Oxidant-induced cell cycle delay of Saccharomyces cerevisiae: the involvement of SWI6

R E S E A R C H A R T I C L E

Oxidant-induced cell-cycle delay inSaccharomyces cerevisiae :the involvementoftheSWI6 transcription factorChii Shyang Fong1, Mark D. Temple1, Nazif Alic1, Joyce Chiu1, Moritz Durchdewald1,Geoffrey W. Thorpe1, Vincent J. Higgins2 & Ian W. Dawes1

1Ramaciotti Centre for Gene Function Analysis and School of Biotechnology and Biomolecular Sciences, UNSW, NSW, Australia;

and 2School of Biomedical and Health Sciences, University of Western Sydney, Penrith South DC, NSW, Australia

Correspondence: Ian W. Dawes, Ramaciotti

Centre for Gene Function Analysis and School

of Biotechnology and Biomolecular Sciences,

UNSW, NSW 2052, Australia. Tel.: 161 2

9385 2089; fax: 161 2 9385 1050;

e-mail: [email protected]

Received 21 August 2007; revised 4 December

2007; accepted 6 December 2007.

First published online 16 January 2008.

DOI:10.1111/j.1567-1364.2007.00349.x

Editor: Terrance Cooper

Keywords

oxidative stress; lipid peroxidation;

cell-cycle delay.

Abstract

Cells treated with low doses of linoleic acid hydroperoxide (LoaOOH) exhibit a

cell-cycle delay that may provide a mechanism to overcome oxidative stress.

Strains sensitive to LoaOOH from the genome-wide deletion collection were

screened to identify deletants in which the cell-cycle delay phenotype was reduced.

Forty-seven deletants were identified that were unable to mount the normal delay

response, implicating the product of the deleted gene in the oxidant-mediated cell-

cycle delay of the wild-type. Of these genes, SWI6 was of particular interest due to

its role in cell-cycle progression through Start. The swi6 deletant strain was delayed

on entry into the cell cycle in the absence of an oxidant, and oxidant addition

caused no further delay. Transforming the swi6 deletant with SWI6 on a plasmid

restored the G1 arrest in response to LoaOOH, indicating that Swi6p is involved in

oxidant sensing leading to cell division delay. Micro-array studies identified genes

whose expression in response to LoaOOH depended on SWI6. The screening

identified 77 genes that were upregulated in the wild-type strain and concurrently

downregulated in the swi6 deletant treated with LoaOOH. These data show that

functions such as heat shock response, and glucose transport are involved in the

response.

Introduction

Cells exhibit a range of responses to oxidative stress, includ-

ing adaptation to increased resistance (Collinson & Dawes,

1992), cell-cycle progression delay (Flattery-O’Brien &

Dawes, 1998) and widespread changes in gene expression

(Gasch et al., 2000) while at higher concentrations they are

no longer able to survive and undergo a form of apoptosis

(Ludovico et al., 2005). They also show similar responses to

lipid peroxides formed as a result of reactive oxygen species

(ROS) damaging unsaturated lipids. Here, the authors

report on genes involved in the response of Saccharomyces

cerevisiae to linoleic acid hydroperoxide (13-hydroperoxyli-

noleic acid; LoaOOH), which is an oxidant that is an

oxidative breakdown product of lipid peroxidation, and

one of the most toxic peroxides to yeast cells (Evans et al.,

1998; Aoshima et al., 1999). LoaOOH is a product of free

radical attack on a long-chain unsaturated fatty acid, and it

has maximal toxicity to cells undergoing respiration. Like

other lipid peroxides, it is reactive and, in the cell, breaks

down into other highly reactive products including epoxides

and reactive aldehydes such as malondialdehyde and 4-

hydroxynonenal (Evans et al., 1998; Dickinson & Forman,

2002).

LoaOOH treatment of wild-type cells leads to a G1 delay

in cell-cycle progression before the Start checkpoint. During

this delay cells may mount antioxidant defences and systems

to elicit adequate repair of oxidant damage before re-

engaging in the cell cycle (Shackelford et al., 2000). This

leads to accumulation of unbudded cells in asynchronous

populations, together with a budding and replication delay

in synchronous ones (Mendenhall & Hodge, 1998; Alic et al.,

2001). Currently, little is known about the genes or processes

that are involved in this cell-cycle delay. Only two genes were

previously implicated in the G1 delay in response to

LoaOOH or other oxidants. These were OCA1 and PEX17

because strains deleted for these genes did not show cell-

cycle delay in response to LoaOOH (Alic et al., 2001).

For cells to pass Start and commit to cell-cycle progres-

sion, the two transcription factor complexes SBF (Swi4p/

FEMS Yeast Res 8 (2008) 386–399c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Swi6p-dependent cell-cycle box-binding factor) and MBF

(Mbp1p/Swi6p-dependent cell-cycle box-binding factor) are

required. These form part of an underlying transcriptional

regulatory network of nine transcription factors that control

the expression of the cyclins and oscillations of the cyclin/

CDK activities during the cell cycle (Simon et al., 2001). The

activation of Swi4p is dependent on Cln3p/Cdc28p activity

as cells reach a critical size (Koch et al., 1996) and Swi4p is

unable to bind to promoters in the absence of Swi6p (Baetz

& Andrews, 1999). Together, SBF and MBF control the

activation of many G1/S phase specific-genes. The cyclin

genes Cln1p and Cln2p that associate with the major cyclin-

dependent kinase Cdc28p are targets of SBF/MBF and SBF,

respectively. Predominantly, the relevant genes involved

in cell-cycle control are activated by either SBF or MBF,

whereas genes involved in cell-wall biogenesis, budding,

cytokinesis, histones, chromatin modifiers and telomere

length are activated by SBF alone, and those involved in

DNA replication are activated by MBF (Simon et al., 2001).

Interestingly, a systematic screen of the genome-wide set

of deletion mutants each with a deletion in one nonessential

gene (Winzeler et al., 1999) revealed that 256 deletants were

sensitive to LoaOOH (Thorpe et al., 2004). These studies

showed that lipid and carbohydrate metabolism is crucial

for LoaOOH tolerance, the peroxisome may be the site of

LoaOOH detoxification and that energy from the tricar-

boxylic acid cycle is required to maintain active defenses

against lipid peroxidation (Thorpe et al., 2004). Interest-

ingly, they also showed that the swi6 deletion strain was

sensitive to LoaOOH.

While the involvement of SWI6 indicates that one or both

of the canonical transcription factor complexes SBF and

MBF may be involved in maintaining resistance to

LoaOOH, it is not known whether it is involved in the G1

checkpoint arrest and what other systems may be involved.

Because cell-division delay is a phenotype that is not easy to

determine in a high-throughput screening, a systematic

evaluation has been carried out of LoaOOH-induced cell

division arrest in deletants of S. cerevisiae that are sensitive

to LoaOOH treatment because such mutants are most likely

to be the ones affected in one or more of the cellular

responses to the ROS.

Materials and methods

Strain and plasmids

The S. cerevisiae deletants used in this study were in the

homozygous diploid strain BY4743 genetic background

(MATa/MATa; his3D1/his3D1; leu2D0/leu2D0; met15D0/

MET15; LYS2/lys2D0; ura3D0/ura3D0) (Brachmann et al.,

1998). These deletants were provided by the European

S. cerevisiae Archive of Functional Analysis (EUROSCARF).

For microarray analysis, the wild-type S. cerevisiae haploid

strain BY4741 (MATa his3D1 leu2D0 met15D0 ura3D0;

Euroscarf) (Brachmann et al., 1998) was used, together with

the swi6 deletant strain (MATa his3D1 leu2D0 met15D0

ura3D0 swi6<kanMX4).

SWI6 coding sequence and its flanking sequences (1 kb

upstream and 0.5 kb downstream) were amplified by PCR

and cloned into the pRS415 CEN LEU2 vector. The resulting

plasmid pSWI6 and the vector control pRS415 were trans-

formed into the swi6 deletant and LEU21 transformants

were selected. The vector control pRS415 was also trans-

formed into swi6 deletant strain as a control.

Media and chemicals

YEPD medium contained 2% (w/v) glucose, 2% (w/v)

bactopeptone and 1% yeast extract. YEPG medium con-

tained 2% (w/v) bactopeptone, 1% yeast extract and 3%

(v/v) glycerol. Synthetic complete (SC) medium contained

2% (w/v) glucose, 0.17% yeast nitrogen base, 0.5% ammo-

nium sulphate, 0.074% complete supplement mixture with-

out tryptophan (CSM-TRP) and 0.01% tryptophan; 2%

(w/v) agar was added to solidify the media. Cultures were

shaken at 300 r.p.m. and incubated at 30 1C, unless other-

wise noted. Linoleic acid hydroperoxide (LoaOOH) stock

was prepared and assayed as described by Evans et al. (1998)

and stored at � 20 1C in methanol. Synchronization of

MATa strains using a-factor, oxidant treatment and release,

and determination of a-factor-resistant cells was performed

as described previously (Alic et al., 2001).

Screening of mutants

SC medium (100 mL) was inoculated with 10mL of an

overnight YEPD starter culture and incubated at 30 1C to

an OD600 nm of 0.2. Cultures were equally divided into two

flasks and incubated until the OD600 nm reached 0.3–0.4.

LoaOOH (to 0.01 mM) was added to one of the flasks,

leaving the other as an untreated control. Samples were

collected 60 min after treatment for budding index (BI)

estimation. Cell culture (1 mL) was collected and cells were

fixed in 3 mL of ice-chilled ethanol. Cells were then washed

in 1 mL of 0.2 M Tris-HCl (pH7.5), resuspended in 20 mL of

0.2 M Tris-HCl (pH7.5) and maintained at 4 1C until count-

ing. Where clumping occurred, washed cells were sonicated

with six pulses of 5 s each at 30% power in a M250 Branson

digital sonifier (Branson) before resuspension. Before

counting, 5 mL of fixed, resuspended cells were mixed with

40, 6-diamidino-2-phenylindole, dihydrochloride (DAPI)

and were viewed under phase contrast in a fluorescence

microscope (Olympus BX60) using a 330–385 nm filter to

detect DAPI fluorescence. At least 400 cells were counted for

each sample.

FEMS Yeast Res 8 (2008) 386–399 c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

387Oxidant-induced cycle-delay: the involvement of SWI6

Cell-cycle analysis

Strains harbouring plasmids pRS415 or pSWI6 were cul-

tured in SC-leu medium at 30 1C until the OD600 nm reached

0.2. a-Factor was added to 1mg mL�1 and cultures were

synchronized by further incubation at 30 1C for 2 h. Cells

were subsequently washed and resuspended in phosphate-

buffered saline (PBS; 8 g L�1 NaCl, 0.2 g L�1 KCl, 1.44 g L�1

Na2HPO4, 0.24 g L�1 KH2PO4 adjusted to pH 7.4 with HCl).

The cultures were divided into equal aliquots and LoaOOH

was added to a final concentration of 0.02 mM in one, while

an equal volume of methanol was added to the other culture.

Cultures were incubated with shaking at 30 1C for 30 min

and were then washed once with PBS. Cells were resus-

pended in 20 mL SC-leu medium and 1 mL samples were

taken at 15-min intervals for 135 min. Cell samples were

spun down and fixed with 1 mL of ice-cold 70% (v/v)

ethanol and resuspended in 1 mL of 50 mM Tris-HCl, pH

7.5. Cells were subsequently sonicated in an M250 Branson

digital sonifier (Branson) for 20 s at 30% amplitude to

disperse clumps, washed once with 1 mL of 50 mM Tris-

HCl pH 7.5 and resuspended in 20mL of the same buffer.

Five microliters of cells were mixed with 1 mL of 100 mg mL�1

DAPI and were observed under � 100 phase contrast using

a BX60 Olympus microscope (Olympus, Japan).

Microarray analysis

Cultures were grown overnight to an OD600 nm of 0.2,

divided into 200 mL aliquots into prewarmed flasks and

allowed to grow to an OD600 nm of 0.4. The aliquots were

then treated with 0.03 mM LoaOOH for 60 min because this

was found to be the optimal treatment to measure the

responsive phenotype previously (Alic et al., 2003). The

reference RNA pool was obtained from five separate 200 mL

aliquots of untreated cells harvested at time zero. The

control culture was treated with methanol only, equivalent

to the volume required for LoaOOH treatment. In compar-

ing the transcriptional response of the swi6 deletant with the

wild type, biological duplicates were obtained for each

strain. Samples (200 mL) were harvested by mixing with

40 g of � 80 1C ice in prechilled tubes and centrifuged

(5 min at 3345 g) at 4 1C. The cells were then rapidly frozen

in an ethanol bath at � 80 1C. The RNA was isolated by

breaking cells in Trizol reagent (Gibco BRL, Life Technolo-

gies, MD) in a mini-bead beater in the presence of acid-

washed glass beads at 4 1C, and extracted according to the

manufacturer’s instructions. The RNA was further purified

using the RNeasy kit (Qiagen, Dusseldorf, Germany), in-

cluding DNAse I treatment. RNA quality was checked by

agarose gel electrophoresis and OD260 nm/280 nm determina-

tion, after which the reference samples were pooled.

Spotted oligonucleotide microarrays were obtained from

the Clive and Vera Ramaciotti Centre for Gene Function

Analysis (Sydney, Australia). Oligonucleotide probes (MWG

Biotech, Ebersberg, Germany) addressing 6250 yeast ORFs

were printed in duplicate on epoxy-coated glass substrates

(Eppendorf, Hamburg, Germany) and were blocked imme-

diately before use with ethanediol according to the manu-

facturer’s instructions. Each comparison was performed in

duplicate.

Labelling and hybridization were carried out according to

a modification of the protocol described by Hughes et al.

(2001). Twenty grams of total RNA was reverse-transcribed

incorporating 5-(3-aminoallyl)-dUTP (Sigma). The cDNA

was labelled by coupling the aminoallyl-dUTP to N-hydroxy

succinimide esters of either Cy3 or Cy5 (Amersham Bios-

ciences). The labelled cDNA of the samples to be compared

was mixed and hybridized to the array overnight at 37 1C

in DIG Easy Hyb (Roche Applied Science) containing

0.5 mg mL�1 Escherichia coli tRNA and 0.5 mg mL�1 dena-

tured herring sperm DNA. The slides were then washed

three times for 20 min in SSC (3 M sodium chloride, 0.3 M

trisodium citrate, pH 7.5) containing 0.1% sodium dodecyl

sulphate at 50 1C, rinsed several times in SSC and dried by

centrifugation. The slides were scanned using an Array-

WoRx E Biochip Reader (Applied Precision, WA) and

technical duplicates were performed including a dye-swap.

Data processing

Image analysis was performed in GENEPIX PRO 3.0 (Axon

Instruments). The signal for a gene was deemed ‘present’ if

no artefacts were associated with the spot, and the program

could identify the spot intensity above the background

intensity. Data were imported into GENESPRING 5.0 (Silicon

Genetics), where all further analysis was performed. Data were

normalized by the LOWESS normalization method. Only the

genes ‘present’ on all six slides were considered further. To

identify genes with twofold different expression, only the

genes ‘present’ on both duplicates of a given condition were

considered. Functional enrichment in the sets obtained was

determined using FUNSPEC (Robinson et al., 2002) and all

available databases. For information on individual gene

products, the SGD database was consulted. Gene lists derived

from the microarray data are given in supplementary Table S2.

Results

Genes involved in cell-cycle delay followingLoaOOH treatment

Delay of cell-cycle progression is thought to allow cells time

to mount defence and repair systems (Hartwell & Weinert,

1989; Shackelford et al., 2000); hence, it was reasoned that

the inability of cells to delay would be likely to contribute to

LoaOOH sensitivity. Because budding is an inherent prop-

erty of S. cerevisiae that occurs only after cells have passed


388 C.S. Fong et al.

through Start and after initiation of cell division (Zettel

et al., 2003), a systematic evaluation of the BI of 111 of the

most sensitive deletants (Thorpe et al., 2004) in response to

a sublethal dose of LoaOOH was carried out to assess their

cell-cycle response phenotype.

Exponentially growing cells of each sensitive mutant

were treated with a range of concentrations of LoaOOH

(0–0.04 mM) and samples were harvested for estimation of

BI at 30-min intervals over a 3-h period. Harvested cells

were fixed in ethanol, stained with DAPI and the BI of each

mutant was determined using fluorescence microscopy.

Representative images used for these analyses are shown in

Fig. 1. The BI (BI = proportion of cells that are budded) was

calculated for the wild type and each deletant strain.

Untreated wild-type cells had a roughly equal distribution

of budded and unbudded cells (BI = 0.52), whereas wild-

type cells treated with 0.01 mM LoaOOH exhibited an

accumulation of 69% unbudded cells (BI = 0.69) as shown

in Fig. 2a. At this dose, the viability of LoaOOH-treated cells

remained high during the treatment as shown in Fig. 2b.

From these data, it is clear that cells treated with 0.01 mM

LoaOOH exhibited both high accumulation (4 70%) of G1

unbudded cells at c. 60 min and high cell viability relative to

the untreated sample. The increase in unbudded cells is

therefore an indicator of cell-cycle delay that is caused by

treatment with LoaOOH, and hence the percentage increase

in unbudded cells on LoaOOH (DUBLoaOOH) was deter-

mined for all strains. The data obtained from the mutant

screen are shown in Fig. 3 to illustrate the distribution of

budding percentages obtained for the mutant strains. From

this it can be seen that some mutants have a substantially

greater percentage of unbudded cells relative to the wild-

type strain without any treatment, indicating that the

mutations may be causing some delay in cell-cycle progres-

sion through Start into the S phase. What is of greater

interest here is whether any of these mutants showed a

significant change in the percentage of unbudded cells after

treatment with LoaOOH.

The average DUBLoaOOH of the wild type (seven repli-

cates) was 17.5% (SD 4.75). This large increase in the

percentage of unbudded cells reflects the extent of delayed

passage through Start in response to LoaOOH treatment. A

deletant strain was considered to be affected in this cell-cycle

delay if the measured DUBLoaOOH was at least two SDs

(9.5%) lower than the wild type response, i.e. all deletants

that exhibited a DUBLoaOOH of o 8. Table 1 lists the 47

deletants that exhibited a significantly lower DUBLoaOOH

than the wild type, together with the response of the wild-

type for comparison. It also lists the sensitivity of the

individual deletants to LoaOOH as determined by Thorpe

et al. (2004).

Cellular functions involved in the cell-cycleresponse to LoaOOH

For each gene identified from the 47 deletant strains, the

gene descriptions, over-represented gene ontologies (GO

terms) (Dwight et al., 2002) and functional categories

(Robinson et al., 2002) were obtained to identify the cellular

processes that may be involved in the cell-cycle delay. These

are listed in supplementary Table S1a. These included PDX3

and THR4 that encode enzymes involved in vitamin B6

metabolism: how this relates to cell-cycle delay remains

speculative. PFK26, RPE1 and TKL1 encode key enzymes

Fig. 1. Microscopic analysis of BY4743 swi6 deletant cell budding. (a)

Phase-contrast microscopy image of cells. (b) Cells stained with DAPI and

viewed under fluorescence. Cells were scored as budded cells if, under

phase-contrast microscopy, both the mother and the bud were visible

and under fluorescence microscopy only the mother cell but not the bud

exhibited DAPI staining of the nucleus.

Fig. 2. Response of wild-type cells to LoaOOH

treatment. (a) The percentage budding of

exponential phase wild-type cells treated with

various concentrations of LoaOOH for 180 min.

Harvested cells were fixed, stained with DAPI

and buds were counted for each treatment.

Data are means and SD of duplicate experi-

ments. (b) A representative experiment showing

the viability of wild-type cells. Samples were

diluted and plated onto YEPD plates to monitor

cell viability.



involved in glucose metabolism. The pfk26 deletant lacks 6-

phosphofructose-2-kinase, which is critical in maintaining

the rate of glycolysis. In addition, Rpe1p and Tkl1p catalyse

consecutive steps in the conversion of ribulose 5-phosphate

into glyceraldehyde 3-phosphate, which are involved in a

reversible shuttle of metabolites between the pentose phos-

phate pathway (PPP) and glycolysis. NADPH produced by

the PPP is required for reduction of oxidized glutathione

(GSSG) and oxidized thioredoxin to confer protection

against damage caused by ROS (Jamieson, 1998). Deletion

of TKL1 confers cross-sensitivity to all the major oxidative

stresses (Sundstrom et al., 1993) and the PPP has been

shown to play protective roles during oxidative stress

(Kletzien et al., 1994; Slekar et al., 1996).

It has been noted previously that the putative protein

tyrosine phosphatase, Oca1p, is involved in cell-cycle delay

in response to LoaOOH (Alic et al., 2001). It has been

proposed recently that Oca1p, Ycr095p, Siw14p and two

additional proteins Ynl056p and Yhl029p constitute a

physical interaction network required for protection against

superoxide stress and that these proteins be named Oca1-5p,

respectively (Warringer et al., 2005; Ericson, 2006). The

occurrence of three mutants of the putative Oca complex in

these data indicate that the complex plays a significant role

in the cell-cycle delay. Interestingly, Warringer et al., using

Synthetic Genetic Array (SGA) analysis, noted strong genet-

ic interactions between members of the Oca complex and

Tkl1p, indicating that the Oca complex has an important

role in carbohydrate utilization and production of NADH/

NADPH, highlighting the potential role of NADPH genera-

tion in some aspect of cell-cycle sensing of oxidative

Fig. 3. Graph of the percentage of unbudded cells for LoaOOH-stressed

(ordinate) and unstressed (abscissa) conditions. Individual deletion strains

are represented by open circles, and representative wild-type ones by

closed circles indicated by arrows.

Table 1. Deletant strains of Saccharomyces cerevisiae that exhibited an

absence of G1 checkpoint in response to LoaOOH treatment

ORF Gene DUBLoaOOH DBIuntreated

LoaOOH

sensitivity

BY4743 Wild-type 17.5 0 Not sensitive

YMR169C ALD3 2.79 � 3.27 4

YJL115W ASF1 5.05 6.51 4

YER177W BMH1 5.88 4.19 4

YER141W COX15 0.38 2.11 6

YGR036C CWH8 0.31 10.13 6

YIL065C FIS1 2.29 � 4.2 6

YOL051W GAL11 � 4.44 4.24 3

YGR163W GTR2 0.09 5.65 6

YLR192C HCR1 2.33 8.3 7

YDR174W HMO1 4.36 8.33 3

YDL115C IWR1 6.56 10.96 4

YLR244C MAP1 7.72 4.46 2

YLR320W MMS22 5.51 � 15.91 5

YKL009W MRT4 4.15 14.49 6

YNL099C OCA1 1.18 2.36 7

YJR073C OPI3 3.02 3.93 3

YBR035C PDX3 2.1 13.3 2

YLR148W PEP3 0.82 4.29 6

YDL065C PEX19 0.32 � 1.25 6

YIL107C PFK26 3.43 � 2.1 2

YGL025C PGD1 � 1.74 � 0.13 4

YDL006W PTC1 3.36 5.89 6

YJL121C RPE1 7.8 � 1.36 2

YLL002W RTT109 � 2.46 � 9.45 6

YNL032W SIW14 4.33 1.63 7

YBR289W SNF5 3.36 8.1 1

YHL025W SNF6 6.49 0.93 3

YGR104C SRB5 � 1.85 6.92 4

YPL057C SUR1 2.66 9.44 6

YJL176C SWI3 1.33 10.63 1

YLR182W SWI6 3.08 13.75 7

YBR069C TAT1 1.52 21.51 2

YLR237W THI7 2.33 5.97 7

YCR053W THR4 6.09 � 2.06 3

YPR163C TIF3 4.1 9.91 6

YPR074C TKL1 6.78 5.9 1

YOR332W VMA4 4.63 4.06 4

YML007W YAP1 4.11 7.3 3

YBR285W YBR285W 2.33 1.63 7

YCR061W YCR061W 2 2.3 7

YCR095C YCR095C 2 3.63 7

YDL173W YDL173W 5.34 1.18 3

YIL077C YIL077C � 6.08 � 0.8 6

YLR456W YLR456W 4.12 1.95 4

YNL080C YNL080C 1.38 � 3.54 5

YOR135C YOR135C 6.58 1.12 3

Overlap YGR064W 4.61 2.34 4

SPT4

Data are given as sensitivity to the change in percent budding as a result

of treatment with 0.01 mM LoaOOH for one hour (DUBLoaOOH). The

difference in percentage buds of the untreated wild-type (DBIuntreated) is

also given, together with the sensitivity of the strains to LoaOOH on a

scale 1 (most sensitive) to 7 (least sensitive).



damage. It is also interesting that the Oca complex exhibits

a strong genetic interaction with Ald6p (Warringer et al.,

2005; Ericson, 2006). While the ald6 deletant was not

detected as being sensitive to LoaOOH and therefore was

not tested, the ald3 mutant was sensitive to LoaOOH and

exhibited an altered cell-cycle response to LoaOOH. ALD3

encodes a stress-induced NAD1-dependent aldehyde dehy-

drogenase that is homologous to Ald6p. Ald3p may play a

role in the detoxification of cytotoxic decomposition pro-

ducts of LoaOOH, such as malondialdehyde and 4-hydroxy-

2-nonenal (Gutteridge, 1995; Oberschall et al., 2000).

Also prominent in the cellular functions required for cell-

cycle delay were eight transcription factors. These included

components of Kornberg’s mediator (SRB) complex (en-

coded by PGD1, SRB5 and GAL11) and the SWI/SNF

transcription activator complex (SNF5 and SNF6) (supple-

mentary Table S1b). These transcription complexes are a

part of the general transcription machinery required for

basal and responsive gene expression. The product of SPT4

participates in transcription elongation.

Two specific transcription factors, encoded by SWI6 and

YAP1, were also shown to be involved. Of these, the

participation of YAP1 is not surprising, given the central

role played by Yap1p in the transcriptional response to

oxidants and xenobiotics (Coleman et al., 1999). The role

of SWI6 is very interesting given the function of Swi6p in

cell-cycle progression from the G1 to the S phase, and this

aspect is discussed in more detail below. These data indicate

that transcription plays an important role in the cell-cycle

response to oxidants, in keeping with previous studies that

have shown that cell-cycle delay is regulated at the level of

transcription (Sidorova & Breeden, 1997).

Interactions between genes involved in thecell-cycle response to LoaOOH

The connections between the functions of many of the 47

genes listed in Table 1 involved in oxidant-induced cell-cycle

delay are not obvious from direct inspection of their

individual functions. In order to try to identify components

of this response that share a common mechanism, all direct

known interactions between the products of the genes with

reference to the BioGRID database were identified (Stark

et al., 2006). The interactions, including protein–protein as

well as synthetic lethality and genetic interactions, are

represented in Fig. 4. These interactions included three of

the over-represented functions from gene ontology data: the

SWI/SNF transcription activator complex (Snf5p, Snf6p and

Swi3p); Kornberg’s mediator (SRB) complex (Gal11p,

Pgd1p and Srb5p); and phosphatases (Oca1p, Ycr095p and

Siw14p). Of these, the two transcription factor complexes

(Kornberg’s mediator and SWI/SNF activator complexes)

are linked via a set of direct interactions. Yap1p directly

interacts with two components of the SWI/SNF transcrip-

tion activator complex. Yap1p also interacts with other

components of the Mediator complex (Med2p, Rox3p and

Srb6p) which in turn interact with Gal11p, Pgd1p and Srb5p

identified from the data for nonresponsive mutants. These

interaction data indicate that Yap1p, which is known to play

a role in regulating oxidative stress response genes, acts in

conjunction with the mediator and SWI/SNF transcription

activator complexes to affect cell-cycle progression. Swi6p

also interacts directly with Pgd1p and Srb5 of the Mediator

complex.

These interaction data raise the possibility of an interac-

tion between Swi6p and Yap1p, linking both oxidant-

responsive and cell-cycle responsive gene regulation. Further

support for an indirect association between Swi6p and

Yap1p is indicated by their common synthetic growth defect

interaction with Mms22p, a protein involved in resistance to

ionizing radiation (Bennett et al., 2001). Mms22p is a key

protein in this interaction network (Fig. 4) because it is the

most highly connected member, exhibiting additional syn-

thetic lethality or growth defect interactions with the

chromatin-associated high mobility group protein Hmo1p,

the type 2C protein phosphatase Ptc1p, the vacuolar per-

ipheral membrane protein Pep3p and the nucleosome-

assembly factor Asf1p. Because synthetic genetic analyses

often indicate the existence of parallel compensatory path-

ways, these data may indicate that the cell-cycle response to

oxidants can in part be compensated by the Mms22p repair

pathway that may resolve replication blocks (Longhese et al.,

2003) or by up-regulation of the high osmolarity glycerol

(HOG) signalling pathway, which is constitutively active in a

strain lacking Ptc1p.

Regulation of cell-cycle progression by theSwi6p transcription factor

The swi6 deletant was sensitive to LoaOOH (Fig. 5a) and it

also showed very little change in bud index on treatment

with LoaOOH (Table 1). This inability of the swi6 deletant

to arrest in the G1/S phase upon treatment with oxidant is

particularly interesting, given the well-characterized role of

Swi6p as a cell-cycle transcription factor. Swi6p complexes

with Swi4p to form the SBF complex, and with Mbp1 to

form MBF, either of which can activate cyclin genes required

for cell-cycle progression and cell growth (Iyer et al., 2001).

In addition, Swi6p interacts with the chromatin-remodel-

ling factors SWI/SNF and Mediator complex to commit the

cell to enter the S phase (Li et al., 2005).

In order to investigate the possible role of Swi6p in the

oxidant regulation of cell-cycle progression, the authors

characterized in greater detail the cell-cycle phenotype of

the wild-type and swi6D strains following LoaOOH treat-

ment. Cells were synchronized with a-factor and subjected



to treatment with 0.02 mM LoaOOH. The concentration of

0.02 mM was chosen for this experiment after optimization

because the conditions used to treat the cells and genetics or

background differed from the previous analysis. As shown in

Fig. 5b, LoaOOH-treated wild-type cells again exhibited a

cell-cycle delay of 15–20 min relative to the wild-type strain.

Interestingly, the swi6 deletant showed a delayed entry into

the S-phase for 15–20 min after its release from a-factor in

the absence of an oxidant. When treated with an oxidant, it

did not show any further cell-cycle delay as observed for the

wild type. This experiment was repeated before performing

the microarray experiment with similar results. To ensure

that this loss of cell-cycle delay phenotype was a result of

SWI6 deletion, the SWI6-coding sequence and its upstream

and downstream regulatory elements on a centromeric

plasmid were cloned and transformed into the swi6 deletant.

The resulting strain was found to arrest in the G1 phase

when treated with LoaOOH (shown in Fig. 5c) in the same

way as the wild type. While it is possible that LoaOOH in

the swi6 deletion may have slowed release from the a-factor

cell-cycle arrest, it is unlikely because the initial screen that

identified the swi6 deletant was performed in asynchronous

cells (in the absence of a-factor). In addition, while the swi6

deletion is slower to release from a-factor, there is still a

differential response between the WT and the swi6 deletant

that is restored by the expression of the pSWI6, which

is fully consistent with the initial screen data. These data

show that the alternative Skn7p-dependent mechanism

that allows the swi6D strain to progress in the cell cycle

(Barrera & Ren, 2006) only does so after a delay in G1 and

that this alternative system is not sensitive to LoaOOH

treatment.

Because Swi6p is involved with the separate partners

Swi4p and Mbp1 in the SBF and MBF complexes,

Fig. 4. An interaction network of deletants

(nodes) that exhibit the cell-cycle delay pheno-

type and interaction data derived from the

BioGRID database (links). Each interaction type

is indicated. Three sets of genes have been

boxed and labelled according to their common

GO term.



respectively, the responses of swi4D and mbp1D strains to

LoaOOH were also analysed to determine whether either of

these complexes was solely responsible for the delay process.

Like the wild type, both swi4D and mbp1D strains accumu-

lated unbudded cells having a DUBLoaOOH of 15.30 (SD

0.97) and 13.66 (SD 1.62), respectively, and were thus

arrested normally in the G1 phase in response to LoaOOH

stress. This indicates that either of the SBF or MBF com-

plexes can function in the wild-type response to treatment

with the oxidant, or that there is some unique function or

partner of Swi6p that has yet to be identified.

Swi6p involvement in the transcriptionalresponse to LoaOOH

From the above data, it is clear that Swi6p plays a funda-

mental role in the process of LoaOOH-induced cell-cycle

delay. Given the role Swi6p plays in cell-cycle regulation via

Fig. 5. The LoaOOH sensitivity of swi6D and its

inability to arrest upon treatment is recovered

by transforming a single copy of SWI6 on a

plasmid. (a) LoaOOH sensitivity of wild-type,

swi6D and swi6D carrying pSWI6 were tested on

plates containing 0.1 mM LoaOOH. Stationary

phase cell cultures were used for the spot test

and photographed after a 48-h incubation.

(b) Cell-cycle progression of a-factor synchro-

nized wild-type (top) and swi6 deletant (bottom)

strains in response to 0.02 mM LoaOOH. Cell

were arrested with a-factor and then washed

and treated with LoaOOH. Cells were resus-

pended in fresh media and aliquots were taken

every 15 min to determine the percentage of

budded cells. Progression through the cell cycle

was determined via the proportion of budded

cells viewed under the microscope. Closed

circles (�) represent untreated cells and open

circles (�) represent LoaOOH-treated cells.

(c) The cell-cycle progression of SWI6 deletion

transformed with a control centromeric plasmid

(top) and the plasmid containing the cloned

SWI6-coding sequence and its upstream and

downstream regulatory elements (bottom).

Representative graphs are shown.



transcriptional regulation of G1/S phase-specific genes (Iyer

et al., 2001), the authors examined the extent

to which SWI6 contributed to transcriptional responses to

LoaOOH by microarray analyses of the transcripts in the

wild-type and swi6 deletant strains before and after

LoaOOH treatment.

In order to identify genes whose response to LoaOOH

treatment depends on Swi6p, the ratio of the expression level

of each gene in treated cells compared with untreated cells

for the wild-type strain was plotted against the same

expression ratio in the swi6 deletant (Fig. 6). In this plot,

most genes were clustered along the diagonal, showing that

their response to LoaOOH was the same in the wild type and

the mutant and was unlikely to be regulated via Swi6p. The

absence of SWI6 did, however, have a profound influence

on some transcriptional changes induced by LoaOOH. The

ratio of treated/untreated transcript levels for 474 genes

exhibited at least a twofold difference between the mutant

and the wild-type strains; 217 genes were upregulated and

257 were downregulated. The complete set of genes whose

expression ratio was perturbed by a greater than twofold in

response to LoaOOH in the swi6 deletant relative to the wild

type is given in supplementary Table S2. These genes were

examined for statistically significant over-represented func-

tional categories using the FUNSPEC program (Robinson et al.,

2002). The results are given in supplementary Table 3a and

3b. Within these tables, genes whose expression was per-

turbed by greater than fourfold, and functional categories

that are over-represented using only the fourfold data have

been highlighted.

These analyses revealed that genes with a reduced response to

LoaOOH in the swi6 deletant were enriched for categories

representing molecular chaperones, response to stress, large

subunit ribosomal proteins and to a lesser extent glucose

metabolism (PPP, glycolysis, glucose transporters, positive

regulation of glycolysis and gluconeogenesis). However, these

genes are not normally associated with regulation by SWI6 and

the repression of ribosomal protein genes has been observed

previously as part of the (general) environmental stress response

(Gasch et al., 2000). The set of genes whose transcripts were

upregulated in the absence of SWI6 were enriched for functions

including purine ribonucleotide metabolism and to a lesser

extent DNA mismatch repair, cell division and the transfer of

pentosyl groups. The overall sets of transcripts misregulated in

the swi6 deletant relative to the wild-type strain included those

whose ratio changed less than twofold in the wild-type strain.

This implied that some of the changes observed on deletion of

SWI6 occurred to compensate for its absence.

Each of the perturbed gene sets identified from the

LoaOOH-treated swi6 deletant was additionally inspected for

the occurrence of genes regulated by common transcription

factors. This was performed using overrepresentation analysis

against gene sets that have common transcription factor-

binding interactions at their promoters and correlated gene

expression profiles across over 500 microarray experiments

(Bar-Joseph et al., 2003). These analyses identify transcription

factors involved in regulation, determined by reference to

published data describing actual yeast transcription factor

binding as opposed to theoretical promoter sequence

analyses. This analysis revealed that only five transcriptional

regulatory motifs were statistically over-represented

(Po 0.05) in the perturbed gene-sets. Furthermore, these five

sets of potentially coregulated genes were segregated in either

the up- or downregulated microarray gene sets as shown

in Table 2. Additionally, each of the up- and downregulated

gene set from the LoaOOH-treated swi6 deletant was searched

for genes that were representative of biochemical pathways

(Kanehisa et al., 2004). These analyses revealed that the

downregulated gene set was over-represented with genes

from seven KEGG pathways (Po 0.01) and only a single

gene of these entire pathways occurred in the inversely

upregulated gene set.

The above analysis revealed that eight genes regulated by

Bas1p were induced in the swi6 deletant relative to the wild

type. Bas1p is a transcription factor involved in the regulation

of enzymes of histidine, purine, and pyrimidine biosynthetic

pathways and one-carbon metabolism. Of these eight genes

Fig. 6. Genome-wide transcriptional response to LoaOOH in the swi6

deletant strain compared with the wild type. The genome-wide tran-

scriptional changes resulting from 1 h of treatment with 0.03 mM

LoaOOH in the exponentially growing wild type or swi6 deletant cells

were determined as described. The normalized ratio (treated/untreated)

in the wild type is given on the x-axis, while for the mutant it is shown on

the y-axis. The genes whose transcripts were deemed to be present on all

slides are shown. The twofold difference boundary of induction in the

mutant vs. the wild type is indicated by the outer diagonal lines. The

twofold change boundary for the expression in the wild type alone is

given by the two outer vertical lines.



(see Table 2), SHM2 and MTD1 exhibit greater than fourfold

expression whereas the others exhibit a lesser twofold

expression. This relative expression profile is consistent

with previous studies of the Bas1p mediated transcriptional

regulon (Gelling et al., 2004; Subramanian et al., 2005).

These genes are important in the generation of purine

nucleotides and may reflect an increased demand for these

precursors for DNA repair when progression in the cell-cycle

mediated by Swi6p is inhibited. As may be expected, the

KEGG metabolic pathways of purine metabolism and

one carbon metabolism are also over-represented in this

gene set. These data indicate that Swi6p has a direct or an

indirect negative regulatory effect on Bas1p. In addition,

three genes, MSH6, GIN4 and TOF1, that have been shown

to bind MBP1 and SWI6 (Bar-Joseph et al., 2003) are

upregulated in the swi6 deletant (as shown in Table 2). This

is surprising because SWI6 is an activator of these genes and

therefore in its absence these genes should be repressed relative

to the wild type. Possibly some compensatory effect of the

gene deletion has led to this occurrence. It is also interesting to

note that components of the MAPK signalling pathway were

down-regulated in the swi6 deletant, indicating a possible

involvement of this pathway in the cell-cycle response to

oxidant treatment.

A common paradigm of cellular responses is that relevant

transcription factors generally function downstream of a

signalling pathway or cascade. If this is true for the Swi6p

transcription factor-regulated component of the LoaOOH

response, then the question arises as to which signalling

pathway is involved.

Components of the signalling pathway arepoorly represented in the responsive gene-sets

A comparison of the LoaOOH-responsive genes of the wild-

type strain (Alic et al., 2004) and the swi6 deletant microarray

experiment was performed. There is a high degree of overlap

(77 genes) between the upregulated genes of the wild-type

strain and those downregulated in the swi6 deletant. These

genes are of interest because they represent those activated in

the wild type in response to LoaOOH that are conversely

downregulated in the swi6 deletant. It is interesting to note

that seven out of eight heat shock proteins from the wild-

type upregulated set and three out of four proteins involved

in glucose transport from the swi6 deletant downregulated

set were included in these common 77 genes (Table 3). In

addition, there were two instances whereby genes known to

be bound by a specific transcription factor (Harbison et al.,

2004) occurred almost exclusively within the 77 intersecting

genes, for instance 4/5 genes bound by Msn4p and 3/3 genes

bound by Stb4p, with respect to the swi6 deletant down-

regulated gene set, were present in the intersection.

Twenty-five genes were common to those downregulated

in the wild-type strain and upregulated in the swi6 deletant.

These are genes that exhibit opposing expression profiles in

response to LoaOOH and these changes are dependent on

the deletion of Swi6p. These genes were poorly enriched for

any GO terms; however, further analysis revealed over-

representation of genes that show stringent binding

(Po 0.001) of both Swi4p and Swi6p (HO, GIC2, MNN, 1

ERG3 and SVS1) and Rlm1p transcription factors (GIC2 and

Table 2. Over-represented genes from various functional categories are highly segregated according to their expression profile in the microarray data

from the swi6 deletant treated with LoaOOH

Regulatory motif (bound transcription factors)

Expression profile

Over-represented genes in Gene ModuleDown Up

BAS1 0 8 HIS4 ‘‘ADE5,7’’ MTD1 SHM2 ADE13 ADE17 ADE4 ADE12

MBP1, SWI6 0 3 MSH6 GIN4 TOF1

IME4, SWI5 2 0 HBT1 YHR138C

HSF1 10 1 HSP26 HSP42 SSA4 BTN2 YLL023C HSP104 CPR6 YLR327C SIS1 STI1

MSN4 5 1 HSP26 SSA4 BTN2 YHR087W HSP104

KEGG Pathway Down Up Over-represented genes in KEGG Biochemical Pathway

Glycolysis/gluconeogenesis 10 0 CDC19 GLK1 PGK1 HXK1 TDH3 ENO1 ENO2 TDH2 PDC5 PGM2

Ribosome 14 1 RPL4A RPL4B RPS13 SMC2 RPS4B RPL2B RPP0 RPL18B RPP2A RPL18A RPL3 RPL5 RPL7B RPL1A

Riboflavin metabolism 4 0 PHO3 FAD1 RHR2 RIB4

Pentose phosphate cycle 5 0 YGR043C SOL4 GND1 PGM2 TKL1

Propanoate metabolism 3 0 LSC1 ALD4 ALD6

MAPK signaling (high osmolarity) 3 0 CTT1 STE20 MSN4

Purine metabolism 4 8 CDC2 ADK2 ‘‘ADE5,7’’ MET14 ADE13 ADE17 ADE4 ADE12

One carbon pool by folate 0 3 MTD1 SHM2 ADE17

Cell cycle 3 8 MBP1 CLB3 DBF4 GIN4 MCM6 CDC5 PHO80 DBF20

Genes whose expression differed by greater than fourfold are highlighted as bold underlined text.



MNN1) (Harbison et al., 2004). This is interesting because

these transcription factors are the downstream effectors of

the Mpk1p (Slt2p)-mediated MAPK signalling pathway

defined by the KEGG metabolic pathway 04010sce (Kanehi-

sa et al., 2004). These data indicate that the wild-type

response to LoaOOH involves the Swi6p-mediated down-

regulation of the Mpk1p-mediated MAPK signalling path-

way because in the swi6 deletant genes regulated by this

pathway are no longer repressed. Furthermore, the intersec-

tion of the downregulated genes of the wild-type strain and

similarly responsive downregulated genes of the swi6 dele-

tant gave rise to only nine genes; however, two of these

genes, PMA1 and GND1, encode proteins that physically

interact with Mpk1p. Again, these data indicate a role for

Mpk1p in the cellular response to LoaOOH. This is con-

sistent with the finding that Mpk1p is required for the full

resistance to LoaOOH, and that the protein is rapidly and

transiently activated in response to LoaOOH (Alic et al.,

2003). It was, however, shown by these authors that Mpk1p

is not directly involved in cell-cycle delay induced by

LoaOOH, despite the fact that oxidative stress due to

hydrogen peroxide has been shown to activate Mpk1p

(Vilella et al., 2005).

Discussion

This study has identified 47 deletant strains that exhibit an

inability to delay at Start, in response to assault by LoaOOH.

These data implicate that the deleted gene products covering

a broad range of cellular functions are required for the G1

checkpoint to operate, whether by a direct or an indirect

mechanism. This nonresponsive phenotype is highly pre-

valent in the subset of deletants that are highly sensitive to

LoaOOH (Thorpe et al., 2004), leading to the conclusion

that the inability of strains to mount a checkpoint response

to low doses is strongly associated with the sensitivity of

these strains at higher doses.

The screening data show that components of the Media-

tor complex are required for the phenotypic response. It is

known that the Mediator complex is required for diverse

aspects of transcription, such as activation, repression and

stimulation of basal transcription. The diversity of these

functions has raised the possibility that the Mediator com-

plex contains functionally distinct modules. The existence of

such modules was demonstrated previously with the finding

of the Rgr1p and Srb4p subcomplexes and the Gal11p

module within the Rgr1p subcomplex as an activator bind-

ing module (Kang et al., 2001).

The Yap1p transcription factor is crucial for the normal

response of cells to a variety of stress conditions including

oxidative stress, stress mediated by many drugs and heat

shock. Importantly, Yap1p is primarily cytosolic, but after

exposure of cells to a number of different oxidizing agents,

such as H2O2, diamide and diethylmaleate, the protein

rapidly accumulates in the nucleus (Kuge et al., 1997) to

increase the expression of a diverse range of oxidant defence

genes. The finding that Yap1p may play a crucial role in the

phenotypic response to LoaOOH indicates a connection

between a major stress-regulated transcription factor and

the regulation of the cell cycle. It remains to be investigated

which genes are directly controlled through the Yap1p

transcription factor in response to LoaOOH in order to

understand its precise role in the pathway of the stress-

induced cell-cycle modulation.

It is interesting that the swi6 deletant exhibited the

no-delay phenotypic response; however, deletion of its

canonical binding partners Swi4p and Mbp1p did

not. These data indicated that there is a sufficient degree of

redundancy between the SBF and MBF complexes for

cells to exhibit wild-type characteristics. It would be

interesting to determine the phenotypic response of the

swi4 mbp1 double mutant to LoaOOH; however, this

mutant was not viable in the BY strain background, which

is consistent with previous attempts to create this strain

(Koch et al., 1993). Alternatively, SWI6 may interact with

Table 3. A comparison of the distribution of molecular function and transcription factor binding of the genes down-regulated following exposure to

LoaOOH in the swi6 deletant and upregulated in the wild type and the genes common to both sets (the intersection)

GO term:

molecular function P-value

Intersecting

genes

Wild-type

upregulated

swi6

downregulated Genes of intersect

Heat shock protein 8.33E-10 7 8 12 SSA3 HSP26 HSP30 HSP78 SSA4 HSP104 HSP82

Glucose transporter 1.03E-03 3 5 4 HXT7 HXT6 HXT2

Binding Data

MSN4 3.32E-03 4 8 5 HSP26 SSA4 YHR087W HSP104

STB4 5.96E-03 3 3 3 ZTA1 YLL056C GTT2

SUT1 3.67E-02 3 9 9 HXT6 CWP1 HXT2

SNT2 4.01E-02 2 2 3 SSA3 YHR138C

YAP1 4.18E-02 3 13 5 SNQ2 OYE2 GTT2

The P-value is quoted for the genes of the intersection.

Genes whose expression differed by greater than fourfold are highlighted as underlined text.



another factor in response to LoaOOH to activate the

cell-cycle delay.

Five genes identified in the cell-cycle screen exhibited

perturbed regulation in the swi6 deletion strain after

LoaOOH treatment. Of these, ASF1 occurred in the

up-regulated set while TAT1, ALD3, SUR1 and TKL1

occurred in the downregulated set, as shown in Fig. 6. Asf1p,

which is known to be involved in the induction of apoptosis,

may be suppressed by Swi6p because its expression is higher

in the swi6 deletant than in the wild type after the LoaOOH

treatment. The gene encoding Ald3p, a aldehyde dehydro-

genase was expressed up to fourfold higher in the wild type

after LoaOOH treatment compared with its expression in

the swi6 deletant; these data indicate that Swi6p is required

for Ald3p induction in response to LoaOOH. ALD3 is one of

five genes known to encode aldehyde dehydrogenases in

S. cerevisiae. The expression of ALD2 and ALD3 is depen-

dent on the general stress transcription factors Msn2p and

Msn4p but independent of the HOG MAP kinase pathway.

Ald3p is induced by a variety of stresses, including osmotic

shock, heat shock, glucose exhaustion, oxidative stress and

drugs. ALD2 is only induced by osmotic stress and glucose

exhaustion (Navarro-Avino et al., 1999).

Because Swi6p has no DNA-binding domain and yet is

central to the two G1/S phase transcription complexes, it has

been suggested that the Swi6p is a target of regulation. While

components of the MAPK-signalling pathway have been

identified in these data, there is no further indication that

components upstream of Mpk1p are involved and Mpk1p is

not required for the cell-cycle delay in response to LoaOOH.

One possibility is that Swi6p is itself able to respond directly

to oxidative stress without the need for, or additional to, a

canonical signal transduction pathway. In addition, while 77

genes were upregulated in the wild-type strain and down-

regulated in the swi6 deletant when treated with LoaOOH, it

is interesting that many other gene expression profiles were

not significantly different between the two strains. Hence,

the deletion of SWI6 does not account for all the changes

observed in the wild type, indicating the existence of other

mechanisms regulating cell-cycle arrest induced by oxidative

stress.

Acknowledgements

This work was supported by grants from the Australian

Research Council and by an Australian Postgraduate Award

to N.A.

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Supplementarymaterial

The following material is available for this article online:

Table S1a. The complete set of deletants that exhibit the

altered cell cycle response phenotype after LoaOOH treat-

ment.

Table S1b. Functions over-represented in the set of

deletants showing no delay after LoaOOH treatment.

Table S2. Set of genes down- and up-regulated in the

swi6 deletant microarray after LoaOOH treatment.

Table S3a. Functions over-represented in the down-

regulated microarray data of the swi6 deletant after

LoaOOH treatment.

Table S3b. Functions over-represented in the micro-

array data for genes up-regulated in the swi6 deletant after

LoaOOH treatment.

This material is available as part of the online article

from: http://www.blackwell-synergy.com/doi/abs/10.1111/

j.1567-1364.2007.00349.x (This link will take you to the

article abstract).

Please note: Blackwell Publishing are not responsible

for the content or functionality of any supplementary

materials supplied by the authors. Any queries (other than

missing material) should be directed to the corresponding

author for the article.



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Oxidant-induced cell cycle delay of Saccharomyces cerevisiae: the involvement of SWI6

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