Saccharomyces cerevisiae Rif1 cooperates withembor.embopress.org/content/embor/15/6/695.full.pdf · Scientific Report Saccharomyces cerevisiae Rif1 cooperates with MRX-Sae2 in promoting

Scientific Report

Saccharomyces cerevisiae Rif1 cooperates withMRX-Sae2 in promoting DNA-end resectionMarina Martina†, Diego Bonetti†, Matteo Villa, Giovanna Lucchini & Maria Pia Longhese*

Abstract

Diverse roles in DNA metabolism have been envisaged for buddingyeast and mammalian Rif1. In particular, yeast Rif1 is involved intelomere homeostasis, while its mammalian counterpart partici-pates in the cellular response to DNA double-strand breaks (DSBs).Here, we show that Saccharomyces cerevisiae Rif1 supports cellsurvival to DNA lesions in the absence of MRX or Sae2. Further-more, it contributes to the nucleolytic processing (resection) ofDSBs. This Rif1-dependent control of DSB resection becomesimportant for DSB repair by homologous recombination whenresection activities are suboptimal.

Keywords double-strand break; Rad9; resection; Rif1; Saccharomyces

cerevisiae

Subject Categories DNA Replication, Repair & Recombination

DOI 10.1002/embr.201338338 | Received 6 December 2013 | Revised 28

February 2014 | Accepted 3 March 2014 | Published online 1 April 2014

EMBO Reports (2014) 15, 695–704

See also: G Ira & A Nussenzweig (June 2014)

Introduction

Rif1 has been identified in Saccharomyces cerevisiae as a negative

regulator of telomere length and transcriptional silencing [1,2].

Although Rif1 physically interacts with the telomeric proteins Rap1

and Rif2, these proteins regulate telomere metabolism by different

mechanisms. In fact, Rap1 and Rif2 inhibit both nucleolytic process-

ing and non-homologous end joining (NHEJ) at telomeres, while

Rif1 is not involved in these processes [3–5]. Instead, Rif1 plays a

unique role in supporting cells’ viability [6,7] and in preventing

nucleolytic degradation in situations where telomere protection is

altered, such as in mutants affecting the CST (Cdc13-Stn1-Ten1)

complex [6]. On the other hand, both Rif1 and Rif2 prevent short

telomeric ends from causing a checkpoint-mediated cell cycle arrest

by inhibiting the recruitment of the checkpoint proteins Rad9, Mec1

and Rad24 to these ends [7,8].

Mammalian Rif1 is not part of the telomeric complex, while it is

involved in the response to DNA double-strand breaks (DSBs). DSBs

can be repaired by homologous recombination (HR), which requires

the formation of RPA-coated single-stranded DNA (ssDNA) that

arises from 50 to 30 nucleolytic degradation (resection) of DNA

ends [9]. DSB resection in mammals is promoted by BRCA1, which

forms a complex with CtIP and MRN (orthologs of S. cerevisiae Sae2

and MRX, respectively) [10]. Rif1 has been recently shown to

prevent DSB resection in G1 by blocking the accumulation of BRCA1

at the sites of damage [11–14].

Whether budding yeast Rif1 functions exclusively at telomeres or

it plays a role also at DSBs like its mammalian counterpart remains

to be determined. Here, we show that S. cerevisiae Rif1 functions

together with Sae2 and MRX in DSB repair by HR.

Results and Discussion

Rif1 supports cell viability in the absence of the MRX complex

In S. cerevisiae, the MRX (Mre11-Rad50-Xrs2) complex initiates DSB

end resection by acting in concert with Sae2 [9]. To investigate

whether Rif1 is involved in the DNA damage response, we analysed

the effects of its absence in cells either lacking Mre11 or Sae2 or

carrying the nuclease-defective mre11-H125N allele. When meiotic

tetrads from diploid strains heterozygous for the rif1Δ and mre11Δalleles were analysed for spore viability on YEPD plates, all rif1Δmre11Δ double-mutant spores formed much smaller colonies than

each single-mutant spore (Fig 1A). The smaller colony size was due

to the loss of viability, as rif1Δ mre11Δ spore clones contained a

much lower number of colony-forming units than each single-

mutant spore clone (Supplementary Fig S1). By contrast, RIF1 dele-

tion did not significantly affect the size of the colonies formed by

mre11-H125N and reduced only slightly the size of the colonies of

sae2Δ spores (Fig 1B,C).

It is known that mre11Δ cells suffer a more severe resection

defect than sae2Δ or mre11-H125N cells. In fact, the MRX complex,

but not its nuclease activity, is required to recruit to DSBs the

50–30 exonuclease Exo1 that can substitute for MRX-Sae2 nuclease

function in resection [15]. We then asked whether rif1Δ sae2Δ cells’

viability depended on EXO1. When tetrads from the appropriate

diploid were dissected on YEPD, all the rif1Δ sae2Δ exo1Δ triple-

mutant spores formed much smaller colonies than rif1Δ sae2Δdouble-mutant spores (Fig 1D). Thus, Rif1 appears to support cell

viability when the MRX complex is not functional.

Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Milan, Italy*Corresponding author. Tel: +39 0264483543; Fax: +39 0264483565; E-mail: [email protected]†These two authors have contributed equally to the work.

ª 2014 The Authors EMBO reports Vol 15 | No 6 | 2014 695

Published online: April 1, 2014

http://dx.doi.org/10.1002/embr.201438825

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EMBO reports Rif1 and DSB processing Marina Martina et al

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On the other hand, rif1Δ sae2Δ and rif1Δ mre11-H125N double

mutants were more sensitive to phleomycin (phleo) and methyl

methanesulfonate (MMS) than each single mutant (Fig 1E,F). This

Rif1 function reflects cooperation between Rif1 and Sae2-MRX in

repairing DNA lesions by HR. In fact, RIF1 deletion did not exacer-

bate the sensitivity to hydroxyurea (HU), phleomycin and campto-

thecin (CPT) of cells carrying the deletion of the HR genes RAD51

and RAD52 (Fig 1G), indicating that Rif1 acts in the same pathway

of Rad51 and Rad52. This function in HR is specific for Rif1, as the

lack of its interacting protein Rif2 did not affect the colony size of

mre11Δ spores (Fig 1H) and did not exacerbate the sensitivity to

DNA-damaging agents of mre11Δ and sae2Δ mutants (Supplemen-

tary Fig S2A).

As the genetic interactions between Rif1 and MRX-Sae2 might

underlie a possible Rif1 involvement in DSB resection, we asked

whether EXO1 overexpression suppressed the sick phenotype of

rif1Δ mre11Δ cells and the hypersensitivity to DNA-damaging agents

of rif1Δ sae2Δ cells. A rif1Δ homozygous diploid strain that was

heterozygous for the mre11Δ allele was then transformed with a 2 lhigh copy number plasmid either empty or carrying the EXO1 gene

or the nuclease-defective exo1-D171A allele. After sporulation and

tetrad dissection of the transformed strains, all rif1Δ mre11Δ spores

with high copy number EXO1 formed colonies of almost wild-type

size, whereas all rif1Δ mre11Δ spores carrying either the empty

vector or high copy number exo1-D171A allele still formed small

colonies (Fig 1I,L). Furthermore, overexpression of EXO1, but not

of the exo1-D171A allele, partially suppressed the hypersensitivity to

phleomycin and MMS of rif1Δ sae2Δ double mutants (Fig 1M). Alto-

gether, these results suggest that Rif1 can cooperate with MRX-Sae2

in DSB resection.

Rif1 promotes DSB resection

To further investigate the possible role of Rif1 in the generation of

30-ended ssDNA at a DSB, we deleted RIF1 in a haploid strain where

a DSB can be generated at the MAT locus by inducing the expres-

sion of the HO endonuclease from a galactose-inducible promoter

[16]. This strain cannot repair the HO-induced DSB because the

HML and HMR homologous donor sequences have been deleted.

Because ssDNA is resistant to cleavage by restriction enzymes, we

directly monitored the formation of ssDNA at the HO-induced DSB

by following the loss of SspI restriction sites by Southern blot analy-

sis under alkaline conditions (Fig 2A). HO was induced by galactose

addition to a-factor or nocodazole-arrested wild-type and rif1Δ cells

that were kept arrested in G1 or G2, respectively. To induce a

persistent G1 arrest, all the strains carried the deletion of the BAR1

gene that encodes an a-factor-degrading protease. The quality and

persistence of the cell cycle arrest was assessed by FACS analysis

and by determining the percentage of unbudded and budded cells

(Fig 2B). Although DSB resection occurs primarily in S/G2 when

Clb-Cdk1 activity is high [16,17], a certain amount of ssDNA can be

detected even in G1-arrested wild-type cells (Fig 2C,D). This finding

is in agreement with previous studies that showed that resection in

G1 is inhibited compared to G2, but not completely abolished

[16,17]. In G1-arrested cells, the HO-cut DNA fragment was

converted into the r2 resection product with similar kinetics in both

wild-type and rif1Δ (Fig 2C,D). However, the appearance of r3

resection product was delayed in rif1Δ cells compared to wild-type

(Fig 2C,D), indicating that the lack of Rif1 impairs resection beyond

the SspI restriction site located 1.7 kb from the HO cutting site. By

contrast, all the resection products accumulated with similar kinet-

ics in G2-arrested wild-type and rif1Δ cells (Fig 2C,D).

As the long resection products are not easily detectable with the

assay used above, we investigated the requirement of Rif1 in

specific DNA repair pathways that are known to strictly rely on

resection, in order to uncover a possible role of Rif1 in promoting

extensive DSB resection in G2. Single-strand annealing (SSA) is the

main repair pathway of a DSB that is flanked by direct repeats and

requires degradation of the 50 DSB ends to reach the complementary

DNA sequences that can then anneal (Fig 2E) [18]. Subsequent

nucleolytic removal of the protruding single-stranded tails results in

deletion of the intervening DNA sequence and one of the repeats

(Fig 2E). We deleted RIF1 in YMV45 and YMV80 strains that carry

tandem repeats of the LEU2 gene separated by 4.6 kb and 25 kb,

respectively, with a recognition site for the HO endonuclease adja-

cent to one of the repeats (Supplementary Fig S3) [18]. Because

repair of this DSB can occur by either SSA or break-induced replica-

tion (BIR), all the strains carried the RAD51 deletion, which abol-

ishes BIR but does not affect SSA [19]. HO was expressed by

galactose addition to nocodazole-arrested cells that were kept

arrested in G2 with nocodazole. SSA-mediated DSB repair showed

equivalent efficiency in both wild-type and rif1Δ strains carrying one

of the flanking LEU2 repeats at 4.6 kb from the DSB (Fig 2F,G).

However, when one of the flanking LEU2 repeats was located at

25 kb from the DSB, accumulation of the repair product was reduced

in G2-arrested rif1Δ cells compared to wild-type (Fig 2F,G). These

data indicate that the lack of Rif1 impairs extensive DSB resection

even in G2, and this defect can reduce the efficiency of SSA-mediated

repair of a DSB between distant flanking homologous sequences.

We then asked whether Rif1-mediated regulation of DSB resec-

tion in G2 was partially redundant with other resection activities.

When a DSB occurs, MRX and Sae2 initiate resection of the 50

strand, while extensive resection relies on two pathways, which

depend on the nucleases Exo1 and Dna2, respectively, with the

latter acting together with the helicase Sgs1 [20,21]. As shown in

Fig 3, G2-arrested rif1Δ sae2Δ (Fig 3A,B) and rif1Δ exo1Δ double

Figure 1. Functional interactions of Rif1 with MRX and Sae2.

A–D Synthetic effects of different genetic combinations. Meiotic tetrads were dissected on YEPD plates that were incubated at 25°C, followed by spore genotyping.E–G Sensitivity to genotoxic drugs. (E, G) Drop test. Exponentially growing cells were serially diluted (1:10), and each dilution was spotted out onto YEPD plates with or

without MMS, HU, phleomycin or CPT. (F) Survival curves. Exponentially growing cell cultures were incubated for two hours with the indicated amounts ofphleomycin or MMS, and proper dilutions were then plated on YEPD to determine the colony-forming units. The mean values are represented with error barsdenoting s.d. (n = 3). Statistically significance differences are indicated: *P < 0.01, Student’s t-test.

H, I Meiotic tetrads were dissected on YEPD plates that were incubated at 25°C, followed by spore genotyping.L Spore clones from plates in (I) were serially diluted (1:10), and each dilution was spotted out on a synthetic dextrose plate lacking leucine (SD-Leu).M Survival curves. The experiment has been performed as in (F).

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mutants (Fig 3C,D) showed more severe resection defects than each

corresponding single mutant. Furthermore, when EXO1 was deleted

in rif1Δ YMV45 strain, G2-arrested rif1Δ exo1Δ cells repaired the

DSB by SSA less efficiently than rif1Δ and exo1Δ single mutants

(Supplementary Fig S4).

Altogether, these results indicate that Rif1 promotes DSB resec-

tion not only in G1, but also in G2 by controlling a pathway that is

partially redundant with the one involving Sae2 and Exo1. The

epistatic relationships in resection between Rif1 and Sgs1/Dna2

could not be investigated because of the dramatic growth defects of

rif1Δ sgs1Δ double-mutant cells. Furthermore, the rif1Δ dna2Δcombination was synthetically lethal even when the essential func-

tion of Dna2 was bypassed by the pif1-M2 mutation (Supplementary

Fig S2B), which reduces the formation of long flaps that are

substrates for Dna2.

Finally, we investigated whether Rif1 was recruited in the

surroundings of the HO-induced DSB. After HO induction by galac-

tose addition, chromatin immunoprecipitation (ChIP) experiments

detected Rif1 binding close to the cut site as early as 1 h after HO

induction (Fig 4A), indicating that Rif1 is recruited to the DSB site.

Consistent with a stronger effect of RIF1 deletion on DSB resection in

G1 than in G2, Rif1 binding was higher in G1 than in G2 (Fig 4A).

The lack of Rad9 restores resection in G1-arrested rif1Δ cells

We quantified by ChIP analysis the effect of the lack of Rif1 on the

binding at the HO-induced DSB of positive (Mre11, Exo1, Sgs1,

Dna2, and Rpa1) and negative (Rad9) regulators of DSB resection

[20–23]. Association of Mre11, Exo1, Sgs1, Dna2 and Rpa1 near the

HO-induced DSB was not impaired in exponentially growing rif1Δcompared to wild-type cells (Fig 4B), which also showed similar

amount of Exo1 bound to the DSB in G1 (Fig 4B). Thus, the resec-

tion defect of rif1Δ cells is not due to decreased association of these

proteins with the DSB ends. Rather, Mre11 and Dna2 recruitment

was even greater in rif1Δ cells than in wild-type (Fig 4B). Mre11

association with the DSB was increased also in G1-arrested rif1Δcells compared to wild-type (Fig 4B), whereas Dna2 association was

very poor in both G1-arrested wild-type and rif1Δ cells, probably

because Dna2 binds ssDNA, whose amount is reduced in G1. Inter-

estingly, the amount of Rad9 bound near the DSB after HO induction

was higher in both exponentially growing and G1-arrested rif1Δcells compared to wild-type (Fig 4C). Detection of all the above

proteins near the HO cut was not influenced by DSB resection, as all

the ChIP signals were normalized for each time point to the

corresponding input signal that decreased with similar kinetics in

both wild-type and rif1Δ cells at 1.8 kb from the DSB (Fig 4D).

After DNA damage, Rad9 binding to chromatin is promoted by inter-

action with histone H2A that has been phosphorylated at serine 129

(cH2A) by the Mec1 checkpoint kinase [24–27]. Formation of cH2Awas still required in rif1Δ cells to promote Rad9 association with chro-

matin, as the substitution of H2A Ser129 with a non-phosphorylatable

alanine residue (hta1-S129A) reduced Rad9 recruitment at the rif1ΔDSB to the extent observed in the hta1-S129A single mutant (Fig 4E).

The increased Rad9 binding to the DSB in rif1Δ cells might be due to an

increased generation of cH2A at the damaged site byMec1. Indeed, the

binding of Mec1 (Fig 4F) and cH2A (Fig 4G) near the HO-induced DSB

was higher in rif1Δ cells than inwild-type, suggesting that the increased

Mec1 association at the DSB leads to more efficient cH2A generation,

which in turn enhances Rad9 binding at DSBs.

Thus, the lack of Rif1 seems to increase the accessibility of some

proteins to regions around the break site. Whether an excess of

MRX and/or Dna2 association at the break site affects DSB resection

is unknown. On the other hand, enhanced Rad9 association has

been proposed to block resection in cells lacking the chromatin

remodeler Fun30 [28], raising the possibility that robust Rad9 bind-

ing might be responsible for resection inhibition in rif1Δ cells. We

therefore investigated whether RAD9 deletion was capable to

suppress the resection defect of G1-arrested rif1Δ cells. Consistent

with a previous finding that the Rad9 inhibitory effect on DSB resec-

tion in G1 becomes apparent only in the absence of Yku, resection

in G1-arrested rad9Δ cells did not increase compared to wild-type

[29]. However, RAD9 deletion abolished the resection defect of

G1-arrested rif1Δ cells (Fig 5A,B), indicating that Rad9 exerts its

function in inhibiting DSB resection in rif1Δ G1 cells even in the

presence of Ku. Rad9 binding at the DSB was increased not only in

G1-arrested but also in exponentially growing rif1Δ cells (Fig 4C),

suggesting that this Rad9 excess in rif1Δ cells is sufficient to impair

resection in G1, but not in G2, where DSB resection occurs much

more efficiently than in G1. Notably, RAD9 deletion suppressed the

growth defect of rif1Δ mre11Δ cells (Fig 5C), further supporting the

hypothesis that their synthetic sickness is due to resection defects

that impair DSB repair by HR. This suppression did not depend on

the Rad9 checkpoint function, as the same growth defect was not

suppressed by the deletion of MEC3 (Fig 5D), whose function is

necessary for the checkpoint response to DSBs.

This Rif1 function on DSB resection recalls the role of Fun30,

which has been shown to promote extensive resection probably by

counteracting Rad9 [28]. We found that RIF1 deletion exacerbated

the resection defect of G2-arrested fun30Δ cells (Fig 5E,F), indicat-

ing that Rif1 and Fun30 act in two different pathways.

In summary, we demonstrate a role for S. cerevisiae Rif1 in the

response to DNA damage, where it promotes DSB nucleolytic

Figure 2. DSB resection in rif1Δ cells.

A System used to detect DSB resection. Gel blots of SspI-digested genomic DNA separated on alkaline agarose gel were hybridized with a single-stranded MAT probethat anneals to the unresected strand. 50–30 resection progressively eliminates SspI sites (S), producing larger SspI fragments (r1 through r7) detected by the probe.

B FACS analysis of DNA content. The percentage of unbudded and budded cells is indicated on the right.C, D G1- or G2-arrested YEPR cell cultures of JKM139 derivative strains were transferred to YEPRG at time zero in the presence of a-factor or nocodazole, respectively.

Genomic DNA was analysed for ssDNA formation (C) as described in (A). Resection products were analysed by densitometry (D). The experiment as in (C) has beenindependently repeated three times, and the mean values are represented with error bars denoting s.d. (n = 3).

E System used to detect SSA. The HO-cut site is flanked by homologous leu2 sequences (grey boxes) that are 4.6 kb (YMV45) or 25 kb (YMV80) apart (double redarrow). Degradation of the 50 DSB ends reaches the complementary DNA sequences that can then anneal.

F, G DSB repair by SSA. HO was induced in nocodazole-arrested cell cultures of YMV45 and YMV80 derivative wild-type and rif1Δ strains. (F) Southern blot analysis ofKpnI-digested genomic DNA. (G) Densitometric analysis of the SSA band signals. The mean values are represented with error bars denoting s.d. (n = 3). Theintensity of each band was normalized with respect to a loading control (not shown).

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Figure 3. RIF1 deletion exacerbates the resection defects of sae2Δ and exo1Δ cells in G2.

A–D Epistasis analysis. (A, C) DSB resection. G2-arrested YEPR cell cultures of JKM139 derivative strains were transferred to YEPRG at time zero in the presence ofnocodazole. Genomic DNA was analysed for ssDNA formation as described in Figure 2. (B, D) Densitometric analysis of resection products. The experiments as in (A)and (C) have been independently repeated three times, and the mean values are represented with error bars denoting s.d. (n = 3).



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Figure 4. Recruitment at DSBs of proteins involved in resection.

A ChIP analysis of Rif1 at the HO-induced DSB. G1- or G2-arrested YEPR cell cultures of JKM139 derivative strains were transferred to YEPRG in the presence of a-factor or nocodazole, respectively. ChIP analysis of recruitment of Rif1-Myc at the indicated distance from the HO cut compared to untagged Rif1 (no tag). Themean values are represented with error bars denoting s.d. (n = 3).

B–D Recruitment of resection regulators at the HO-induced DSB. HO expression was induced in exponentially growing (exp) or G1-arrested (G1) cell cultures of strainswith the indicated genotypes and expressing the indicated untagged (no tag) or fully functional Myc- or HA-tagged proteins. ChIP analysis of the recruitment ofthe indicated proteins at the indicated distances from the HO-induced DSB. The mean values are represented with error bars denoting s.d. (n = 3). *P < 0.01,Student’s t-test. (D) Input DNA used for the ChIP analysis in (B) and (C) normalized to the ARO locus. The mean values are represented with error bars denoting s.d.(n = 3).

E–G ChIP analysis. The experiments have been performed as in (B–D). All strains carrying the hta1-S129A allele carried also HTA2 deletion. In all diagrams, the ChIPsignals were normalized for each time point to the corresponding input signal.



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Figure 5. RAD9 deletion suppresses the resection defect of rif1Δ cells.

A, B G1-arrested YEPR cell cultures of JKM139 derivative strains were transferred to YEPRG at time zero in the presence of a-factor. Genomic DNA was analysed forssDNA formation (A) as described in Figure 2. Resection products were analysed by densitometry (B). The experiment in (A) has been independently repeated threetimes, and the mean values are represented with error bars denoting s.d. (n = 3).

C, D Meiotic tetrads were dissected on YEPD plates that were incubated at 25°C, followed by spore genotyping.E, F G2-arrested YEPR cell cultures of JKM139 derivative strains were transferred to YEPRG at time zero in the presence of nocodazole. Genomic DNA was analysed for

ssDNA formation (E) as described in Figure 2. Resection products were analysed by densitometry (F). The experiment in (E) has been independently repeated threetimes, and the mean values are represented with error bars denoting s.d. (n = 3).



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processing possibly by limiting the action of the resection inhibitor

Rad9. This Rif1 control on Rad9 loading becomes crucial for DSB

repair when resection activities are suboptimal, such as in mre11Δ,sae2Δ and exo1Δ mutants. This function is different from that of

mammalian Rif1, which inhibits resection in G1 by excluding

BRCA1 from the DSBs [11–14]. As budding yeast lacks a BRCA1

ortholog, mammalian Rif1 may have acquired this function during

evolution to regulate resection in a BRCA1 context.

How Rif1 influences protein binding to DSBs remains to be deter-

mined. Budding yeast Rif1 blocks checkpoint activation at telomeres

by limiting the association of checkpoint proteins [7,8]. Further-

more, Rif1 has been shown to negatively control the firing of a

subset of replication origins by modulating the binding of replication

factors in both yeast and mammals [30]. Interestingly, both budding

yeast and mammalian Rif1 localize at the nuclear periphery [31,32],

and also DSBs, replication origins and telomeres are clustered and

tethered to the nuclear membrane [33]. We therefore speculate that

Rif1 may act at all these DNA regions possibly by directly control-

ling their chromatin structure and therefore their accessibility to

regulatory proteins. In this view, modulation of chromatin accessi-

bility might be the evolutionarily conserved function of Rif1.

Materials and Methods

Yeast strains

Strain genotypes are listed in Supplementary Table S1. Strains

JKM139, YMV45 and YMV80 were kindly provided by J. Haber

(Brandeis University, USA). Strains carrying MEC1-MYC allele have

been constructed as described in 34. Cells were grown in YEP medium

(1% yeast extract, 2% peptone) supplemented with 2% glucose

(YEPD), 2% raffinose (YEPR) or 2% raffinose and 3% galactose

(YEPRG). Synthetic dextrose plates lacking leucine (SD-Leu) were used

tomaintain the selective pressure for the 2 l LEU2 plasmids.

DSB resection

Double-strand breaks end resection at the MAT locus was analysed

on alkaline agarose gels as described in 29. Quantitative analysis of

DSB resection was performed by calculating the ratio of band inten-

sities for ssDNA and total amount of DSB products.

Other techniques

ChIP analysis was performed as described in 29. Data are expressed

as fold enrichment at the HO-induced DSB over that at the non-

cleaved ARO1 locus, after normalization of each ChIP signals to the

corresponding input for each time point. Fold enrichment was then

normalized to the efficiency of DSB induction.

Supplementary information for this article is available online:

http://embor.embopress.org

AcknowledgmentsWe thank J. Haber for strains, Arianna Lockhart for preliminary results and

Michela Clerici for critical reading of the manuscript. This work was supported

by grants from Associazione Italiana per la Ricerca sul Cancro (AIRC) (Grant

IG11407) and Cofinanziamento 2010-2011 MIUR/Università di Milano-Bicocca

to MPL.

Author contributionsMM, DB and MPL conceived and designed the experiments. MM, DB and MV

performed the experiments. MM, DB, MV, GL and MPL analysed the data. MPL

and GL wrote the paper.

Conflict of interestThe authors declare that they have no conflict of interest.

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704 EMBO reports Vol 15 | No 6 | 2014 ª 2014 The Authors



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