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
EMBO reports Vol 15 | No 6 | 2014 ª 2014 The Authors
EMBO reports Rif1 and DSB processing Marina Martina et al
696
Published online: April 1, 2014
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).
◂
ª 2014 The Authors EMBO reports Vol 15 | No 6 | 2014
Marina Martina et al Rif1 and DSB processing EMBO reports
697
Published online: April 1, 2014
EMBO reports Vol 15 | No 6 | 2014 ª 2014 The Authors
EMBO reports Rif1 and DSB processing Marina Martina et al
698
Published online: April 1, 2014
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).
◂
ª 2014 The Authors EMBO reports Vol 15 | No 6 | 2014
Marina Martina et al Rif1 and DSB processing EMBO reports
699
Published online: April 1, 2014
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).
EMBO reports Vol 15 | No 6 | 2014 ª 2014 The Authors
EMBO reports Rif1 and DSB processing Marina Martina et al
700
Published online: April 1, 2014
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.
ª 2014 The Authors EMBO reports Vol 15 | No 6 | 2014
Marina Martina et al Rif1 and DSB processing EMBO reports
701
Published online: April 1, 2014
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).
EMBO reports Vol 15 | No 6 | 2014 ª 2014 The Authors
EMBO reports Rif1 and DSB processing Marina Martina et al
702
Published online: April 1, 2014
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.
References
1. Hardy CF, Sussel L, Shore D (1992) A RAP1-interacting protein involved
in transcriptional silencing and telomere length regulation. Genes Dev 6:
801 – 814
2. Marcand S, Gilson E, Shore D (1997) A protein-counting mechanism for
telomere length regulation in yeast. Science 275: 986 – 990
3. Marcand S, Pardo B, Gratias A, Cahun S, Callebaut I (2008) Multiple
pathways inhibit NHEJ at telomeres. Genes Dev 22: 1153 – 1158
4. Bonetti D, Clerici M, Anbalagan S, Martina M, Lucchini G, Longhese MP
(2010) Shelterin-like proteins and Yku inhibit nucleolytic processing of
Saccharomyces cerevisiae telomeres. PLoS Genet 6: e1000966
5. Vodenicharov MD, Laterreur N, Wellinger RJ (2010) Telomere capping in
non-dividing yeast cells requires Yku and Rap1. EMBO J 29: 3007 – 3019
6. Anbalagan S, Bonetti D, Lucchini G, Longhese MP (2011) Rif1 supports
the function of the CST complex in yeast telomere capping. PLoS Genet
7: e1002024
7. Xue Y, Rushton MD, Maringele L (2011) A novel checkpoint and RPA
inhibitory pathway regulated by Rif1. PLoS Genet 7: e1002417
8. Ribeyre C, Shore D (2012) Anticheckpoint pathways at telomeres in
yeast. Nat Struct Mol Biol 19: 307 – 313
9. Longhese MP, Bonetti D, Manfrini N, Clerici M (2010) Mechanisms and
regulation of DNA end resection. EMBO J 29: 2864 – 2874
10. Zimmermann M, de Lange T (2013) 53BP1: pro choice in DNA repair.
Trends Cell Biol 24: 108 – 117.
11. Chapman JR, Barral P, Vannier JB, Borel V, Steger M, Tomas-Loba A,
Sartori AA, Adams IR, Batista FD, Boulton SJ (2013) RIF1 is essential for
53BP1-dependent nonhomologous end joining and suppression of DNA
double-strand break resection. Mol Cell 49: 858 – 871
12. Di Virgilio M, Callen E, Yamane A, Zhang W, Jankovic M, Gitlin AD, Feld-
hahn N, Resch W, Oliveira TY, Chait BT et al (2013) Rif1 prevents resec-
tion of DNA breaks and promotes immunoglobulin class switching.
Science 339: 711 – 715
13. Escribano-Díaz C, Orthwein A, Fradet-Turcotte A, Xing M, Young JT, Tká�c
J, Cook MA, Rosebrock AP, Munro M, Canny MD et al (2013) A cell
cycle-dependent regulatory circuit composed of 53BP1-RIF1 and BRCA1-
CtIP controls DNA repair pathway choice. Mol Cell 49: 872 – 883
14. Zimmermann M, Lottersberger F, Buonomo SB, Sfeir A, de Lange T
(2013) 53BP1 regulates DSB repair using Rif1 to control 5’ end resection.
Science 339: 700 – 704
15. Shim EY, Chung WH, Nicolette ML, Zhang Y, Davis M, Zhu Z, Paull TT,
Ira G, Lee SE (2010) Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku
proteins regulate association of Exo1 and Dna2 with DNA breaks. EMBO
J 29: 3370 – 3380
16. Ira G, Pellicioli A, Balijja A, Wang X, Fiorani S, Carotenuto W, Liberi G,
Bressan D, Wan L, Hollingsworth NM et al (2004) DNA end resection,
ª 2014 The Authors EMBO reports Vol 15 | No 6 | 2014
Marina Martina et al Rif1 and DSB processing EMBO reports
703
Published online: April 1, 2014
homologous recombination and DNA damage checkpoint activation
require CDK1. Nature 431: 1011 – 1017
17. Aylon Y, Liefshitz B, Kupiec M (2004) The CDK regulates repair of
double-strand breaks by homologous recombination during the cell
cycle. EMBO J 23: 4868 – 4875
18. Vaze MB, Pellicioli A, Lee SE, Ira G, Liberi G, Arbel-Eden A, Foiani
M, Haber JE (2002) Recovery from checkpoint-mediated arrest after
repair of a double-strand break requires Srs2 helicase. Mol Cell 10:
373 – 385
19. Ivanov EL, Sugawara N, Fishman-Lobell J, Haber JE (1996) Genetic
requirements for the single-strand annealing pathway of double-strand
break repair in Saccharomyces cerevisiae. Genetics 142: 693 – 704
20. Mimitou EP, Symington LS (2008) Sae2, Exo1 and Sgs1 collaborate in
DNA double-strand break processing. Nature 455: 770 – 774
21. Zhu Z, Chung WH, Shim EY, Lee SE, Ira G (2008) Sgs1 helicase and two
nucleases Dna2 and Exo1 resect DNA double-strand break ends. Cell
134: 981 – 994
22. Lydall D, Weinert T (1995) Yeast checkpoint genes in DNA damage
processing: implications for repair and arrest. Science 270: 1488 – 1491
23. Lazzaro F, Sapountzi V, Granata M, Pellicioli A, Vaze M, Haber JE, Plevani
P, Lydall D, Muzi-Falconi M (2008) Histone methyltransferase Dot1 and
Rad9 inhibit single-stranded DNA accumulation at DSBs and uncapped
telomeres. EMBO J 27: 1502 – 1512
24. Toh GW, O’Shaughnessy AM, Jimeno S, Dobbie IM, Grenon M, Maffini S,
O’Rorke A, Lowndes NF (2006) Histone H2A phosphorylation and H3
methylation are required for a novel Rad9 DSB repair function allowing
checkpoint activation. DNA Repair 5: 693 – 703
25. Downs JA, Lowndes NF, Jackson SP (2000) A role for Saccharomyces
cerevisiae histone H2A in DNA repair. Nature 408: 1001 – 1004
26. Javaheri A, Wysocki R, Jobin-Robitaille O, Altaf M, Côté J, Kron SJ (2006)
Yeast G1 DNA damage checkpoint regulation by H2A phosphorylation is
independent of chromatin remodeling. Proc Natl Acad Sci USA 103:
13771 – 13776
27. Hammet A, Magill C, Heierhorst J, Jackson SP (2007) Rad9 BRCT domain
interaction with phosphorylated H2AX regulates the G1 checkpoint in
budding yeast. EMBO Rep 8: 851 – 857
28. Chen X, Cui D, Papusha A, Zhang X, Chu CD, Tang J, Chen K, Pan X, Ira G
(2012) The Fun30 nucleosome remodeller promotes resection of DNA
double-strand break ends. Nature 489: 576 – 580
29. Trovesi C, Falcettoni M, Lucchini G, Clerici M, Longhese MP (2011)
Distinct Cdk1 requirements during single-strand annealing, crossover,
and noncrossover recombination. PLoS Genet 7: e1002263
30. Yamazaki S, Hayano M, Masai H (2013) Replication timing regulation of
eukaryotic replicons: Rif1 as a global regulator of replication timing.
Trends Genet 29: 449 – 460
31. Buonomo SB, Wu Y, Ferguson D, de Lange T (2009) Mammalian Rif1
contributes to replication stress survival and homology-directed repair.
J Cell Biol 187: 385 – 398
32. Park S, Patterson EE, Cobb J, Audhya A, Gartenberg MR, Fox CA (2011)
Palmitoylation controls the dynamics of budding-yeast heterochromatin
via the telomere-binding protein Rif1. Proc Natl Acad Sci USA 108:
14572 – 1457
33. Taddei A, Gasser SM (2012) Structure and function in the budding yeast
nucleus. Genetics 192: 107 – 129
34. Paciotti V, Clerici M, Lucchini G, Longhese MP (2000) The checkpoint
protein Ddc2, functionally related to S. pombe Rad26, interacts with
Mec1 and is regulated by Mec1-dependent phosphorylation in budding
yeast. Genes Dev 14: 2046 – 2059
704 EMBO reports Vol 15 | No 6 | 2014 ª 2014 The Authors
EMBO reports Rif1 and DSB processing Marina Martina et al
Published online: April 1, 2014