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DNA Repair 9 (2010) 394–402

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DNA Repair
journal homepage: www.elsevier.com/locate/dnarepair

Functional overlap between the structure-specific nucleases Yen1 and Mus81-Mms4 for DNA-damage repair in S. cerevisiae
Miguel G. Blanco, Joao Matos, Ulrich Rass, Stephen C.Y. Ip 1 , Stephen C. West ∗
London Research Institute, Clare Hall Laboratories, Cancer Research UK, South Mimms, Herts EN6 3LD, UK

a r t i c l e

i n f o

a b s t r a c t
In eukaryotic cells, multiple DNA repair mechanisms respond to a wide variety of DNA lesions. Homologous recombination-dependent repair provides a pathway for dealing with DNA double-strand breaks and replication fork demise. A key step in this process is the resolution of recombination intermediates such as Holliday junctions (HJs). Recently, nucleases from yeast (Yen1) and human cells (GEN1) were identified that can resolve HJ intermediates, in a manner analogous to the E. coli HJ resolvase RuvC. Here, we have analyzed the role of Yen1 in DNA repair in S. cerevisiae, and show that while yen1 mutants are repair-proficient, yen1 mus81 double mutants are exquisitely sensitive to a variety of DNAdamaging agents that disturb replication fork progression. This phenotype is dependent upon RAD52, indicating that toxic recombination intermediates accumulate in the absence of Yen1 and Mus81. After MMS treatment, yen1 mus81 double mutants arrest with a G2 DNA content and unsegregated chromosomes. These findings indicate that Yen1 can act upon recombination/repair intermediates that arise in MUS81-defective cells following replication fork damage. © 2009 Elsevier B.V. All rights reserved.

Article history: Received 29 July 2009 Received in revised form 23 November 2009 Accepted 21 December 2009 Available online 27 January 2010 Keywords: Recombination Recombination intermediates DNA repair GEN1 Rad2/XPG Holliday junction resolvase Yeast

1. Introduction Enzymes that process unusual DNA structures such as DNA flaps and Holliday junctions are required for the restart of stalled DNA replication forks and the resolution of recombination intermediates. Eukaryotic cells possess a number of structure-specific endonucleases that carry out such functions. In Saccharomyces cerevisiae these include Mus81-Mms4 and Slx1-Slx4, two heterodimeric endonucleases that cleave a wide range of branched DNA substrates [1–4]. Human orthologs of these proteins have been identified and catalyze similar reactions [5–11]. In addition to these structure-specific endonucleases, specialized Holliday junction (HJ) resolvases have been identified in a variety of organisms including bacteriophage (T4 endonuclease VII, T7 endonuclease I), E. coli (RuvC), yeast (Cce1 and Ydc2, albeit these proteins function in the mitochondria) and archaea (Hjc and Hje) [12]. These nucleases promote HJ resolution by the introduction of

Abbreviations: HJ, Holliday junction; HR, homologous recombination; MMS, methyl methanesulfonate; HU, hydroxyurea; 4NQO, 4-nitroquinoline 1-oxide; CPT, camptothecin; IR, ionizing radiation. ∗ Corresponding author. Tel.: +44 1707 625868; fax: +44 1707 625801. E-mail addresses: miguel.blanco@cancer.org.uk (M.G. Blanco), joao.matos@cancer.org.uk (J. Matos), uli.rass@cancer.org.uk (U. Rass), cyip@govtlab.gov.hk (S.C.Y. Ip), stephen.west@cancer.org.uk (S.C. West). 1 Current address: Forensic Science Division, Hong Kong Government Laboratory, Ho Man Tin Government Offices, 88 Chung Hau Street, Kowloon, Hong Kong, China. 1568-7864/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2009.12.017

symmetrically related nicks in two strands of like polarity, leading to the formation of nicked duplex products that can be readily ligated [13,14]. The search for nuclear eukaryotic HJ resolvases that fit this paradigm recently resulted in the identification of Yen1 from S. cerevisiae, together with its ortholog GEN1 from human cells [15,16]. Yen1/GEN1 are members of a subgroup (class IV) of the Rad2/XPG family of structure-specific nucleases, which includes a number of proteins involved in DNA replication, recombination and repair, such as Rad27 (FEN1 in humans), Rad2 (XPG) and Exo1 (EXO1) [17]. In addition to HJ resolution activity, Yen1/GEN1 retain the characteristic 5 -flap endonuclease activity of the Rad2/XPG family. The cellular functions of Yen1/GEN1 are presently unknown. In contrast to Yen1/GEN1, Mus81 belongs to the conserved Rad1/XPF family of 3 -flap endonucleases that are implicated in repairing DNA lesions induced by exposure to UV-light, alkylating or cross-linking agents [4]. All proteins in the Rad1/XPF family are heterodimeric endonucleases and yeast Mus81, in combination with Mms4 (or Eme1 in S. pombe), resolves recombination intermediates and promotes essential repair reactions in response to DNA damage, in particular those necessary for the repair and restart of stalled replication forks [3]. Loss of Mus81 sensitizes cells to a variety of DNA-damaging agents such as UV-light, HU, MMS and CPT, all of which induce replication fork stalling or collapse [18–20]. Moreover, synthetic interactions between MUS81 and genes involved in DNA replication have been detected in fission and budding yeast [21–23]. Synthetic lethality is also observed for mus81 in combination with sgs1 (rqh1 in S. pombe) [24]. Sgs1, the only member of

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the RecQ family of helicases in yeast, is required for a variety of reactions relevant to the maintenance of genomic stability, such as the stabilization of replisomes, the processing of stalled replication forks, checkpoint signalling, and the unwinding or dissolution of early and late homologous recombination intermediates [25]. The synthetic lethality of S. cerevisiae mus81 sgs1 double mutants can be suppressed by deletion of recombination functions mediated by RAD51, RAD52 or RAD54 [26], while in S. pombe it can be rescued by over-expression of the cryptic bacterial Holliday junction resolvase RusA [20]. These results indicate that Mus81 may be involved in a parallel, but overlapping pathway with Sgs1 for the removal of potentially toxic recombination intermediates. In an attempt to integrate these data, several models have been proposed where Mus81 could act at early steps in replication fork repair, by cleaving stalled or regressed forks, and/or late steps, by processing recombination intermediates generated during repair [3]. The preferred in vitro DNA substrates of Mus81-Mms4/Eme1 are branched structures with extended 3 -flaps [27–29]. The nuclease is efficient at cleaving Y-shaped structures similar to those that might arise after replication fork arrest (i.e. forks containing leading strand gaps, or partially regressed forks), D-loop structures and nicked Holliday junctions [29–31]. However, the ability of purified Mus81-Mms4/Eme1 to resolve intact HJs is limited and proposals for a role of Mus81 in Holliday junction resolution have been controversial [1,3,32–35]. Recent studies in human cells, however, suggest that the protein may be part of a HJ resolvase complex in combination with other endonucleases such as SLX1-SLX4 and XPF-ERCC1 [8,9]. Certainly, there is compelling genetic evidence showing that mus81 and mms4/eme1 mutants present severe meiotic phenotypes, especially in S. pombe [19,21,36–38], suggesting a role for these proteins in the processing of recombination intermediates. Given that the Yen1/GEN1 proteins were identified on the basis of biochemical assays for HJ resolution, and that there are potential overlaps between these resolvases and Mus81 in terms of their substrate specificities, it is likely that these newly identified enzymes may also play important roles in recombination and DNA repair. As a first step towards their characterization and definition of their cellular functions, we have determined whether Yen1 is involved in DNA-damage repair in vegetative S. cerevisiae cells and uncovered its relationships with Mus81. 2. Materials and methods 2.1. Yeast strains A list of strains is provided in Table 1. MUS81, SGS1 or RAD52 gene deletions were generated by PCR-based gene replacement with HIS3 or URA3. All manipulations were carried out according to standard methods [39]. 2.2. Plasmid construction The RAD27, RAD2, EXO1 and YEN1 coding sequences were amplified from BY4741 genomic DNA and cloned into pDONR221 using the Gateway system (Invitrogen, USA). Further sub-cloning into pYES-DEST52 (Invitrogen, USA) or pAG416GPD-ccdB-HA [40] was carried out to generate pYES-DEST52-RAD27-V5-6xHis, pYES-DEST52-RAD2-V5-6xHis, pYES-DEST52-EXO1-V5-6xHis, pYES-DEST52-YEN1-V5-6xHis and pAG416GPD-YEN1-3xHA. Sitedirected mutagenesis was performed to generate pAG416GPDYEN1E193A/E195A -3xHA, encoding a catalytically inactive version of Yen1-3xHA. The coding sequence of Yen1-V5-6xHis was amplified from pYES-DEST52-YEN1-V5-6xHis and sub-cloned into the BamHI and HindIII sites of p416ADH [41] to generate p416ADH-YEN1-V56xHis. The C-terminal V5-6xHis-tagged versions of Rad2, Rad27

Table 1 List of strains used in this study. Strain BY4741 YWL166a YWL167b YWL168b YWL169b YWL170b YWL229b YWL232b YWL230b YWL239b YWL247a YWL248a YWL249a YWL250a YWL251a
a b a

Genotype MATa his3 1 leu2 0 met15 0 ura3 0 BY4741 yen1 ::kanMX4 BY4741 sgs1 ::HIS3 BY4741 yen1 ::kanMX4 sgs1 ::HIS3 BY4741 mus81 ::HIS3 BY4741 yen1 ::kanMX4 mus81 ::HIS3 BY4741 rad52 ::URA3 BY4741 yen1 ::kanMX4 rad52 ::URA3 BY4741 mus81 ::HIS3 rad52 ::URA3 BY4741 yen1::kanMX4 mus81 ::HIS3 rad52 ::URA3 BY4741 rad27 ::kanMX4 BY4741 rad2 ::kanMX4 BY4741 exo1 ::kanMX4 BY4741 rad52 ::kanMX4 BY4741 pso2 ::kanMX4

Yeast knock-out collection (Open Biosystems, Thermo Fisher Scientific). This study.

and Exo1 were shown to be active, as determined by their ability to suppress mutant phenotypes (data not shown). 2.3. DNA-damage sensitivity assays Cells were grown to mid-logarithmic phase in either YPD or SC-Ura containing 2% glucose. All cultures were normalized to ∼0.5 × 107 cells/ml and 10-fold serial dilutions were spotted onto YPD or SC-Ura + 2% glucose or galactose plates containing different concentrations of MMS, HU, 4NQO, phleomycin or CPT. UV sensitivity was determined by plating cells on YPD after 10-fold dilution, followed by exposure to UV254 light using a CL-1000 UV cross-linker (UVP, USA). To assess ionizing radiation (IR) sensitivity, cells were irradiated in an IBL 437C gamma-irradiator (CisBio International, France) and then spotted onto YPD plates. For the cross-linking agents, nitrogen mustard and cisplatin, cells were incubated with different concentrations of the drugs for 2 h at 30 ◦ C on a rotating platform. The cells were then washed in PBS, serially diluted and spotted onto YPD plates. All plates were incubated at 30 ◦ C in the dark for 2–4 days. All chemicals were purchased from Sigma. 2.4. Doubling time assays Log-phase cultures growing in YPD or SC-Ura + 2% glucose were diluted to an OD600 ∼0.05 and growth was monitored by optical density measurements for 6–12 h. Doubling times were calculated as described [42]. 2.5. Immunoblotting Similar amounts of cells were scraped from plates used for sensitivity assays and resuspended in 1 ml of 50 mM Tris–HCl pH 7.5. Cells were collected, frozen in dry ice and mechanically disrupted with glass beads in 100 l 2% SDS. Lysates were boiled for 3 min and then 100 l 4× SDS-loading buffer was added. Lysates were boiled again for 1 min and centrifuged. Supernatants were recovered and 5 l aliquots were separated by SDS-PAGE through 4–12% NuPAGE Bis–Tris gels (Invitrogen, USA). Proteins were blotted onto Hybond-C super nitrocellulose membranes (GE Healthcare, UK) and detected using anti-V5 or anti-Pgk1 mouse monoclonal antibodies (Invitrogen, USA). 2.6. Cell synchronization and cell cycle analysis Cells were grown to early log-phase and diluted to a density of ∼3 × 106 cells/ml. -Factor was added to a final concentration of 5 g/ml and the cells were incubated at 30 ◦ C for 2.5 h. At this stage

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∼90% of yen1 mus81 mutant cells, and >98% of cells from all other strains tested were unbudded. Cells were washed twice with YPD and resuspended in pre-warmed YPD containing 0–0.01% MMS. Samples were taken every 20 min for 3 h and cells were fixed in 70% ethanol overnight. Fixed cells were washed in 50 mM Tris–HCl pH 7.8, resuspended in the same buffer containing 0.4 mg/ml RNase A and incubated at 37 ◦ C overnight. Cells were then pelleted, resuspended in 55 mM HCl containing 5 mg/ml pepsin, and incubated at 37 ◦ C for 30 min, before being washed with FACS buffer (180 mM Tris–HCl, 190 mM NaCl, 70 MgCl2 ). After resuspension in FACS buffer containing 50 g/ml propidium iodide and dilution in 1 ml 50 mM Tris–HCl pH 7.8, the DNA content of cells was analyzed using a Becton Dickinson FACScan. All chemicals and enzymes were purchased from Sigma. 2.7. Microscopy For immunofluorescence analysis, cells were spheroplasted and stained following methods described elsewhere [43]. Tubulin was detected using rat monoclonal anti-alpha tubulin IgG (1:250 dilution; Serotec) and Alexa Fluor 488-conjugated goat anti-rat IgG (Invitrogen, USA). Images were acquired using a Zeiss AXIO Imager M1 with a 40× EC-PLAN-NEOFLUOR lens and Hamamatsu photonics camera under the control of Volocity software. Images were processed using Adobe Photoshop. 3. Results 3.1. Deletion of YEN1 in a mus81 DNA-damage sensitivity background increases

Since yen1 cells did not exhibit any obvious sensitivity to DNA-damaging agents such as the alkylating agent methyl

methanesulfonate (MMS) (Fig. 1A), we analyzed the effect of Yen1 loss in a mus81 mutant background. Previous studies have shown that mus81 cells are sensitive to MMS [18], which has been suggested to induce the formation of single-strand DNA breaks (SSBs) and stalled replication forks [44]. We found that the yen1 mus81 double mutant displayed a more severe repair-defective phenotype than the mus81 mutant (Fig. 1A). Indeed, approximately 4-fold higher doses of MMS were required to produce comparable levels of growth inhibition in the mus81 cells compared with the yen1 mus81 double mutant. These results indicate that Yen1 and Mus81-Mms4 provide alternative and/or overlapping pathways for the repair of MMS-induced DNA lesions. To expand our view of the repair-deficiency associated with the loss of Yen1 and Mus81-Mms4 activities, the same strains were subjected to treatment with a wide variety of DNA-damaging agents (Fig. 1B). We found that yen1 mus81 cells showed a hypersensitivity to all agents for which the mus81 single mutant was sensitive: HU, 4NQO, phleomycin, CPT, UV-light, nitrogen mustard and cisplatin. In contrast, the damage resistance of yen1 cells was similar to wild-type. All these agents induce DNA lesions that disturb replication fork progression, leading to fork arrest or collapse. Neither the yen1 or mus81 single mutants nor the yen1 mus81 double mutant showed any sensitivity to ionizing radiation up to 200 Gy, a dose that directly generates DSBs. These results suggest that Yen1 acts in repair pathways that relate primarily to replication fork processing/restoration rather than directly in DSB repair. The SGS1 gene encodes a RecQ family helicase that is thought to play multiple roles in DNA repair and recombination, including the dissolution of double HJs [25]. Since it is known that inactivating mutations in MUS81 and SGS1 are synthetically lethal [26,27], we investigated whether deletion of YEN1 in an sgs1 background would result in increased sensitivity to DNA-damaging agents. We

Fig. 1. Synergistic effects between YEN1 and MUS81 for resistance to DNA-damaging agents. (A) BY4741 (WT), yen1 , mus81 and yen1 mus81 strains were grown to mid-log-phase, normalized and spotted in 10-fold serial dilutions onto YPD plates containing the indicated amounts of MMS. (B) As (A), but using the indicated DNA-damaging agents. The rad52 and pso2 strains serve as positive controls. (C) As (B), but using sgs1 and yen1 sgs1 strains.

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found that yen1 sgs1 cells were viable and exhibited a similar MMS, HU and 4NQO-sensitivity to that observed with the sgs1 single mutant (Fig. 1C). 3.2. Over-expression of Yen1 partially rescues the MMS sensitivity of mus81 Expression of a bacterial HJ resolvase (RusA) is known to suppress some of the phenotypes associated with the absence of Mus81 or Sgs1 in yeast [20,45,46]. We therefore determined whether expression of Yen1 could suppress the DNA repair defect seen in mus81 or sgs1 cells. To do this, the coding sequence of Yen1 was cloned into a plasmid that allowed constitutive expression of a C-terminally tagged Yen1-3xHA fusion protein. When transformed into mus81 or yen1 mus81 cells, we observed that expression of Yen1 resulted in reduced sensitivity to MMS, comparable to WT cells (Fig. 2A). This effect was dependent upon Yen1 activity, since expression of a catalytically inactive version of the protein, Yen1E193A/E195A [15], failed to rescue MMS sensitivity. Yen1 expression failed to suppress the MMS sensitivity of sgs1 or yen1 sgs1 cells (Fig. 2A). Similar results were obtained with HU, 4NQO and UV-light (data not shown). These experiments show that YEN1 is a multi-copy suppressor of the repair defects associated with loss of MUS81, but not SGS1, and lead us to suggest that Yen1 and Mus81-

Mms4 are capable of processing similar structures in vivo or that Yen1 can process structures that are generated or persist in the absence of Mus81-Mms4. Interestingly, we found that over-expression of the catalytically dead version of Yen1 in the mus81 background aggravated the DNA repair defect (Fig. 2B), indicating that Yen1E193A/E195A exerted a dominant negative effect. This could be due to competition between the mutant and the endogenous form of Yen1 at sites of DNA damage or mutual exclusion from protein complexes required for efficient repair. 3.3. Yen1 is the only member of the Rad2 family of nucleases that can complement mus81 defects Yen1 belongs to the conserved Rad2 family of nucleases, which includes Rad27, Rad2 and Exo1 [15,17]. Members of the family typically exhibit a characteristic 5 -flap endonuclease activity [47–49]. To investigate whether the suppression of DNA repair defects associated with loss of Mus81 is specific to Yen1 or a common feature of the Rad2 nuclease family, we determined whether over-expression of Rad27, Rad2 or Exo1 could reduce the sensitivity of the mus81 strain to DNA-damaging agents. First, we used a pYES-DEST52-based inducible expression system under control of the GAL1 promoter, because constitutive over-expression of Rad2

Fig. 2. Analysis of the effects of Yen1 over-expression on the sensitivity of mus81 , yen1 mus81 , sgs1 or yen1 sgs1 cells to MMS. Wild-type and mutant strains were transformed with empty vector (pAG416GPD-ccdB-HA), or derivatives encoding either active Yen1-3xHA or the catalytically inactive Yen1E193A/E195A -3xHA under the control of the GPD1 promoter. Analyses were as described for Fig. 1A, but using SC-Ura plates containing the indicated doses of MMS.

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Fig. 3. Suppression of the repair phenotype of mus81 cells is specific to Yen1. (A) Wild-type, yen1 , mus81 and yen1 mus81 strains were transformed with either pYES-DEST52, or derivatives of this vector encoding Rad27-V5-6xHis, Rad2-V5-6xHis or Exo1-V5-6xHis (under the control of GAL1 promoter) or a p416ADH derivative encoding Yen1-V5-6xHis (under the control of ADH1 promoter). Drug sensitivity assays were carried out as in Fig. 1A, but on SC-Ura/2% galactose plates containing MMS or 4NQO. (B) Comparison of Rad2 family protein expression levels. Lysates from the cells used in (A) were probed for expression of V5-tagged proteins following SDS-PAGE. Pgk1 was used as a loading control. Arrowheads indicate full-length proteins.

is detrimental [50]. In this experimental set-up, only Yen1 was able to suppress the sensitivity of mus81 and yen1 mus81 cells (data not shown). However, the expression levels of Rad2 and Exo1 were much lower than those of Rad27 and Yen1, so it was difficult to exclude the possibility that suppression by Yen1 was due to its high level expression. We therefore placed the coding sequence for Yen1-V5-6xHis under the control of the ADH1 promoter, in an attempt to bring Yen1 expression down to the levels of Rad2 and Exo1 when expressed using the GAL1-system. Again, we only observed suppression of the MMS- and 4NQO-sensitivity of mus81 and yen1 mus81 mutant cells by over-expression of Yen1 (Fig. 3A). However, we now observed that Yen1 was the least abundant of the four proteins tested (Fig. 3B), indicating that suppression of the mus81 phenotype is Yen1-specific and not a generic feature of Rad2 family nucleases. 3.4. yen1 mus81 intermediates cells accumulate toxic recombination

support to the notion that toxic HR intermediates accumulate in response to DNA damage when Mus81 and Yen1 are absent. 3.5. yen1 mus81 double mutants are synthetically sick

Given that both Mus81 and Yen1 are capable of resolving recombination intermediates, we hypothesized that the repair defect of the yen1 mus81 double mutant may be due to an accumulation of such DNA structures. If so, the sensitivity of yen1 mus81 mutants should be suppressed by mutations that abrogate homologous recombination. To investigate this we deleted RAD52, a gene essential for all types of HR-mediated DNA repair in S. cerevisiae [51], in the wild-type, yen1 , mus81 and yen1 mus81 strains and determined their sensitivity to different DNA-damaging agents (Fig. 4). As expected, deletion of RAD52 from otherwise wild-type cells led to sensitivity to MMS, 4NQO and HU. Importantly, the sensitivity of rad52 cells to MMS and 4NQO was milder than that of the yen1 mus81 double mutant. This allowed us to show that deletion of RAD52 in the yen1 mus81 background alleviated the MMS- and 4NQO-sensitivity of this strain to levels comparable to those found with the rad52 single mutant. These results indicate an epistatic relationship of RAD52 with YEN1 and MUS81 and lend

Defects in DNA repair are often associated with a prolonged cell cycle. This may reflect a need for additional time to restore genomic integrity after DNA damage and cell cycle checkpoint activation. When we measured the doubling time of yen1 mus81 cells under unperturbed conditions, we observed a 15–20 min increase in the duration of the cell cycle compared with wild-type, yen1 or mus81 cells growing in YPD (Fig. 5A). This delay was similar to that observed for rad52 cells. Constitutive expression of Yen13xHA, but not the catalytically dead version of Yen1, reduced the doubling time of the yen1 mus81 mutant to wild-type levels (Fig. 5B). Since the slow-growth phenotype was dependent upon loss of both Yen1 and Mus81-Mms4, these results show that the activity of either nuclease alone is sufficient for normal cell cycle progression. Interestingly, we found that the presence of inactive Yen1 resulted in an increased doubling time of the mus81 single mutant, reminiscent of the dominant negative effect observed in the sensitivity assays (Fig. 2B).

Fig. 4. RAD52 is epistatic to MUS81 and YEN1 for DNA-damage resistance. Assays were carried out as in Fig. 1A.

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3.6. After MMS treatment yen1 with G2 DNA content

mus81

double mutants arrest

Fig. 5. Slow-growth phenotype of yen1 mus81 cells. (A) The indicated strains were grown in YPD and their doubling times measured. (B) The indicated strains were transformed with active and inactive versions of Yen1, as described for Fig. 2A, and their doubling times were measured during growth in SC-Ura. Mean values and standard deviations for three independent experiments are given. Note that the time scales are different in (A) and (B).

In order to pinpoint the stage at which normal cell cycle progression was perturbed in yen1 mus81 mutants, we analyzed G1 synchronized cells following their release into YPD in the presence or absence of MMS. In the course of a 3-h experiment, FACS analysis did not reveal any marked differences between the yen1 mus81 double mutants, the yen1 and mus81 single mutants, and the wild-type. These experiments were carried out at a range of MMS concentrations up to 0.01%, and a representative example is shown in Fig. 6A. This result seemed surprising given that chronic exposure to much lower levels of MMS in spot assays led to severe growth inhibition of yen1 mus81 cells. We therefore analyzed asynchronous cells during prolonged exposure to MMS (0.0012%) by FACS analysis (Fig. 6B). In the absence of MMS, the wild-type, yen1 , mus81 and yen1 mus81 strains showed an approximate 1:1 distribution of cells with G1 (1N) or G2 (2N) DNA content after incubation for 8 h. In the presence of MMS, the FACS profile for the wild-type and the single mutants showed a slight shift of cells to G2, but a large proportion of cells were in G1. In contrast, yen1 mus81 cells shifted entirely to G2, indicative of defects in the G2/M transition. Moreover, the yen1 mus81 culture became stationary after an incubation period of 8–12 h (∼3 cell divisions; data not shown), while all other strains were able to continue growth under these conditions. We next analyzed DNA segregation in wild-type, yen1 , mus81 and yen1 mus81 cells by microscopy. To do this, cells that had completed DNA replication were identified and assessed for effective separation of their genomic DNA into two distinct masses (Fig. 6C). In the absence of MMS, 50% of wild-type and

Fig. 6. yen1 mus81 cells arrest with G2 DNA content after MMS treatment. (A) The indicated strains were synchronized in G1 using -factor and released into YPD containing 0.0012% MMS. DNA replication was monitored by FACS for 3 h. (B) Asynchronous cells were analyzed by FACS after 8 h growth in YPD with or without MMS. (C) An aliquot of the cells used in (B) were spheroplasted, stained with DAPI and anti-tubulin antibodies, and scored for DNA segregation by microscopy. Cells (200 for each strain) that have completed DNA replication (identified as those having a big bud and an evident bipolar spindle) were scored for the presence of one single DNA mass or two separate DNA masses, as revealed by DAPI staining. The percentage of cells with segregated DNA (red) is shown. Examples of cells scored as segregated or unsegregated DNA are shown. (D) Examples of yen1 mus81 cells that failed to segregate their DNA.

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yen1 cells, 44% of mus81 cells and 39% of yen1 mus81 cells contained two distinct DNA masses. In the presence of 0.0012% MMS, the number of cells containing segregated DNA masses remained unchanged in case of the wild-type and yen1 mutant strain. In contrast, segregation of the mus81 and yen1 mus81 cells dropped to 34% and 14%, respectively. Morphological examination showed that most yen1 mus81 cells had buds similar in size to the mother cell, and that they contained a single DNA mass (Fig. 6D). These results show that yen1 mus81 mutants have a cell cycle defect that manifests as a failure in DNA segregation after MMS treatment. 4. Discussion The recent identification of S. cerevisiae Yen1 and its ortholog GEN1 from human cells, as enzymes that promote Holliday junction resolution in vitro, raises questions relating to their in vivo functions. Structure-specific nucleases are known to play important roles in a variety of DNA repair processes. In yeast, Mus81-Mms4 has been shown to be involved in repair reactions associated with DNA replication, such as the restart of stalled or broken replication forks, inter-strand DNA cross-link repair, and recombination, by the resolution of recombination intermediate structures [3,4]. Here we provide the first genetic evidence for a cellular role for Yen1 in DNA repair in mus81 cells, and in particular show that Yen1 and Mus81-Mms4 provide complementary functions that are necessary for DNA-damage resistance. We have demonstrated that deletion of YEN1 in a mus81 background results in a dramatic increase in sensitivity to a wide range of DNA-damaging agents that impede normal replication fork progression. Conversely, over-expression of Yen1, but not other Rad2/XPG family members (Rad27, Rad2 or Exo1), suppresses the damage sensitivity of both mus81 and yen1 mus81 mutants, indicating that Mus81-Mms4 and Yen1 can utilize similar DNA substrates to facilitate repair. Although yen1 mutants have been shown to be mildly sensitive to MMS under competitive growth conditions [52], we failed to detect marked DNA-damage sensitivity in yen1 single mutants in our assays. This suggests that Mus81-Mms4-catalyzed reactions are likely to provide the primary repair pathway at stalled replication forks or DNA structures resulting from fork demise in wild-type cells. However, Yen1 may be required in particular circumstances, such as in the absence of Mus81 or following the formation of structures that Mus81 finds difficult to process effectively. The precise substrates that are recognized and acted upon by Mus81-Mms4 and Yen1 in vivo remain to be determined. In vitro studies have shown that Mus81-Mms4 acts preferentially upon branched DNA structures [27,28]. Such structures include nicked Holliday junctions, D-loops, replication forks and 3 -flaps [3,4]. The ability of Mus81-Mms4 to cleave intact Holliday junctions in vitro is more limited [19,20,29,35], but is thought to occur in a cellular context, possibly in association with other factors such as Slx4 [8,9,53]. However, the preferred in vitro substrates for Yen1 and its human ortholog GEN1 are HJs, and to a lesser extent replication forks and 5 -flaps [15]. It is therefore possible that Mus81-Mms4 and Yen1 process somewhat different DNA structures that arise at various stages of the repair process. Indeed, Yen1 may act upon structures that are formed in the absence of Mus81-Mms4 (e.g. fully ligated Holliday junctions), and it is possible that the action of Mus81-Mms4 and Yen1 may lead to the same biological outcome. The Sgs1 helicase provides a nuclease-independent pathway for processing recombination intermediates. Loss of Sgs1 sensitizes cells to DNA-damaging agents, but we did not detect hypersensitivity in sgs1 yen1 cells, probably because the absence of Yen1 activity is mostly inconsequential when Mus81 is present. Unfor-

tunately, yen1 sgs1 mus81 cells cannot be analyzed because mus81 and sgs1 exhibit synthetic lethal interactions. This lethality, however, can be suppressed by constitutive over-expression of Yen1, giving rise to synthetically sick but viable colonies (unpublished observations). We take this as evidence that Yen1 provides an alternative means for repair in the absence of Sgs1 and Mus81, ruling out epistasis between SGS1 and YEN1. The DNA-damaging agents used in the present study produce a wide range of lesions in DNA, so it is interesting that mus81 and yen1 mus81 mutants display a qualitatively equivalent spectrum of sensitivities. Agents that inhibit cell proliferation include HU, MMS, UV-light, 4NQO, CPT, cisplatin, nitrogen mustard and phleomycin. HU is an inhibitor of nucleotide reductase and induces replication fork stalling by depletion of the dNTP pool. MMS and UV-light generate lesions that interfere with the progression of replication forks. Although frequently described as a UV-light mimicking chemical, 4NQO induces a wide range of damage to DNA in the form of covalent adducts, oxidative damage and single-strand breaks, and it has been proposed that 4NQO-induced DNA adducts may irreversibly trap topoisomerase I (Top1) cleavage complexes [54–56]. CPT is also a Top1 poison that leads to the formation of replication-induced DNA breaks [57]. Cisplatin and nitrogen mustard are chemicals that generate DNA adducts that may be converted into DNA intra- and inter-strand cross-links [58], the latter posing a physical barrier to replisome progression. Phleomycin, like the structurally related bleomycin, can induce DNA breaks [59]. Our results obtained with mus81 cells are mostly consistent with previous reports detailing their sensitivity to DNA-damaging agents [18,24,27,60–62], but in addition we have shown that mus81 cells are sensitive to nitrogen mustard and phleomycin. The sensitivity to nitrogen mustard is in accord with our results obtained with cisplatin and previous reports of the sensitivity of Mus81-defective cells to mitomycin C [61], consistent with a role for Mus81 in interstrand cross-link repair. Interestingly, no IR-sensitivity was found in yen1 , mus81 , or yen1 mus81 mutants. This is in agreement with previous work reporting that Mus81-deficient yeast cells are not sensitive to IR or bleomycin [18,63], and suggests that Mus81 and Yen1 act in repair pathways that relate primarily to replication fork processing/restoration rather than by playing a direct role in DSB repair. In this regard, the effect of phleomycin is surprising. One possible explanation may be that phleomycin, unlike bleomycin, is not a perfect radiomimetic drug and may induce a wider range of DNA lesions. Consistent with this suggestion, phleomycin is more cytotoxic to yeast cells than bleomycin [64]. Abrogation of HR results in a higher sensitivity to agents that perturb replication fork progression, indicating that some of the repair mechanisms that cells employ to repair and/or restart stalled or damaged forks involve HR. In this sense, the epistatic relationship of RAD52 with MUS81 and YEN1, demonstrated by an increase in the MMS or 4NQO resistance in the triple yen1 mus81 rad52 mutant, provides evidence that it is indeed HR-dependent repair that generates the target structures for the Yen1 and Mus81 nucleases. A defect in cell cycle progression in the yen1 mus81 mutants was revealed both by their mild slow-growth phenotype under normal conditions and the apparent arrest of a large fraction of cells in G2/M that failed to segregate DNA after MMS treatment. Unresolved recombination intermediates formed in S-phase may lead to G2/M checkpoint activation or obstruct chromosome segregation in anaphase due to the persistence of inter-sister joint molecules. Our observations could be consistent with any of these two hypotheses and further experiments will be required to address these issues in greater detail. In conclusion, we have shown that Yen1 is a multi-copy suppressor of the mus81 mutant phenotype. The sensitivity of mus81

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and yen1 mus81 cells to a variety of replication fork stalling agents, but not to IR, indicates that Yen1 and Mus81-Mms4 process DNA structures that arise from replication fork demise rather than those that are generated during the repair of single- and/or doublestrand breaks. However, it is important to note that yen1 single mutants fail to exhibit a repair-defective phenotype, suggesting that Yen1 and Mus81-Mms4 can provide alternative, overlapping pathways for the removal of toxic HR intermediates generated during the repair of damaged replication forks. These processing reactions are essential for chromosome segregation and normal progression through the cell cycle. In accord with the present experiments, data presented elsewhere shows that expression of the human ortholog of Yen1, GEN1, can rescue the hypersensitivity of S. pombe mus81 mutants to both MMS and CPT [65]. Moreover, ectopic expression of GEN1 was found to promote crossover formation during meiosis in a mus81 mutant. In S. pombe, which appears to lack Yen1, there is compelling evidence that Mus81-Eme1 is the primary activity responsible for the resolution of meiotic recombination intermediates, so rescue of the mus81 phenotype by GEN1 is suggestive of a role for Yen1/GEN1 in the resolution of Holliday junctions that accumulate in the absence of Mus81. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgements We thank Jim Haber and Michael Lichten for valuable discussions and input. We also thank John Diffley and Helle Ulrich for sharing plasmids and strains. This work was supported by Cancer Research UK, the Louis-Jeantet Foundation, the EU Consortium on DNA Repair and the Swiss Bridge Foundation. M.G.B. and U.R. are ˜ supported by the Angeles Alvarino program of the Xunta de Galicia (Spain) and the Breast Cancer Campaign (UK), respectively. References
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