Mutation Research 698 (2010) 52–59

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Mutation Research/Genetic Toxicology and
Environmental Mutagenesis
journal homepage: www.elsevier.com/locate/gentox
Community address: www.elsevier.com/locate/mutres

Differential participation of homologous recombination and nucleotide excision

repair in yeast survival to ultraviolet light radiation
Martin Toussaint, Raymund J. Wellinger, Antonio Conconi ∗
Département de Microbiologie et d’Infectiologie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, QC J1H 5N4, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Aims: The purpose of this research was to assess the ultraviolet light (UV) phenotype of yeast sir cells vs.
Received 3 September 2009 WT cells, and to determine whether de-silenced chromatin or the intrinsic pseudoploidy of sir mutants
Received in revised form 16 February 2010 contributes to their response to UV. Additional aims were to study the participation of HR and NER in
Accepted 20 March 2010
promoting UV survival during the cell cycle, and to define the extent of the co-participation for both
Available online 27 March 2010
repair pathways.
Main methods: The sensitivity of yeast Saccharomyces cerevisiae to UV light was determined using a
Keywords:
method based on automatic measurements of optical densities of very small (100 ␮l) liquid cell cultures.
Cell survival
Mating type
Key findings: We show that pseudo-diploidy of sir strains promotes resistance to UV irradiation and that
Homologous recombination HR is the main mechanism that is responsible for this phenotype. In addition, HR together with GG-NER
Nucleotide excision repair renders cells in the G2-phase of the cell cycle more resistant to UV irradiation than cells in the G1-phase,
Sir proteins which underscore the importance of HR when two copies of the chromosomes are present. Nevertheless,
Pseudo-diploidy in asynchronously growing cells NER is the main repair pathway that responds to UV induced DNA
Ultraviolet light radiation damage.
Significance: This study provides detailed and quantitative information on the co-participation of HR and
NER in UV survival of yeast cells.
Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.

1. Introduction DNA lesions that are repaired by HR are first processed by the 5 –3
nucleolytic activities, which create a single-stranded DNA (ssDNA)
In living cells, DNA is prone to damage that can be induced tail that is bound by Rad51p. Then, recombination proceeds in three
endogenously by cellular metabolites, or exogenously by a plethora steps: strand invasion, whereby the invading 3 end is extended by
of chemical and physical environmental agents. If not repaired, DNA synthesis; branch migration, followed by second end capture;
DNA damage can lead to mutations, carcinogenesis or cell death and double Holliday junction formation that are resolved with or
[1,2]. Cells have developed complex DNA repair pathways that are without crossovers [8].
specific for each class of DNA lesion, although extensive cross-talk Another important type of DNA lesion is the cis-syn cyclobu-
between repair pathways can occur [3]. One type of harmful DNA tane pyrimidine dimer (CPD) that is generated by ultraviolet light
lesions is DNA double strand breaks (DSB) that result from ioniz- (UV). In general, this lesion is removed by nucleotide excision repair
ing radiation [4] or can form during the process of DNA replication (NER). NER proceeds in five steps: recognition of the damage, inci-
[5,6]. Homologous recombination (HR) and non-homologous end- sion of the DNA strand on both sides of the lesion, DNA unwinding
joining (NHEJ) are the main pathways that repair these lesions and excision of a ∼30 nucleotides long DNA fragment containing
[7]. In Saccharomyces cerevisiae, HR is mediated by the products the lesion with the formation of a ssDNA gap, filling of the gap by
of genes from the RAD52 epistasis group (MRE11, XRS2, RAD50, DNA synthesis, and DNA ligation [1,9]. In yeast, genes involved in
RAD51, RAD52, RAD54, RAD55, RAD57, RAD59 and RDH54/TID1) [8]. NER can be classified into two groups: mutations in group-I (RAD1,
2, 3, 4, 10, 14 and 25) confer a high degree of sensitivity to UV,
whereas mutations in group-II (RAD7, 16, 23 and MMS19) confer
a moderate degree of sensitivity [9]. In addition, CPDs are rapidly
Abbreviations: CPD, cis-sin cyclobutane pyrimidine dimer; DSB, double strand
break; FACS, fluorescence-activated cell sorting; GG-NER, global genome NER; HR, removed from the transcribed strand (TS) of actively transcribed
homologous recombination; HM, cryptic mating type; MAT, mating type; NER, RNA polymerase II and I genes, a process that is referred to as
nucleotide excision repair; SIR, silent information regulator; TC-NER, transcription- transcription-coupled NER (TC-NER) [10–15]. Rad26p and Rad34p
coupled NER; UV, ultraviolet light.
participate in TC-NER driven by RNA polymerase II and I, respec-
∗ Corresponding author at: Département de Microbiologie et d’Infectiologie, Fac-
tively [16–18]. The non-transcribed DNA strands are repaired by
ulté de Médecine, Poste 7446, Université de Sherbrooke, 3001 12th Ave Nord,
Sherbrooke, QC J1H 5N4, Canada. Tel.: +1 819 564 5360; fax: +1 819 564 5392. global genome NER (GG-NER), which requires the Rad7p-Rad16p
E-mail address: [email protected] (A. Conconi). and Abf1 complexes [19].

1383-5718/$ – see front matter. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.mrgentox.2010.03.013
 M. Toussaint et al. / Mutation Research 698 (2010) 52–59 53

DNA repair processes are not mutually exclusive. For instance, MTO4 was constructed by one step PCR deletion strategy [56] using BY4737 (Mat˛
cross-talk occurs between HR and NER [20,21], and budding yeast ade2::hisG his3200 met150 trp163 ura30) [56], with sir2-fwd and sir2-
rev primers (Table 2) and 1 ng of pRS303 [57]. The diploid MTO6 (homozygous for all
cells with defective HR (e.g. rad52) are more sensitive to UV irra-
SIR loci) was constructed by mating BY4741 (Mata his31 leu20 met150 ura30)
diation [22,23]. Furthermore, the heterodimer Rad1p/Rad10p takes [56] with BY4737 (Mat˛ ade2::hisG his3200 met150 trp163 ura30), and
part in both NER and HR [24] and, in at least some situations, HR selected on synthetic medium YC-LEU-TRP. The strains MTO131 to MTO135 were
repairs CPDs that are present on the TS of active genes [20]. On the constructed by one step PCR deletion strategy [56] with rad50-F and rad50-R
other hand, NER and the stalling of replication forks at CPDs create primers (Table 2) and 1 ng of pRS305 [57]. The strains MTO24 to MTO28 were con-
structed using rad52-fwd and rad52-rev primers (Table 2) with 1 ng of pRS305
DNA nicks and single-stranded gaps, which promote recombination [57], and MTO44 to MTO48 were constructed with rad54-fwd and rad54-rev
in mitotic cells [25–29]. Consequently, UV irradiation can induce primers (Table 2) and 1 ng of pRS305 [57]. Correct insertion of PCR fragments into
DNA recombination [25,30–34]. However, to what extent HR con- the genome was confirmed by Southern blot analysis of total genomic DNA derived
tributes, together with NER, to cell survival after UV irradiation is from the respective strains.
not clear.
2.2. Media, growth conditions and UV irradiation
DNA repair is affected by the structure of chromatin [35–38],
whereby arrays of nucleosomes inhibit the recognition of the dam- Yeast were grown exponentially (∼0.7 × 107 to 107 cells/ml) in yeast
age and/or its removal [39,40]. In yeast, transcriptionally silenced extract–peptone–dextrose (YEPD) at 26 ◦ C and under continuous rotation. After
chromatin is functionally analogous to heterochromatin in higher centrifugation, cells were re-suspended in sterile water to 0.8 × 107 cells/ml and
UV irradiated (see below). For each strain, three wells of a 96-well non-coated
eukaryotes. It forms at some DNA regions, including the cryptic
polystyrene microplates (Corning incorporated) containing 95 ␮l of fresh YEPD were
mating type loci (HM loci) and telomeres [41–44]. The establish- inoculated with 5 ␮l of irradiated, or mock treated, yeast cultures (corresponding to
ment of these domains is dependent on the histone deacetylase 4 × 104 cells), and cell growth was monitored with a PowerWave micoplate scan-
Sir2p and on structural proteins such as Sir3p and Sir4p. At the HM ning spectrophotometer (Bio-Tek). The optical density was automatically recorded
using KC4 microplate data analysis software (Bio-Tek), and the software was pro-
loci, the recruitment of the structural Sir proteins requires Sir1p,
grammed with the following settings: the optical density was measured at 660 nm,
whereas at telomeres Rap1p and Ku70/Ku80, but not Sir1p, are incubation was kept at 30 ◦ C, ±0.1 ◦ C, the microplates were subjected to continuous
required [45–47]. It is thought that the presence of the Sir-complex shaking at intensity 2, during 595 s. Readings were done every 10 min during a 48-h
represses transcription by compacting chromatin [48,49]. Hence, period.
deletion of SIR2, SIR3 or SIR4 abolishes both HM repression [50] and For UV irradiation, yeast cells, 300 ␮l of 0.8 × 107 cells/ml in sterile water,
were placed on a glass plate and UV irradiated (primary 254 nm) with 50, 80,
telomeric silencing [42]. Loss of HM silencing results in the expres-
100 and 150 J/m2 , as measured with a UVX radiometer (Ultra-Violet Products,
sion of the sexual mating type “a” and “␣” transcription-regulators Upland, US). To study repair of UVC induced DNA lesions in yeast, doses in the
genes, which reside at the HML and HMR loci, respectively. Thus, range of 100–200 J/m2 are often used (e.g. [15,58]). For the colony-formation assays
in addition to the active MAT locus, haploid sir mutant strains described in Fig. 1, 5 ␮l of 10-fold serial dilutions (0.8 × 107 , 0.8 × 106 , 0.8 × 105 ,
0.8 × 104 and 0.8 × 103 cells/ml), from the same mock treated or UV irradiated cells
express both HMRa and HML˛ genes. Since they display proper-
used for the growth curve assay, were spotted on YEPD plates. The plates were
ties of MATa/MAT˛ diploids, haploid sir mutants are defined as incubated in the dark at 30 ◦ C for 48 h.
pseudo-diploid cells [50,51].
Similar to the process of transcription, silenced chromatin 2.3. Data collection and analysis of growth curves
inhibits DNA repair. For example, NER removes CPDs more slowly
Analysis of cell recovery after UV irradiation is described in detail in previ-
from the silenced HML locus than from the active MAT˛ locus, and
ous studies [59,60]. Briefly, the curves obtained were analyzed by an algorithm to
this preferential repair is abolished in sir3 mutants [52,53]. More- obtain two growth parameters: the lag time (), which represents the time required
over, deletion of SIR2 increases NER efficiency in the HML locus [37]. for a population of cells to reach its maximal growth rate, and the recovery time
Analogously, the efficiency of repair of CPDs was examined in the (RT), which corresponds to the time needed to recover from UV irradiation. RT is
URA3 gene that was engineered near the right telomere end of chro- calculated by subtracting the  at 0 J/m2 from the  at 150 J/m2 (RT150 = 150 − 0 ).

mosome V and, therefore, it is subject to telomeric transcriptional 2.4. Cell synchronization and FACS analyses
silencing. It was found that NER in the URA3 gene operates more
efficiently in sir3 cells than in the WT, conversely over expres- Cells were grown exponentially (∼0.7 × 107 to 107 cells/ml) in YEPD at 30 ◦ C
sion of Sir3p reduces the efficiency of DNA repair even further under continuous rotation, collected and re-suspended in 1.5 ml fresh YEPD at
0.4 × 107 cells/ml (asynchronized cultures) or in 1.5 ml fresh YEPD with 1 ␮g/ml of
[54]. Therefore, sir cells could be more resistant to UV irradiation,
alpha factor (sigma cat# T6901) (G1 synchronized cultures) or in 1.5 ml fresh YEPD
although this condition has not been thoroughly tested. with 10 ␮g/ml of nocodazol (sigma cat# M1404) (G2 synchronized cultures). There-
In this study we analyzed whether sir mutants are more resis- after, cultures were incubated at 30 ◦ C for 2 h under continuous rotation, collected
tant to UV irradiation and, if so, whether this phenotype results by centrifugation and re-suspended in 1.5 ml sterile water. The washing step was
from the activity of HR in pseudo-diploid cells. In addition, the repeated four times and 300 ␮l of cell suspension were irradiated as described in
Section 2.2. In parallel, 800 ␮l of washed cells was prepared for FACScan as following:
impact of HR and NER in cell recovery from UV irradiation was collected, re-suspend in 1 ml ethanol 70% (v/v), and incubated for 1 h at room tem-
investigated in G2/M-synchronized cells, and the co-participation perature. Cells were harvested, washed in 1 ml PBS (phosphate buffer saline; 37 mM
of HR and NER was followed in different phases of the cell cycle. NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate
Finally, the contribution of HR vs. NER to cell survival after UV irra- monobasic, pH of 7.4) and incubated for 1 h at 37 ◦ C in the dark in 1 ml staining solu-
tion [0.5 mg RNAse A (Roche applied science cat# 1 119 915) and 5 mg propidium
diation was determined in exponentially (asynchronous) growing
iodide (Sigma cat# P4170) per ml PBS], followed by gentle sonication (2 s, at set-
cells. ting 1 on a Visonic 50, Virtis) to disrupt cell clumps. Cell acquisition was made with
a Becton-Dickinson FACScan cytometer and the data analyzed with the CellQuest
2. Materials and methods software (Becton-Dickinson).

2.1. Yeast strains 3. Results and discussion

All strains are listed in Table 1. Additional information for the BY4741-
related strains is available in the Saccharomyces Genome Deletion Project web 3.1. Yeast sir haploid strains recover faster than the WT from
site [http://www-sequence.stanford.edu/group/yeast deletion project/deletions3. UV irradiation
html]. Diploid MTO5 (sir2::HIS3/sir2::KanMX4) was constructed by mating for 6 h
at 30 ◦ C YDL042C (Mata sir2::KanMX4 his31 leu20 met150 ura30) [55] with Wild type (WT), sir1, sir2, sir3, sir4 and rad7 (here used
MTO4 (Mat˛ sir2::HIS3 ade2::hisG his3200 met150 trp163 ura30), both
containing pDR589 (SIR2 inserted in pRS426, kindly provided by Dr Rivier). Selec-
as control) cells were irradiated with 150 J/m2 of UV light. Their
tion for sir2/sir2 diploid cells without pDR589 was made on synthetic medium sensitivities to UV irradiation were compared by spotting serial
YC-LEU-TRP containing 1 g/L of 5-FOA (5-fluoroorotic acid; Bioshop cat# FOA555). dilutions of yeast suspensions on solid medium, or by using the
54 M. Toussaint et al. / Mutation Research 698 (2010) 52–59

Table 1
Strains.

Name Genotype References

BY4741 MATa his31 leu20 met150 ura30 [56]

YKR101W Same as BY4741 sir1::KanMX4 [55]
YDL042C Same as BY4741 sir2::KanMX4 [55]
YLR442C Same as BY4741 sir3::KanMX4 [55]
YDR227W Same as BY4741 sir4::KanMX4 [55]
YJR052W Same as BY4741 rad7::KanMX4 [55]
JRY2334 Isogenic to W303-1a; Mata ade2-1 can1-100 his3-11,15 [51]
leu2-3,112 trp1-1 ura3-1 rad5-535
JRY4563 Same as JRY2334 sir2::TRP1 [51]
JRY3289 Same as JRY2334 sir3::TRP1 [51]
JRY4580 Same as JRY2334 sir4::TRP1 [51]
JRY3658 Same as JRY2334 hmla p mata p hmra p [51]
JRY6348 Same as JRY2334 hmla p mata p hmra p sir2::TRP1 [51]
JRY3606 Same as JRY2334 hmla p mata p hmra p sir3::TRP1 [51]
JRY6349 Same as JRY2334 hmla p mata p hmra p sir4::TRP1 [51]
JRY5384 Mata/Mat␣ [51]
JRY6328 Mata p/Mat␣ [51]
MTO5 sir2::KanMX4/sir2::HIS3 (YDL042C X MTO4) This study
MTO6 Mata/Mat␣ (BY4741 X BY4737) This study
MTO64 Same as BY4741 rad26::LEU2 This study
MTO65 Same as YKR101W ra26::LEU2 This study
MTO66 Same as YDL042C rad26::LEU2 This study
MTO67 Same as YLR442C rad26::LEU2 This study
MTO68 Same as YDR227W rad26::LEU2 This study
MTO131 Same as BY4741 rad50::LEU2 This study
MTO133 Same as YDL042C rad50::LEU2 This study
MTO134 Same as YLR442C rad50::LEU2 This study
MTO135 Same as YDR227W rad50::LEU2 This study
MTO24 Same as BY4741 rad52::LEU2 [60]
MTO25 Same as YKR101W rad52::LEU2 This study
MTO26 Same as YDL042C rad52::LEU2 This study
MTO27 Same as YLR442C rad52::LEU2 This study
MTO28 Same as YDR227W rad52::LEU2 This study
MTO44 Same as BY4741 rad54::LEU2 This study
MTO45 Same as YKR101W rad54::LEU2 This study
MTO46 Same as YDL042C rad54::LEU2 This study
MTO47 Same as YLR442C rad54::LEU2 This study
MTO48 Same as YDR227W rad54::LEU2 This study
W3749-1A Isogenic to W303; Mata can1-100 his3-11,15 leu2-3,112 [69]
trp1-1 ura3-1 bar1::LEU2
YJR052W Same as BY4741 rad7::KanMX4 [55]
YBR114W Same as BY4741 rad16::KanMX4 [55]
YJR035W Same as BY4741 rad26::KanMX4 [55]
YDR314C Same as BY4741 rad34::KanMX4 [55]
YMR224C Same as BY4741 mre11::KanMX4 [55]
YNL250W Same as BY4741 rad50::KanMX4 [55]
YDR369C Same as BY4741 xrs2::KanMX4 [55]
YER095W Same as BY4741 rad51::KanMX4 [55]
YGL163C Same as BY4741 rad54::KanMX4 [55]
YDR076W Same as BY4741 rad55::KanMX4 [55]

Table 2
Oligos.

Name Sequence

sir2-fwd 5 -AGACACATTCAAACCATTTTTCCCTCATCGGCACATTAAAGAGATTGTACTGAGAGTGCAC-3
sir2-rev 5 -AATGTCATCACAGTACCCCAAAAGAGATAAATCAAATTCTGCTGTGCGGTATTTCACACCG-3
rad50D-f 5 -GCAGACAATTGACGCAAGTTGTACCTGCTCAGATCCGATAAGATTGTACTGAGAGTGCAC-3
rad50D-r 5 -CCTTGTTGTTCGCGAAGGCAAGCCCTTGGTTATAAATAGGCTGTGCGGTATTTCACACCG-3
rad52-fwd 5 -GGAGGTTGCCAAGAACTGCTGAAGGTTCTGGTGGCTTTGGAGATTGTACTGAGAGTGCAC-3
rad52-rev 5 -GTTTCGGCCAGGAAGCGTTTCAAGTAGGCTTGCGTGCATGCTGTGCGGTATTTCACACCG-3
rad54-fwd 5 -GAGCAGCAACTTTTCTCTTTCTTCACTAAAGCTGCTACGAAGATTGTACTGAGAGTGCAC-3
rad54-rev 5 -CCATGCCCATCATTTAAAATAAATATGTAATGACCCCCCGCTGTGCGGTATTTCACACCG-3

growth curve assay of yeast grown in liquid medium [59,60]. Both tion. Compared to sir mutants, the higher UV sensitivity of WT is
assays show that all yeast strains replicate similarly under normal revealed by the longer time needed to reach OD660 ∼ 0.4 (Fig. 1A,
growth conditions (Fig. 1A and B, upper panels), and that rad7 is lower panel). These results show that the growth curve assay can
highly sensitive to UV irradiation at 150 J/m2 (Fig. 1A and B, lower reveal changes in UV sensitivity when yeast strains are only weakly
panels). After UV irradiation, the spot test does not show any sig- affected in DNA repair proficiency. Thus, the growth curve assay
nificant differences in colony formation between sir and WT cells was used in the experiments that follow. The OD curves (Fig. 1A)
(Fig. 1B, lower panel). Conversely, the growth curve assay clearly were analyzed by an algorithm to calculate the lag time () that
shows that sir mutants recover faster than WT from UV irradia- represents the time required for a population of cells to reach its
 M. Toussaint et al. / Mutation Research 698 (2010) 52–59 55

Fig. 1. Exposure of WT, rad7, sir1, sir2, sir3, and sir4 cells to UV irradiation.
(A) Analysis of cell concentration by optical density (OD660 ) measurements. Yeast
were grown exponentially and mock treated (0 J/m2 , upper panel) or UV irradiated
(150 J/m2 , lower panel), and 4 × 104 cells were deposited in triplicate in 96-well
plates. Cell densities were measured every 10 min for 48 h. Curves 1, 2, 3, 4, 5 and 6
are WT, rad7, sir1, sir2, sir3, or sir4 cells, respectively. (B) Spot test assay.
Ten-fold serial dilutions of the same cultures were spotted on YEPD plates and incu-
bated in the dark at 30 ◦ C for 48 h. (C) The lag times () in hours obtained from 24
independent growth curve experiments were calculated and used to generate the
recovery time (RT150 = 150 − 0 ). Values represent the mean ± S.E., p = 0.0003 for
sir1 and p < 0.0001 for sir2, sir3 and sir4.

Fig. 2. Exposure of haploid, pseudo-diploid and diploid cells to UV irradiation. Yeast

maximal growth rate, and the recovery time (RT) that corresponds
were grown exponentially, mock treated (0 J/m2 ) or UV irradiated (150 J/m2 ). Anal-
to the time needed to recover from UV irradiation [59,60]. RT is yses of cell concentration by optical density (OD660 ) were done as in Fig. 1A. (A)
calculated by subtracting the  at 0 J/m2 from the  at 150 J/m2 Analyses of UV recovery for WT, sir2, sir3 and sir4 in which all mating type
(RT150 = 150 − 0 ). A comparison of the RT150 values for the sir genes were inactivated by promoter deletion (hmla p, mata p, hmra p) (curves 1, 2,
3 and 4, respectively). (B) Analyses of UV recovery for an a/␣ diploid strain (WT/WT;
mutants with those obtained for WT clearly shows that the sir
curves 1) and a− /␣ diploid strain (WT p/WT: hmla p, mata p, hmra p/HML˛,
strains recover significantly faster than the WT (Fig. 1C). MAT˛, HMRa; curves 2). (C) Analyses of UV recovery for WT diploid cells (WT/WT;
curves 1) and homozygous sir2 diploid cells (sir2/sir2; curves 2).
3.2. Fast recovery of diploid and pseudo-diploid yeast cells after
UV irradiation
for the hmla p mata p hmra p SIR+ strain and all hmla p mata p
Haploid sir mutants present characteristics of a/␣ diploid hmra p sir strains are very similar (Fig. 2A, 150 J/m2 ; compare 1
yeast and are described as pseudo-diploid cells [50,51]. It was with 2, 3 and 4). Thereafter, we followed recovery from UV irra-
shown that diploid and pseudo-diploid cells present increased diation of two SIR+ diploid strains: an a/␣ diploid (WT) and an
resistance to ␥-ray radiation and radiomimetic drugs [61–63]. a− /␣ diploid (mata p/MAT˛) in which only the ␣ information is
Whether the UV resistance phenotype of sir cells reported here expressed. The a− /␣ strain, albeit being diploid and having two
(Fig. 1) results from pseudo-diploidy or from increased DNA repair copies of the genome, it behaves like ␣-mating haploid and is typ-
activity in de-silenced chromatin is not known. In order to dis- ified as pseudo-haploid. The data show that both strains grow at
tinguish between these possibilities, we used a strain in which similar rates before irradiation (Fig. 2B, 0 J/m2 ). After irradiation
a deletion of SIR-genes does not induce pseudo-diploidy. In this with 150 J/m2 of UV light, the a/␣ diploid cells recover faster than
strain, all genes for mating type specificity (HML, MAT, HMR) are the a− /␣ pseudo-haploid cells (Fig. 2B, compare curves 1 and 2).
inactivated by promoter deletions (hmla p mata p hmra p) [51]. These results support the notion that changes in sexual mating type,
Therefore, the hmla p mata p hmra p cells behave like a-mating rather than changes in repair of DNA in silent chromatin regions,
haploid cells even when the SIR genes are deleted [51]. control the recovery speed of sir mutants after UV irradiation.
Cells recovery from UV irradiation was analyzed as described Finally, UV sensitivity was followed for the diploids WT (SIR2/SIR2)
in the previous section. The results show that all strains grow at and the homozygous sir2 cells (sir2/sir2). Both diploid strains
a similar rate (Fig. 2A, 0 J/m2 ), and that sir strains in this genetic grow at a similar rate (Fig. 2C, 0 J/m2 ) and show similar recovery
background recover faster than the WT cells from UV irradiation lag-times after UV irradiation (Fig. 2C, 150 J/m2 ). Thus, diploid cells
at 150 J/m2 (data not shown). Conversely, the times of recovery lacking the SIR2 gene are not affected in the recuperation from
56 M. Toussaint et al. / Mutation Research 698 (2010) 52–59

Fig. 3. Exposure of WT, sir1, sir2, sir3, sir4, rad50, rad52, rad54, rad26 and sir rad double mutants to UV irradiation. Yeast strains were grown and UV
irradiated as described in materials and methods. Analyses of cells recovery were done as in Fig. 1A and the RT150 values were calculated as in Fig. 1C. Values represent the
mean ± 1 S.E. of three independent experiments. ND: not determined.

UV irradiation. These data show that the absence of SIR-genes, 150 J/m2 and above (60). These same results were obtained with
and therefore of silenced chromatin domains, is not the predomi- sir1, 3 and 4 strains (data not shown). At these doses yeast that
nant cause for fast recovery from UV irradiation. Consequently, the are deleted for one of the group-I or II NER genes are highly sen-
pseudo-diploidy phenotype of haploid sir mutants promotes this sitive to UV irradiation and do not grow (Fig. 6D). Thus, only the
fast recovery. rad26 strain could be used to overcome this obstacle, although
the implication of only one of the two NER sub-pathways is tested].
3.3. Fast recovery of sir haploid cells (pseudo-diploids) after UV
UV irradiations with a dose of 150 J/m2 and measurements of cell
irradiation depends on the HR RAD50, RAD52 and RAD54 genes,
recovery for WT, rad26, and sir rad26 double mutants were
but not on the NER RAD26 gene
done as described above. In the rad26 strain TC-NER is impaired
and, as expected, the rad26 strain is more sensitive to UV irra-
To further investigate the relative fast recovery of sir pseudo-
diation than the WT (Fig. 3; compare filled bars), that is; it needs
diploid cells from UV irradiation, we ascertained the contribution
an additional ∼10 h to recover from the insult. The sir1, sir2,
of genes involved in HR and NER pathways. In haploid cells, HR uses
sir3 and sir4 double mutants with rad26 recover faster than
DNA sequences on sister chromatids (in late S and G2/M phase of
the rad26 single mutant. This result implies that TC-NER does not
the cell cycle) to ensure faultless repair of DSBs [8]. UV irradia-
participate in the accelerated recovery of pseudo-diploid cells from
tions and measurements of cell recovery for WT, rad50, rad52,
UV induced DNA damage.
rad54, sir, and their respective double mutants were performed
as described in the previous sections. As expected from the results
shown in Fig. 1, sir strains recover faster than WT cells: the RT150 3.4. Haploid cells in G2-phase recover faster than cells in
for sir1, sir2 and sir3 being about 14 h, the RT150 for sir4 G1-phase from UV irradiation
being about 12 h, and the RT150 for the WT being about 19 h (Fig. 3).
Compared to WT, the rad50, rad52 and rad54 strains are con- Since in haploid cells, HR takes place during the S/G2/M phases
siderably more sensitive to UV irradiation (Fig. 3; compare filled of the cell cycle [8], whereas NER is active throughout the cell cycle
bars), underscoring the implication of HR in cell recovery from UV
irradiation. On average, these rad strains need an additional 6.5 h
to recover from the insult. Moreover, rad50, rad52 and rad54
show similar recovery times and, therefore, are about equally sen-
sitive to UV irradiation. The sir1, sir3 and sir4 double mutants
with rad50, rad52 and rad54 have similar recovery times as
the single rad50, rad52 and rad54 mutants; the sir3rad50
being somewhat more sensitive. These results imply that HR partic-
ipates in the accelerated recovery of pseudo-diploid cells from UV
induced DNA damage. The Sir2p could have additional functions,
since the sir2 rad double mutants are generally more sensi-
tive than the corresponding rad single mutants. Given that Sir2p
belongs to a large family of NAD-dependent enzymes, which influ-
ences the expression of a number of RNA polymerase II transcripts
[64], Sir2p may have broader effects on cell metabolism. Therefore,
we cannot exclude the possibility that Sir2p affects UV sensitivity Fig. 4. Exposure of non-synchronized, G2 synchronized and G1 synchronized WT
in complex and indirect ways. cells to UV irradiation. (A) WT cells were grown exponentially and suspended in
To test if NER participates in the fast recovery of pseudo- fresh YEPD (non-synchronized), in fresh YEPD with 10 ␮g/ml of nocodazol (G2 syn-
diploid cells from UV induced DNA damage, we repeated the same chronization), and in fresh YEPD with 1 ␮g/ml of alpha factor (G1 synchronization),
incubated and prepared for FACS analyses as described in Section 2. (B) Aliquot of
experiments with sir rad26 double mutants. [Note: in previous
synchronized and non-synchronized cells were mock treated (0 J/m2 ) or UV irradi-
experiments we have shown that fast recovery after UV irradiation ated with 150 J/m2 . Then, 4 × 104 cells were deposited in triplicate in 96-well plates.
of sir2 cells compared to WT can be measured only at doses of Cell densities were measured every 10 min for 48 h.
 M. Toussaint et al. / Mutation Research 698 (2010) 52–59 57

[65–68], we investigated the recovery of synchronized cells from

UV irradiation. Exponentially growing cultures were synchronized
in G2/M by nocodazole or in G1 with alpha factor before irradia-
tion with a dose of 150 J/m2 . The FACS of yeast cultures show that
cells arrested at the predicted phases of the cell cycle (Fig. 4A). Cells
recovery from UV irradiation was analyzed as described in the pre-
vious sections. The assay does not detect any difference between
arrested cells in time of cell growth recovery, before UV irradiation
(Fig. 4B, 0 J/m2 ). [We note that although there is a slight delay in cell
growth for synchronized vs. non-synchronized cultures, this is not
shown by the plots since the scale of the x-axis is 8 h and multiples
thereof]. The cell recovery from UV irradiation is shown in Fig. 4B
(150 J/m2 ). Cells arrested and irradiated in G2-phase recover much
faster than the non-synchronized cells (NS), which contain approx-
imately 60% G1- and 40% G2-cells. Furthermore, cells arrested in G1
phase recover at an even slower pace. These results suggest that in
G2, the activity of the two repair pathways render the cells more
resistant to UV light.
To determine the participation of HR and NER in the accelerated
recovery of G2-cells (Fig. 4B), additional experiments were done
by irradiating strains carrying deletions of one of the genes from
the RAD52 epistasis group with 100 J/m2 , or by irradiating strains
carrying deletions of one of the representative NER genes of group-
II with 80 J/m2 . Strains carrying deletions of group-I NER genes
are highly sensitive to UV and, therefore, could not be used. Both
non-synchronized and synchronized cells in G2/M were irradiated,
their recovery was followed with the growth curve assay and the
corresponding RTs were calculated. As shown in Fig. 5, WT cells

Fig. 5. Exposure of non-synchronized and G2 synchronized HR and NER mutant

cells. (A) WT, mre11, rad50, xrs2, rad51, rad52, rad54, rad55, and (B) WT, Fig. 6. Exposure of WT, rad14, rad7, rad16, rad26, rad52 and rad54
rad7, rad16, rad26 and rad34, were exposed to UV irradiation at the indicated to UV irradiation. Analyses of cells concentrations by optical densities (OD660 )
doses. Yeast strains were grown, synchronized and UV irradiated as described in Fig. for the indicated yeast strains that were (A) mock treated (0 J/m2 ) or irradi-
4. Analyses of cells recovery were done as in Fig. 1A and the RT values were calculated ated with (B) 50 J/m2 , (C) 100 J/m2 , (D) 150 J/m2 . (E) The respective RT values
as in Fig. 1C. White bars: non-synchronized (NS) cells. Grey bars: G2-synchronized were calculated as described in Fig. 1C. White bars: 50 J/m2 ; gray bars: 100 J/m2 ;
(G2/M) cells. Values represent the mean ± 1 S.E. of three independent experiments black bars: 150 J/m2 . Values represent the mean ± 1 S.D. of three independent
for (A) and ±1 S.D. of three independent growth curves for (B). experiments.
58 M. Toussaint et al. / Mutation Research 698 (2010) 52–59

synchronized in G2/M recovered ∼5 h sooner than asynchronous cycle dependent survival to UV irradiation, and this report shows
cells after irradiation with 100 J/m2 , and ∼2 h after irradiation with that cells synchronized in the G2/M phase of the cell cycle are
80 J/m2 (WT; compare filled with empty bars in Fig. 5A and B). As more resistant to UV compared to non-synchronized and G1 syn-
expected, asynchronous and synchronized yeast are more sensi- chronized cells. Therefore, both HR and GG-NER participate in the
tive to UV when HR or NER are impaired (compare WT with HR and recovery of G2/M cells from UV irradiation. We suggest that the
NER depleted strains). Interestingly, recovery of non-synchronized combined activities of HR and GG-NER (when two copies of the
vs. synchronized HR mutants (mre11, rad50, xrs2, rad51, chromosomes are present) explain the higher resistance of G2 cells
rad52, rad54, and rad55) and GG-NER mutants (rad7 and vs. G1 haploid cells. Taken together these results underscore the
rad16) proceeds at similar rates (compare empty with filled bars role of cross-talk that occurs between HR and NER in cells survival.
in Fig. 5A and B), showing that HR and GG-NER co-participate With this study we provide detailed and quantitative information
during the G2/M phases to repair UV induced DNA damage. On on the co-participation of HR and NER in UV survival of yeast cells.
the contrary, recovery of TC-NER mutants (rad26 and rad34) is
faster in G2/M synchronized cells than in asynchronous cells. These Conflict of interest statement
results suggest that HR assists mostly GG-NER in the recovery from
UV induced DNA damage when cells are in the G2/M phase of the There are no conflicts of interest.
cell cycle.
Acknowledgments
3.5. HR and NER participate to different extents in the recovery of
cell growth after UV irradiation We thank Dr Kobrin at the Université de Sherbrooke for critical
reading of the manuscript. Also, we would like to thank Dr Rivier for
Although NER is the main pathway that removes UV photoprod- the gift of the pDR589 plasmid, Dr Rine for the gift of the JRY strains,
ucts [9], HR is also involved [22,23] and it is clearly important for Luc Grenier for the data analyses and Leonid Volkov at the Centre
cell cycle specific recovery after UV irradiation (Figs. 4 and 5). How- de Recherche Clinique Etienne-Le Bel for help with FACS analysis.
ever, to date, it is not described the extent of the participation of This work was supported by the grant from the Nat. Sci. Eng. Res.
the two repair pathways in cells survival after UV irradiation. For Coun. Can. (NSERC) to A.C. M. Toussaint was supported in part by a
this purpose, WT and selected knockout strains of genes involved NSERC fellowship and in part by the NSERC grant to A.C. R.J.W. was
in NER and HR were irradiated with doses of 50, 100 and 150 J/m2 supported by a grant of the Canadian Institutes of Health Research
of UV light, and cell survival was monitored with the growth curve to R.J.W. (CIHR grant MOP 12616).
assay. The UV sensitivities of rad52 and rad54 strains, which
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