-nd strains were exposed to MMS and plated on buffer only (potassium phosphate), lactate- or fumarate-containing agar plates. The exposure to fumarate, but not buffer only or lactate, strongly reduced the DSB susceptibility of the mre11 (H125N)-nd yeast (Fig. 3b, Supplementary Fig S2B). These observa-tions indicate that cytosolic fumarase and the product of its enzymatic reaction are important for the DSB resection process. Furthermore, these results suggest that the effect of fumarase on the resection process is not mediated by Mre11, since overexpression of fumarase or fumarate can suppress the mre11-nd mutant phenotype. Therefore, we speculated that cytosolic fumarase may affect some other DDR factor in the resection process.

The Sae2 endonuclease is also involved in the initial step of the DSB resection (Cannavo and Cejka 2014; Clerici et al. 2005; Mimitou and Symington 2008; Rattray et al. 2001), so we explored whether there is a functional interplay between cytosolic fumarase and Sae2. Similar to the FumM strain, reduction in Sae2 cellular levels has been shown to inhibit the phosphorylation of Rad53 upon DSB induction (Robert et al. 2011). The similarity of the Rad53 phenotypes which are induced by the depletion cytosolic fumarase and the reduction in Sae2 protein level, supports the possibility that these proteins functionally interact. To further investigate this possibility, we overexpressed the Sae2 endonuclease in the FumM strain, and checked the DSB susceptibility of the FumM yeast (Fig. 3c). The overexpression of Sae2 in the FumM strain, indeed reduced DSB sensitivity, as determined by the ability of the cells to grow following exposure to MMS (Fig. 3c). On the other hand, the overexpression of cytosolic fumarase did not affect the DSB susceptibility of the Δsae2 yeast (Fig. 3d. Taken together these observations point to a functional relationship between cytosolic fumarase and Sae2. Notably, similar genetic experiments involving the resection factors Exo1 and Sgs1 did not yield indications of a functional interaction with cytosolic fumarase (Supple-mentary Fig S2C–E).

To directly assess the importance of the cytosolic fuma-rase population for the DNA DSB resection, we induced a single DSB at the MAT locus by HO endonuclease expres-sion. Subsequently, we employed a PCR-based method to evaluate the resection kinetics 0.29kbp from the HO cut site (Fig. 4a) (Chen et al. 2013; Ferrari et al. 2015; Zierhut and Diffley 2008). Using this approach, we amplified a short sequence adjacent to the DSB, which includes a SspI restric-tion enzyme cutting site. Resection of this region abolishes the ability of SspI to cut its recognition site, thereby enabling PCR amplification (Clerici et al. 2005). By following the accumulation of this PCR product, we were able to calculate the percentage of resected DNA. The depletion of cytosolic fumarase strongly inhibited the DSB resection ability of the

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cells (Fig. 4b). However, the resection ability of the FumM strain was completely restored by the re-expression of cyto-solic fumarase or the overexpression of Sae2. These results indicate that cytosolic fumarase and Sae2 functionally coop-erate in the resection process.

In addition to the functional relationship between cyto-solic fumarase and Sae2 endonuclease, we asked whether there is a physical interaction in-vivo. For this, the split-ubiquitin assay was employed (Fig. 5a) (Lehming 2002). The cytosolic form of fumarase (Fum1ΔMTS) was fused to the N-terminal domain of ubiquitin (Nub), while Sae2 was fused to the ubiquitin C-terminal domain (Cub) and the enzyme Ura3 that had been modified to start with an arginine (RUra3). If there is a physical interaction between fumarase and Sae2, the Nub and Cub domains will be in close prox-imity with each other and form a functional ubiquitin-like

moiety. This leads to the cleavage and subsequent degrada-tion of the RUra3 enzyme, thus rendering the yeast incapable of surviving on media lacking uracil. Sae2-Cub-RUra3 was expressed from a single-copy vector under the SAE2 pro-moter. The expression of Sae2–Cub–RUra3 fusion restored growth of cells lacking Sae2 on HU plates, demonstrat-ing that this fusion is fully functional (Supplementary Fig S3A). In addition, cells expressing Sae2–Cub–RUra3 were capable of growing on plates lacking uracil, demonstrat-ing that RUra3 is enzymatically active (Fig. 5b). Cytosolic fumarase fused to Nub (Nub–Fum1ΔMTS) was expressed from a single-copy vector under the control of the ADH1 promoter. Western Blot analysis showed that the expression of Nub–Fum1ΔMTS from the ADH1 promoter was compa-rable to that of endogenous fumarase, while the expression of Nub–Fum1ΔMTS from the endogenous FUM1 promoter

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Fig. 3 Cytosolic fumarase is involved in DSB resection and function-ally interacts with Sae2. a Overnight cultures of the indicated yeast strains were diluted to OD600 = 0.5 and incubated for 90 min at 30 °C. The cultures were then serially diluted and plated on SC-Gal with or without 200 mM hydroxyurea (HU). b The indicated strains were prepared for the experiment as in A, then incubated for 1 h with or without 0.2% (v/v) MMS and plated on YP-Dex containing potas-

sium phosphate buffer, and supplemented with either 25  mM lactic acid (Lactate), 25  mM fumaric acid (Fumarate) or no organic acid (Buffer). c The indicated strains were prepared for the experiment as in a, then incubated for 1 h with or without 0.2% (v/v) MMS and plated on SC-Dex plates. d The experiment was conducted as in c. The indicated strains were plated on SC-Gal plates

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was much lower (Supplementary Fig S3B). The expression of Nub–Fum1ΔMTS in cells lacking FUM1 restored growth on plates containing HU, demonstrating that the fusion is functional (Supplementary Fig. S3C). We found that coex-pression of Nub–Fum1ΔMTS and Sae2–Cub–RUra3 sig-nificantly decreased the ability of the cells to grow on media lacking uracil (Fig. 5b). This result indicates that the cyto-solic fumarase and the Sae2 endonuclease physically interact with each other in-vivo. Notably, this interaction occurs in the presence and also in the absence of DSB induction fol-lowing exposure to HU (Fig. 5b).

The split-ubiquitin assay indicates interaction of fumar-ase and Sae2 in vivo, however, it is possible that this inter-action is mediated by other proteins. To demonstrate that fumarase and Sae2 interact with each other directly, both proteins were first purified from E. coli (Fig. 5c). We used cytosolic versions of fumarase without its mitochondrial targeting signal (Fum1ΔN23, lacking the first 23 amino acids, or Fum1ΔN19, lacking the first 19 amino acids) fused to GST. Sae2 was fused to six histidines and the HA-tag (H6–HA–Sae2). Figure 5c shows that GST–Fum1ΔN23

(lane 7) and GST–Fum1ΔN19 (lane 9), but not GST alone (lanes 6 and 8), were able to retain purified H6–HA–Sae2 on columns packed with glutathione beads, indicating that fumarase interacts directly with Sae2.

To further characterize the functional relationship between cytosolic fumarase and Sae2, we tried to deter-mine whether the depletion of cytosolic fumarase can influ-ence the cellular levels of Sae2. This was interesting, since Sae2 abundance was previously shown to be important for the normal rate of the DSB resection (Robert et al. 2011; Tsabar et al. 2015). First, we addressed the possibility that cytosolic fumarase can regulate Sae2 at the transcript level. We determined the level of Sae2 mRNA in WT and FumM cells by quantitative RT-PCR (Supplementary Fig S4A). The results showed no significant difference in Sae2 tran-script level following the depletion of cytosolic fumarase in the presence or absence of HU. These findings suggest that cytosolic fumarase probably does not regulate Sae2 at the transcript level. Next, we wished to determine whether cyto-solic fumarase depletion can alter Sae2 protein level. Sae2 fused to a 3xHA tag and ten histidines (Sae2–HA3–H10), was

-15%

5%

25%

45%

65%

A

HO

No PCR product

SspI(-0.29kbp)

SspI diges�on

PCR product

Mock diges�on

HO

Unresected

HO

PCR product

SspI(-0.29kbp)

SspI diges�on

Resected

HO

PCR product

Mock diges�on

B

Perc

enta

ge o

f res

ecte

d (s

ingl

e-st

rand

ed) D

NA

Time a�er HO induc�on [min]

*

*****

WT

FumM

FumM pSae2

FumM pΔMTS-Fum

Fig. 4 Cytosolic fumarase affects DSB resection. a Schematic rep-resentation of the quantitative PCR-based, DSB resection assay (Chen et  al. 2013; Ferrari et  al. 2015; Zierhut and Diffley 2008). b HO nuclease was induced [2% (w/v) galactose] in logarithmic cul-

tures of the indicated strains, grown in YP-raffinose. At the indi-cated time points, the fraction of resected (single-stranded) DNA in these cultures was determined by quantitative PCR. *p = 3.66 × 10−4, **p = 2.74 × 10−5 and ***p = 4.87 × 10−4. Error bars SEM

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expressed from a single copy plasmid under its endogenous promoter (Fig. 5d, e). The expression of Sae2–HA3–H10 reduced the DSB sensitivity of cells lacking Sae2, indicating that the fusion is functional (Supplementary Fig S3A). We detected a reduction in the protein level of Sae2 in the FumM strain, compared to the WT (Fig. 5d, compare lanes 3 and 5). The expression of cytosolic fumarase in this strain partially restored the Sae2 protein level (Fig. 5d, compare lanes 1 and 3). Similar effects were observed following the exposure of the cells to HU (Fig. 5d, compare lanes 2, 4 and 6). We also generated WT and FumM strains in which the 3×FLAG tag was inserted at the 3′ end of the SAE2 gene on chromo-some VII (Supplementary Fig. S4B, C). Cytosolic fumarase depleted cells showed reduced Sae2 protein levels compared

to the WT. This reduction was also evident following the treatment of the cells with HU for two hours (Supplementary Fig S4B, compare lanes 4 and 8). Interestingly, the reduc-tion in Sae2 protein level was not statistically significant following the exposure to HU for shorter periods of time (30 and 60 min, Supplementary Fig S4C). These results suggest that cytosolic fumarase can affect Sae2 protein levels. In addition, this implies that cytosolic fumarase probably acts upstream of Sae2, as it can influence its protein abundance.

Taken together, our results suggest that the cytosolic population of fumarase is important for the DSB resec-tion process and the HR repair pathway. The involvement of fumarase in DSB resection is mediated by its functional relationship with the Sae2 endonuclease. This functional

WTFumMStrain

--+pΔMTS-Fum

+-+-+-HU

A

Nub

Nub-Fum1ΔMTSSae2-Cub-RUra3

- Uracil - Uracil + HU

B

Fumarase

Nub

Sae2

CubRUra3

No interac�on

Fumarase

Nub

Sae2

CubRUra3raRU 3RUrra3Ura

Interac�on

D

pSae2-HA3-H10

Control

Control

0 20 40 60 80 100 120

FumM pΔMTS-Fum

FumM pΔMTS-Fum + HU

FumM

FumM + HU

WT

WT + HU

Rela�ve protein level

pSae2-HA3-H10E

C

Fig. 5 Cytosolic fumarase interacts with Sae2 in  vivo and in  vitro, and influences its protein levels. a Schematic diagram of the Split-ubiquitin system. Left: the Sae2–Cub–RUra3 fusion expressed form a single-copy vector under the control of the SAE2 endogenous promo-tor, allows cells to grow on plates lacking uracil. Right: The interac-tion between Sae2–Cub–RUra3 and cytosolic fumarase fused to Nub (Nub–Fum1ΔMTS) leads to the formation of a ubiquitin-like moiety, rapid degradation of RUra3 and incapability to grow on plates lack-ing uracil. b BY4743ΔW cells expressing the indicated fusions were serially diluted, and spotted onto control plates (control), onto plates lacking uracil (-Uracil) and onto plates lacking uracil that contain 100 mM HU (-Uracil + HU). c Cytosolic fumarase without its mito-chondrial targeting signal was fused to GST (Fum1ΔN23 lacks the N-terminal 23 residues while Fum1ΔN19 lacks the N-terminal 19 residues). Sae2 was fused to six histidines and the HA-tag (H6–HA–

Sae2). These fusion proteins were expressed in E. coli and purified. H6–HA–Sae2 was incubated with gluthatione beads containing GST alone, GST–Fum1ΔN23 or GST–Fum1ΔN19. The beads were loaded onto spin columns, the proteins were eluted with SDS loading dye, and analyzed by Western blot using anti-HA and anti-GST antibodies. A 10% input is included for normalization purposes. d Cells harbor-ing a single-copy vector expressing Sae2–HA3–H10 under the control of the SAE2 promoter (pSae2–HA3–H10), or also expressing cytosolic fumarase (pΔMTS–Fum), were grown to mid-log phase and induced (+ HU) or not induced (-HU) with 400  mM HU for 2  h. Sae2 pro-tein levels were determined by Western blot analysis using anti-HA antibody. An anti-CPY (Carboxypeptidase Y) antibody was used as a loading control (Control). e Western blot bands were quantified with ImageQuant (BioRad). Shown are ratios of the bands for Sae2–HA3–H10 normalized to the bands for CPY of the same lane. Error bars SD

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interplay is characterized by a physical interaction between the proteins. In addition, cytosolic fumarase is suggested to act upstream of Sae2, presumably by regulating its protein level.

Discussion

DNA DSBs are one of the most cytotoxic forms of DNA damage. Defective or inappropriate repair of such lesions can lead to gross chromosomal rearrangements, including large deletions, insertions and translocations. Such rear-rangements can induce oncogene misexpression and loss of tumor suppressor genes, both of which have been implicated in cancer induction and progression (Hanahan and Weinberg 2011; Lengauer et al. 1998; O’Driscoll 2012; Richardson and Jasin 2000; Shiloh and Lehmann 2004; van Gent et al. 2001). Therefore, the identification and characterization of new factors that play a role in the cellular response against DSBs, is extremely important.

The human homolog of fumarase, termed fumarate hydratase (FH), has been shown to function as a tumor sup-pressor. Heterozygous mutations in the FH gene were shown to cause the HLRCC syndrome. Individuals with HLRCC can suffer from multiple cutaneous and uterine leiomyomas and are prone to develop type II papillary renal cell carci-noma. HLRCC is a dominantly inherited syndrome charac-terized as a two-hit condition. It has been demonstrated that virtually all tumors of HLRCC patients exhibit deactivation of both FH alleles. This observation stresses the importance of the complete loss of FH activity to the tumorigenesis pro-cess (Kiuru et al. 2001; Launonen et al. 2001; Reed et al. 1973; Tomlinson et al. 2002). In spite of the established involvement of FH in HLRCC, mutations in this gene are rarely detected in sporadic tumors. Nevertheless, biallelic inactivation of FH has been reported in some cases of uter-ine leiomyomas, soft tissue sarcoma and type II papillary renal cell carcinomas (Barker et al. 2002; Gardie et al. 2011; Kiuru et al. 2002; Lehtonen et al. 2004).

Extensive research has been conducted to determine the mechanism by which FH functions as a tumor suppres-sor. The evidence suggests that the loss of FH activity and the accumulation of fumarate inhibits the α-ketoglutarate-dependent prolyl hydroxylase enzymes (PHD) 1, 2 and 3. The inhibition of PHD stabilizes the α subunit of the hypoxia-inducible transcription factor (HIF), resulting in the formation of an active HIF transcription complex. Increased HIF levels have been shown to enhance angiogenesis and glucose metabolism, both of which are known to be essen-tial for tumorigenesis (Hanahan and Weinberg 2011; Isaacs et al. 2005; Pollard et al. 2005; Selak et al. 2005; Vanharanta et al. 2006). Our previous and current studies suggest an

additional mechanism by which the loss of fumarase can contribute to tumor development (Yogev et al. 2010).

Our previous work indicated that fumarase is important for the DSB DDR in human cell lines, and not only in yeast. The cellular levels of FH increased following DSB induc-tion and nuclear translocation of the protein was observed. In addition, FH knockdown has been shown to increase cell susceptibility to HU and ionizing radiation induced DSBs (Yogev et al. 2010). These results have been further sup-ported in a recent study by Jiang et al. which provided evi-dence for the exact role of FH in the DDR against DSBs in human cells (Jiang et al. 2015). As mentioned above, genomic instability is one of the hallmarks of cancer (Hana-han and Weinberg 2011). Therefore, the loss of fumarase as a guardian of genome integrity contributes to the develop-ment of cancer.

The comprehensive study by Jiang et al. suggested that following DSB formation FH is phosphorylated by the DNA-dependent protein kinase (DNA-PK) complex at Thr 236. This modification was shown to be required for the recruitment of FH to the DSB. Following its recruit-ment, FH-mediated fumarate production was suggested to inhibit the α-ketoglutarate-dependent lysine demethylase 2B (KDM2B). KDM2B inhibition increased the H3K36me2 which subsequently led to the enhanced accumulation of DNA–PK complex and the repair of the break by NHEJ. The phosphorylation of FH at Thr 236 was suggested not to be required for the function of the HR repair pathway. Inter-estingly, a mutation of this residue did not affect γ-H2AX levels even though it would be expected due to the reduced DNA–PK accumulation at the DSB (An et al. 2010; Jiang et al. 2015; Stiff et al. 2004). The aberrant kinetics of H2AX phosphorylation following the knockdown of FH was pre-viously described by us and confirmed by Jiang et al. Nev-ertheless, the mutation of Thr 236 did not affect γ-H2AX (Jiang et al. 2015; Yogev et al. 2010). This observation sug-gests that FH function in the DSB DDR is not limited to the NHEJ pathway.

In contrast to the results presented by Jiang et al., indi-cating involvement of fumarase in the human NHEJ repair, our FumM yeast model indicates that fumarase is involved in the HR pathway. The yeast S. cerevisiae exhibits simi-larity to human cells in many aspects and can be used as a model organism for the investigation of numerous cellular mechanisms (Duina et al. 2014; Sarto-Jackson and Tomaska 2016). Before discussing our results in yeast, we would like to point out that transfer of the FUM1 gene to the mitochon-drial genome in the FumM strain does not present a com-pletely normal metabolic phenotype. Fumarase protein levels in the FumM cells was shown to be lower than in the WT, and additional mitochondrial genes, which are located near the FUM1 insertion site in the mitochondrial genome, may also be affected. These aberrations are probably responsible

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for the lower than normal ability of the FumM strain to grow on media which requires respiration (data not shown). Nev-ertheless, it should be noted that in spite of the phenotypes described above, the FumM strain exhibits significant mito-chondrial functionality compared to Δfum1 cells and is the best existing model for cytosolic depletion of fumarase.

Another problem that can arise from using cells with altered mitochondrial metabolism, in DDR research, is the possibility of excessive production of reactive oxygen spe-cies, which can endogenously induce DNA damage (Evans et al. 1997). Notably, our results suggest that endogenous damage is not prominent in the FumM strain, as such damage would presumably increase the percentage of cells that pre-sent foci of all the DDR regulators that have been assessed in Fig. 1a. However, only the percentage of cells that present Mre11 foci was significantly elevated in the FumM strain.

Based on our results, we speculate, that cytosolic fuma-rase in yeast is important for the initial step of the resection process. First, as stated above, we did not detect any genetic interactions with the extensive resection factors (Supple-mentary Fig. S2C–E). Furthermore, previous observations indicated that during the initial step of resection 50–1600 bases of DSB flanking DNA can be processed (Garcia et al. 2011; Mimitou and Symington 2008; Zhu et al. 2008). Our resection assay was designed to measure resection 0.29kbp upstream the HO cut site (Fig. 4a). If following the depletion of cytosolic fumarase only the extensive step of resection is affected, one would detect at least some level of initial resec-tion 0.29 kbp from the DSB. The fact that resection was not detected, suggests that cytosolic fumarase is important for the initial step of the DSB resection process.

This suggestion is supported by the functional interac-tion between cytosolic fumarase and Sae2. The interaction was first implied by the phenotypic similarities between the FumM and the Δsae2 strains. Both strains exhibit delayed dissociation of Mre11 from DSBs, impaired kinetics of DSB repair and decreased resection (Clerici et al. 2005; Ferrari et al. 2015; Lisby et al. 2004). The overexpression of Sae2 partially suppressed the DSB susceptibility of the FumM strain and reconstituted its resection capacity. In addition, based on the split ubiquitin assay, we find that these proteins physically interact in-vivo. The remaining questions were whether cytosolic fumarase acts upstream of Sae2, and if so, by what mechanism does it regulate this endonuclease? One clue was the reduced Sae2 protein levels in cytosolic fumar-ase depleted cells, which suggested that cytosolic fumarase acts upstream of Sae2, presumably by regulating its protein abundance. This regulation is conducted at the protein level, and not the Sae2 mRNA level. However, it is still unclear whether cytosolic fumarase is important for the translation or degradation of Sae2. It is conceivable that cytosolic fuma-rase may have a positive effect on the translation of Sae2 or a negative effect on its degradation. Regarding the possibility

that cytosolic fumarase may be important for shielding Sae2 against degradation, we tried to determine whether fumarase is involved in the mechanism of the autophagy-mediated Sae2 degradation, yet our preliminary genetic experiments were inconclusive (Robert et al. 2011).

Another major question is what is the role of the fumar-ase enzymatic activity and its product fumarate in the func-tional relationship between cytosolic fumarase and Sae2? We showed that fumarate can inhibit the DSB sensitivity of the mre11-nd mutant cells. This suggests that fumarate may be important for the resection process. Nevertheless, it is unclear whether fumarate is important for the activity or the protein level maintenance of Sae2, and if so, what is the mechanism of action.

Fumarase is a highly conserved enzyme that has been proposed to be important for the two main DSB repair path-ways. In this study, we shed some light on the mechanism of cytosolic fumarase involvement in the yeast DSB DDR. Our results suggest that cytosolic fumarase is important for the DSB repair by the HR pathway, and that cytosolic fumarase plays a role in the DSB resection process. Consistently, the function of cytosolic fumarase in resection is mediated by the Sae2 endonuclease, whose levels are regulated by cyto-solic fumarase. Nevertheless, additional enquiries need to be made in order to decipher the complex role of fumarase in the DDR pathway. Deeper comprehension of this role may help us to better understand the function of fumarase as a tumor suppressor.

Materials and methods

Yeast strains

The FumM strain has been previously described (Yogev et al. 2010). The WT (BY4741) strains, in which Mre11, Rad9, Ddc2, Rad24 and Rad54 were fused to YFP and the Mre11-nd mutants, were kindly provided by Prof. Michael Lisby at the University of Copenhagen. The construction of FumM strains which express the same YFP fusions, was performed using a previously described PCR-based method (Reid et al. 2002). All the knockout strains used in this study have been obtained from the European Saccharomyces cerevisiae archive for functional analysis (EUROSCARF) (Winzeler et al. 1999). The FumM ΔyKu70 strain was generated by transforming FumM cells with a PCR product containing the KanMX4 cassette flanked by sequences homologous to the upstream and downstream regions of the YKU70 gene (Bau-din et al. 1993; Wach et al. 1994). The WT (BY4742) and FumM, Sae2-3×FLAG strains were generated by transforma-tion with a PCR product amplified using the pFA6a-6 ×GLY-3×FLAG-hphMX4 vector (Addgene #20755) (Funakoshi and Hochstrasser 2009).

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Growth conditions

Yeast were grown at 30 °C in yeast extract/peptone (YP) or synthetic complete (SC) media. YP media contains 10 g/L yeast extract and 20  g/L peptone. SC media contained 6.7 g/L yeast nitrogen base and a complete supplement mix-ture (CSM). 2% (w/v) dextrose, galactose or raffinose were used as a carbon sources. Solid media contained 18 g/L agar. Logarithmic cultures were obtained by diluting stationary phase cultures to OD600 = 0.5, and incubating at 30 °C until the yeast cultures reached OD600 ≅ 1.0.

Plasmids

The pGAL-HO expression plasmids were kindly provided by Prof. James E Haber (Herskowitz and Jensen 1991; Nickoloff et al. 1989). The pΔMTS–Fum vector has been previously described (Yogev et al. 2010). The pGAL–Sae2 vector was constructed by cloning of the SAE2 gene into the p425GAL1 yeast expression vector. The pGAL–Sae2 vector also underwent a “marker swap” conversion, in order to be used with a uracil selection (Cross 1997). The pGAL–Exo1 vector was constructed by cloning of the EXO1 gene into the p426GAL1 yeast expression vector. The Sae2–HA3–H10 fusion was cloned into the single-copy vector YCplac33, under the endogenous SAE2 promoter. The Sae2-Cub–RUra3 fusion was constructed by cloning of the SAE2 gene and its endogenous promoter, into the CubRUra314 vector (Laser et al. 2000). Fum1ΔN23 and Fum1ΔN19 were cloned into GEX-5X-1 (GE Healthcare) and H6–HA–Sae2 was cloned into pET11a (Novagen).

Western blot analyses and antibodies

Western blots were performed using standard procedures (Regev-Rudzki et al. 2005). Densitometric analyses was performed using ImageJ or TINA software (Schneider et al. 2012). The following antibodies were used during this study: anti-Rad53 [EL7.E1] (abcam #ab166859), anti-fumarase (previously generated) (Yogev et al. 2010), anti-FLAG M2 (Sigma-Aldrich #F3165), anti-aco1 (previously generated) (Regev-Rudzki et al. 2005), anti-HA (Roche #12CA5), anti-CPY (Invitrogen #6428), anti-GST (Novagen #71097), anti-pgk1 (Abcam #113687).

DSB resection assay

The percentage of resected DNA was determined using a previously described method (Chen et al. 2013; Ferrari et al. 2015; Zierhut and Diffley 2008). The genomic DNA was digested by SspI (NEB #R0132S), and amplified using ABsolute™ Blue qPCR SYBR® Green (Thermo Scientific #AB4163) and Mic qPCR Cycler (bio molecular systems).

The percentage of resected (single-stranded) DNA was calculated using the following formula: % ssDNA = (100/(((1 + 2ΔCt)*0.5)/2))/f, where ΔCt = (Ct[SspI digested template] − Ct[SspI undigested template]) and f is the fraction cut by HO, which was determined by quantitative PCR.

Microscopy

For all the experiments cells were grown in SC-Dex media. The yeast live cell fluorescence microscopy was performed as previously described (Eckert-Boulet et al. 2011; Lisby et al. 2004).

DSB repair assay

The assay was performed as previously described (Haber et al. 1993). To determine the kinetics of DSB repair, we preformed densitometric analyses of the agar gels, and cal-culated the repair rate by the linear slope of the reaction (Schneider et al. 2012). The obtained rates were normal-ized to the proportion of MATα cells in the culture and the HO cleavage efficiency, which were determined by semi-quantitative PCR.

Split‑ubiquitin assay

The assay was performed as described previously (Ang et al. 2012). BY4743 cells co-expressing the Nub–Fum1ΔMTS and Sae2–Cub–RUra3 fusion proteins were tenfold serially diluted and spotted onto control plates, onto plates lacking uracil and onto plates lacking uracil that contained 100 mM HU. The plates were incubated at 28 °C for 6 days. Pro-tein–protein interaction between cytosolic fumarase and Sae2 was revealed by the absence of growth on the plate lacking uracil.

Column retention assay

Cytosolic fumarase (fumarase without its mitochondrial targeting signal, lacking either the first 23 amino acids, Fum1ΔN23, or the first 19 amino acids, Fum1ΔN19) was fused to GST, expressed in E. coli and purified from E. coli protein extracts with the help of glutathione beads (GE Healthcare). Sae2 was fused to six histidines and the HA-tag (H6-HA-Sae2), expressed in E. coli and purified from E. coli protein extract with the help of a Ni-column (GE Healthcare). H6–HA–Sae2 was eluted from the col-umn with two times 0.5 ml 500 mM imidazol and incu-bated with glutathione beads containing GST–Fum1ΔN23, GST–Fum1ΔN19 or GST alone in 0.5 ml 10% PBS for one hour in the coldroom on a roller. The glutathione beads were loaded onto spin columns (GE Healthcare), washed five times with 0.5 ml 10% PBS and eluted from the beads

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with 50 μl 1xSDS loading dye. Protein samples, including 10% input, were separated on two 10% polyacrylamide gels and transferred to two nitrocellulose membranes. The mem-branes were incubated with an anti-HA antibody and with an anti-GST antibody, respectively. Protein bands were visual-ized with the help of the ECL system (GE Healthcare).

Acknowledgements We thank Sheera Adar for critical reading of the manuscript. This work was supported by grants to O. Pines from the Israel Science Foundation (ISF) and the German Israeli Project Coop-eration (DIP). N. Lehming and O. Pines were supported by The CRE-ATE Project of the National Research Foundation of Singapore. M. Lisby was supported by the Danish Council for Independent Research and the Villum Foundation.

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