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47 Yeast as a Model System to Study DNA

Damage and DNA Repair


ANTONIO CONCONI

ABSTRACT yeastgenome.org), Yeast Protein Database (YPD; www.proteome.


This chapter provides procedures for determining cell survival com), Munich Information Center for Protein Sequences (MIPS)
and the extent of DNA damage and repair after treatment of yeast Comprehensive Yeast Genome Database (CYGD; mips.gsf.de/
cultures with DNA-damaging agents such as bleomycin, methyl genre/proj/yeast/index.jsp), the Yeast Resource Center (depts.
methanesulfonate, dimethyl sulfate, and ultraviolet light. Compre- washington.edu/yeastrc), and yeast green uorescent protein
hensive protocols for basic methods in yeast biology such as how (GFP) fusion localization database (yeastgfp.ucsf.edu). Further-
to prepare different media, how to grow and store yeast cells, how more, 5916 ORFs were deleted to construct a heterozygous diploid
to isolate nucleic acids, how to transform and prepare yeast mutant collection that contains deletions in both essential and nonessen-
strains, and how to use Internet-based yeast resources are tial genes.13 About 4800 nonessential deletions were generated
available. as haploid MATa and MAT strains or as homozygous diploids,
Key Words: Benzo[a]pyrenediol, Bleomycin, CPD, Dimethyl and these collections can be obtained from Research Genetics
sulfate, Double-stranded breaks, Methyl methanesulfonate, (ResGen) (www.resgen.com/products/) or the Euroscarf consor-
Nucleotide excision repair, Single-stranded breaks, 2,3,5- tium (web.uni-frankfurt.de/fb15/mikro/euroscarf/col_index.html)
Triphenyltetrazolium chloride. in readily usable forms.14 Among numerous applications of the
ORF deletions libraries, they have been employed to understand
INTRODUCTION the genetics that determine cell sensitivity and resistance to drugs,
The budding yeast Saccharomyces cerevisiae is classied as a and some multidrug resistance genes have already been identi-
safe microorganism and has been used for many years as a bio- ed.15 Also, the toxicity of chemicals is currently tested in yeast
logical model.13 It has provided important contributions to our that present metabolic defects similar to those observed in human
current understanding of basic eukaryotic cellular functions. Fur- cancers,16 and high-throughput screenings are employed to deter-
thermore, yeast is employed as a model to investigate mammalian mine the mode of action of DNA-damaging agents such as those
diseases. For instance, at least 31% of yeast proteins have human employed in chemotherapy.17 Finally, studies on cytotoxic com-
homologues and nearly 50% of human genes implicated in pounds such as methyl methanesulfonate (MMS) and hydroxy-
heritable diseases have yeast homologues.4 Although its nuclear urea,18 bleomycin (BLEO),19 and cisplatin,20 on ionizing radiation,21
genome is only 3.5 times larger than that of Escherichia coli, the and on ultraviolet light (UV) radiation22 have revealed genes that
yeast genome displays most of the hallmarks of higher eukaryotic are involved in DNA repair, cell cycle checkpoints, DNA recom-
genomes. The genome of haploid yeast cells is about 1.2 107 bp bination, and DNA replication. The results have highlighted the
and is organized into 16 chromosomes, ranging in size from 200 importance of the nucleotide excision repair (NER) mechanism
to 2200 kb.5 Finally, yeast DNA is found within a nucleus and its together with homologous recombination, postreplication repair,
nucleosomal organization of chromatin is similar to that present and cellular responses to DNA damage.
in higher eukaryotes, except that there is no histone H1.6,7 MMS and DMS are DNA methylating agents that produce a
The powerful yeast genetics and its relatively high rate of variety of damaged bases, of which N7-methylguanines and
recombination facilitate the insertion of DNA sequences at spe- N3-methyladenine constitute about 80% and 10% of the lesions,
cic locations in the genome, allowing the creation of yeast strains respectively.23 In yeast, N-methylpurines are enzymatically
carrying gene knockouts,8 the tagging of proteins with different removed by the base excision repair pathway (BER), and the
epitopes,9 and the engineering of regulated promoters upstream sensitivity to MMS increases in cells lacking genes involved in
of a given open reading frame (ORF).10 In yeast there are 6466 BER. However, homologous recombination (HR) may also par-
ORFs.11,12 Their functions, information on the corresponding pro- ticipate in the repair of methylpurines. Bleomycin is a radiomi-
teins, and their cellular localization are available through data- metic drug that in the presence of cofactors creates single strand
bases such as the Saccharomyces Genome Database (SGD; www. breaks (SSB) and double strand breaks (DSB) by a concerted free
radical attack on the sugar moieties of the DNA backbone.23 DNA
From: Sourcebook of Models for Biomedical Research strand breaks block DNA replication, and the difculty of
(P. M. Conn, ed.), 2008 Humana Press Inc., Totowa, NJ. repairing DSBs is the major source of BLEOs cytotoxicity. The

445
446 SECTION V / MODELS OF BEHAVIOR

recombination and postreplication DNA repair pathways are the survival tests, large quantities of cells are needed for DNA
involved in the repair of BLEO-induced DNA lesions. UV light repair analyses.
induces the formation of two major photoproducts: cis-sin
cyclobutane pyrimidine dimers (CPD) and (6-4) pyrimidine GROWTH OF LARGE YEAST CULTURES AND
pyrimidone dimers [(6-4)PD].23 These lesions block gene tran- PREPARATION OF CELLS FOR TREATMENT WITH
scription and DNA replication, and are repaired by the NER DNA-DAMAGING AGENTS
pathway. Although UV photoproducts induced by UV-C serve as
1. For liquid cultures, add the YEPD medium (10 g yeast
the paradigm to study the mechanisms of NER, this ubiquitous
extract, 20 g peptone, 20 g glucose, and distilled water to 1 liter)
repair pathway operates on a large variety of chemical adducts to
to a ask that holds two to three times the required volume. For
DNA.23
solid plates, prior to autoclaving add 2% (w/v) agar to the YEPD
NER is characterized by the incision of the damaged DNA
in a ask containing a stir bar. Autoclave at 121C and 15 psi
strand on both sides of the lesion, resulting in the removal of an
(1 atm) for 20 min. Gently mix the agar-containing medium on a
oligonucleotide fragment (2530 nucleotides long) containing the
stirrer and let it cool down to 5055C before pouring the plates
damage. This is followed by DNA repair synthesis and ligation.
(2025 ml/Petri dish). Allow the plates to solidify and dry at room
The yeast genes involved in NER can be grouped into two classes;
temperature (RT) for 2 days; store them at 4oC in plastic bags.
the rst consists of RAD1, 2, 3, 4, 10, 14, and 25 genes, and the
2. Streak out the cells on solid YEPD and incubate the plate at
second consists of RAD7, 16, and 23 and the MMS19 genes. Muta-
2830C until colonies form (23 days). Prepare a preculture as
tions in the genes of class 1 cause a very high degree of UV sen-
follows: use one colony to inoculate 7 ml YEPD in a sterile culture
sitivity, whereas mutations in the genes of class 2 cause moderate
tube (e.g., borosilicate glass 16 150 mm, Fisher Scientic catalog
UV sensitivity.24 An additional factor encoded by the RAD26 gene
no. 14-961-31). Usually, cells grow to a stationary phase when incu-
is involved in transcription-coupled repair (TCR), the subpathway
bated overnight at 2830C with continuous rotation or shaking.
of NER that is responsible for removing DNA lesions from the
3. DNA repair experiments are done with exponentially
transcribed strand of active genes.25
growing cultures (2 107 cells/ml). One 2-liter ask containing
Photoreactivation is an alternative mechanism to NER that
500 ml of YEPD is inoculated with 300400 l of preculture cells
specically removes pyrimidine dimers. This process is catalyzed
(nal concentration 105 cells/ml). The culture is incubated at
by one enzyme (photolyase) and is light dependent (blue light).
30C with continuous shaking (300 rpm) overnight. To monitor
Photolyases are widespread in nature, though there is no photo-
cell growth blank the spectrophotometer with sterile medium,
reactivation of DNA damage in human cells.23 To inhibit the
remove an aliquot, and measure the absorbance of the cells at
activity of photolyase, all the experiments done with UV-
600 nm [optical density (OD)600]. Use dilutions to make sure that
irradiated yeast cultures are performed in a dark room, under
measurements are done in the linear range of the spectrophotom-
yellow light (e.g., Rapid Start Fluorescent lamps, Reprographic-
eter, and calculate the number of cells/ml considering that OD600
Gold 40 W 2250 K, Standard Products Inc.; catalog 6050RGO).
0.1 is approximately 2 106 cells/ml. [Note: The correlation
OD vs. number of cells varies not only from strain to strain but
GROWING YEAST CELLS AND INDUCTION OF also for a given strain grown in different media. Therefore it is
DNA DAMAGE important to determine the cell number per OD unit using a count-
ing chamber, and plotting the growth curves to determine the
Culturing yeast is simple, economical, and rapid. They grow
times of cell division.]
in liquid and on solid media, forming a bud that pinches off to
4. Cells are harvested when they are in mid-exponential phase
originate daughter cells. During exponential growth (or log-phase)
(1 to 2 107 cells/ml) in a benchtop centrifuge (20003000 g/5 min),
in rich media, yeast have a short life cycle of about 90 min. As
washed in ice cold phosphate-buffered saline (PBS) (8 g NaCl,
cell density increases, nutrients drop and the rate of cell division
0.2 g KCl, 1.44 g Na2HPO4, 0.24 g of KH2PO4, pH 7.4 per liter),
slows. The log-phase is divided into three stages: early-log phase
and reharvested to prepare them for treatment with DNA-
(<107 cells/ml), mid-log phase (between 1 and 5 107 cells/ml),
damaging agents.
and late-log phase (between 5 107 and 2 108 cells/ml). At a
density of 2 108 cells/ml, yeast enter the stationary (G0) phase.
TREATMENT OF SMALL CULTURES OF CELLS WITH
The growth curve of newly inoculated cells (e.g., at 2 105/ml)
METHYL METHANESULFONATE AND BLEOMYCIN TO
is characterized by a lag phase of two to three cell divisions (5 h),
DETERMINE CELL SURVIVAL
exponential growth for about six cell divisions (9 h), and the
stationary phase (during this last phase, cells undergo about two 1. Yeast strains (10 ml cultures) are grown exponentially (107
more divisions). The optimal growth conditions can vary depend- to 2 107 cells/ml), collected by centrifugation, washed
ing on the yeast genetic background but, in general, yeast grow with 10 ml of sterile water, and resuspended in 5 ml of water to
very rapidly at 2830C in the most commonly used YEPD 0.8 107 cells/ml.
medium. The recipes for rich, minimal, and complete minimal 2. MMS (Sigma, catalog no. M 4016) is used as the stock
dropout media and their uses have been reported.13 solution. BLEO (from MeadJohnson or Sigma) is dissolved in
Below is described how we grow yeast cultures and incubate sterile water to 5 U/ml, and stored in aliquots at 20C.
them with DNA-damaging agents and how we monitor DNA 3. Chronic exposures of yeast cultures to MMS and BLEO are
repair. However, optimal growth conditions and treatments with done in triplicate: 5 l of cell suspensions (0.8 107 cells/ml in
genotoxic compounds could vary from strain to strain. Thus it is sterile water) is used to inoculate the wells of a microplate con-
advisable to carry out pilot experiments to follow the dose sur- taining 95 l of YEPD media and various concentrations of MMS
vival curves. Also, while small cultures provide enough cells for (0 as control, 0.005, 0.01, and 0.02%; v/v) or BLEO (0 as control,
CHAPTER 47 / YEAST AS A MODEL SYSTEM TO STUDY DNA DAMAGE AND DNA REPAIR 447

6 and 10 mU/ml). The microplates are incubated as described in harvested by centrifugation. [Note: If needed, to prevent DNA
the section on Measuring Yeast Sensitivity to Genotoxic Agents replication during repair incubation, hydroxyurea is added to a
in Liquid Cultures. Alternatively, 10-fold serial dilutions of cells nal concentration of 100 mM (e.g., 25 and 26 and references
are prepared in sterile water and 5 l of each dilution is spotted therein).]
on YEPD plates containing different concentrations of MMS or 4. Total genomic DNA is isolated as described in the section
BLEO (see above). The plates are prepared and incubated as on Isolation of Genomic DNA from Yeast Cells.
described in the section on Measuring Yeast Sensitivity to
TREATMENT OF LARGE CULTURES OF CELLS WITH
Genotoxic Agents by Colony Spot Testing.
BLEOMYCIN
4. Acute exposures of yeast cultures to MMS are done in
triplicate: 1 ml of yeast cultures in sterile water (prepared as in 1. Yeast cells are grown at 30C in YEPD to mid-log phase
step 1) is collected by centrifugation, resuspended in 1 ml of fresh (OD600 0.4) as described in the section on Growth of Large Yeast
YEPD (0.8 107 cells/ml) containing different concentrations of Cultures.
MMS (0 as control, 0.01, 0.025, and 0.05%; v/v), and incubated 2. After washing twice with sterile ice-cold water, cells are
at 30C for 90 min under continuous rotation. Thereafter, cells resuspended in 50 mM NaCl, 2 mM MgCl2, and 0.02% glucose to
are harvested by centrifugation, washed twice with sterile water, a cell density of 1 to 2 109 cells/ml.
and resuspended in 1 ml of water. As described above, 5 l of 3. Digitonin (Sigma, 10% stock) and Fe(NH4)2(SO4)2 (10 mM,
cell suspensions (4 104 cells) is used to inoculate the wells freshly dissolved in H2O) are added to the cell suspension to nal
of a microplate containing 95 l of fresh YEPD (without DNA- concentrations of 0.05% and 50 M, respectively.26
damaging agents; MMS). The microplates are incubated as described 4. The cells suspension is divided into ve aliquots and one
in the section on Measuring Yeast Sensitivity to Genotoxic Agents aliquot is kept as the negative control (no treatment with BLEO).
in Liquid Cultures. Alternatively, 10-fold serial dilutions of washed BLEO (Sigma, 20 units/ml stock) is added to nal concentrations
cells are prepared in sterile water and 5 l of each dilution is spotted of 12.5, 25, 50, and 100 mU/ml, followed by a 12-min incubation
on YEPD plates (without DNA-damaging agents). The plates are at 30C.
incubated as described in the section on Measuring Yeast Sensitivity 5. To stop the reaction, the ve cell suspensions are mixed
to Genotoxic Agents by Colony Spot Testing. with 100 volumes of ice-cold YEPD and the cells are collected
by centrifugation. [Note: BLEO can aggregate at cell plasma
ULTRAVIOLET LIGHT IRRADIATION OF SMALL
membranes and even in the presence of ethylenediaminetetraace-
CULTURES OF CELLS TO DETERMINE CELL SURVIVAL
tic acid (EDTA), DNA can be damaged during the extraction
1. In sterile water 350 l of 0.8 107 cells/ml is spotted on an process. BLEO molecules are efciently removed from mamma-
autoclaved glass plate and irradiated (254 nm) with UV doses of lian cell membranes by trypsin,27 and from yeast cells walls by
20, 80, and 150 J/m2, as measured with a UVX radiometer (Ultra- washing the cells with yeast media.26]
Violet Products, Upland, CA). How to monitor UV intensities and 6. Cells are washed a second time in ice-cold YEPD, collected
convert them to UV doses is described in the section on Ultravio- by centrifugation and prepared for total genomic DNA extraction
let Light Irradiation of Large Cell Cultures (step 3). as described in the section on Isolation of Genomic DNA from
2. Triplicates of 5 l of UV irradiated, or mock treated Yeast Cells.
(control), cell suspensions are used to inoculate the wells of a
ULTRAVIOLET LIGHT IRRADIATION OF LARGE CELL
microplate containing 95 l of YEPD media. The microplates are
CULTURES
incubated in the dark to avoid photoreversal of DNA damage by
photolyase as described in the section on Measuring Yeast Sen- 1. Yeast cells are grown at 30C in YEPD to mid-log phase
sitivity to Genotoxic Agents in Liquid Cultures. Alternatively, (OD600 0.4) as described in the section on Growth of Large Yeast
5 l of 10-fold serial dilutions of irradiated and nonirradiated cells Cultures. The cell pellet from a 500-ml culture (total 5 109 cells)
are spotted on YEPD plates. The plates are incubated in the dark is resuspended in 300 ml PBS (1.67 107 cells/ml). [Note: Cells
to avoid photoreversal of DNA damage by photolyase as described are irradiated in PBS, as YEPD lters out 254 nm UV light.]
in the section on Measuring Yeast Sensitivity to Genotoxic Agents 2. An aliquot of 50 ml is kept as the control (UV) and the
by Colony Spot Testing. remaining cells are poured into trays (21.5 14 cm of mat gray
plastic from Rubbermaid) and irradiated with primary 254 nm UV
TREATMENT OF LARGE CULTURES OF CELLS WITH
(UVC) light. [Note: For optimal and equal UV irradiation the
DIMETHYL SULFATE
thickness of yeast suspensions should not be above 2 mm, the
1. Yeast cells are grown at 30C in YEPD to mid-log phase maximal cell density should be kept below 3 107 cells/ml, and
(OD600 0.4) as described in the section on Growth of Large Yeast the UV lamps should be turned on about 5 min before use.]
Cultures. An aliquot of 12 109 cells is kept as control [no treat- 3. Cell suspensions are irradiated with 254-nm emitting
ment with dimethyl sulfate (DMS)]. germicidal lamps (15 W; General Electric). The UV intensity is
2. The rest of the culture is mixed with DMS (Sigma, undi- measured with a radiometer (UVX from Ultra-Violet Products,
luted solution) to give a nal concentration of 0.03% (v/v), and Upland, CA) and converted to UV dose with the following
incubated at RT for 2 min. equation:
3. Cells are washed twice with PBS, resuspended in 200 ml of
Dose (J/m2) = Time of irradiation (seconds)
prewarmed YEPD at 30C, poured into a 500-ml Erlenmeyer
Intensity (W/cm2)/100
ask, and incubated at 30C with continuous shaking (300 rpm)
for different repair incubation times (e.g., 0, 1, 2, and 4 h). Then For instance, to study DNA repair of chromatin in yeast, we
50-ml aliquots (12 109 cells) are collected on ice and cells are routinely irradiate cells at 150 J/m.2
448 SECTION V / MODELS OF BEHAVIOR

4. Soon after irradiation, cells of a 50-ml aliquot are harvested 3. Of each yeast strain 100 l is plated in triplicate for each
by centrifugation and used as control (+UV, no DNA repair). The UV dose and dilution. The plates are allowed to dry for 10 min at
remaining 200 ml of the cell suspension is collected as previously RT before irradiation (without a lid). After irradiation, the plates
described (see the section on Growth of Large Yeast Cultures, are incubated at 30C for 48 h in the dark. The colonies are
step 4), resuspended in 200 ml of 30C prewarmed YEPD, poured counted and plotted as percent survival versus UV doses.
into a 500-ml Erlenmeyer ask, and incubated at 30C with con-
MEASURING YEAST SENSITIVITY TO GENOTOXIC
tinuous shaking (300 rpm) to allow repair. At different time
AGENTS IN LIQUID CULTURES
points (e.g., 30 min, 1 h, 2 h, and 4 h after UV irradiation) 50-ml
aliquots are collected and cells are harvested by centrifugation. 1. Yeast strains (10 ml cultures) are grown exponentially (107
Isolation of total genomic DNA is described in the section on to 2 107 cells/ml) in YEPD, collected by centrifugation, washed
Isolation of Genomic DNA from Yeast Cells. with 10 ml, and resuspended in 5 ml of sterile water to nal con-
centrations of 0.8 107 cells/ml.
ASSESSING YEAST SURVIVAL RATES 2. For a control, the OD600nm of the 5-ml cell suspensions can
To test cytotoxic compounds on yeast from the ORF deletions be remeasured.
libraries, cells carrying deleted genes are plated onto solid media 3. Yeast are treated with DNA-damaging agents as described
containing the drugs, and the strains that are sensitive to the drugs in the section on Treatment of Small Cultures of Cells and the
are recognized by their slow growth. By this assay, sets of multi- section on Ultraviolet Light Irradiation.
drug resistance genes have already been identied.15 However, 4. Prepare a 96-well plate (at bottom, nontreated, low evapo-
analyses of cell growth in liquid media are better suited for the ration lid, polystyrene, sterile; Costar, catalog no. 3370) with 95 l
detection of weak sensitivities, and are thought to be more repre- of sterile YEPD in each well with or without DNA-damaging
sentative than the plating assays.15,28 Finally, methods that measure agent.
cell metabolism are also used to determine the fraction of surviv- 5. Triplicates of 5-l yeast samples (corresponding to 4 104
ing cells.29 One such test is the reduction of 2,3,5-triphenyltetra- cells, treated or mock treated with DNA-damaging agents) are
zolium chloride (TTC) to formazan by the mitochondria. deposited in each well and mixed with the YEPD by pipetting up
and down (it is convenient to use a multichannel pipette).
MEASURING YEAST SENSITIVITY TO GENOTOXIC 6. To avoid evaporation during the incubation period, the 96-
AGENTS BY COLONY SPOT TESTING well plates are sealed with Paralm.
7. Cell growth is monitored with a PowerWave microplate
1. The yeast strains are grown exponentially in YEPD at 30C
scanning spectrophotometer (Bio-Tek). The OD of cell growth is
under continuous rotation. Cells are collected by centrifugation,
recorded using KC4 microplate data analysis software (Bio-Tek),
resuspended in sterile water, and treated with the DNA-damaging
programmed as follows: running time, 48 h; OD reading interval,
agents (see the section on Treatment of Small Cultures of Cells
10 min; OD wavelength, 660 nm; shaking, intensity 2 and duration
and the section on Ultraviolet Light Irradiation).
of 595 sec before every reading; temperature, 30C.
2a. For chemical agents 10-fold serial dilutions of yeast cul-
8. The data are exported to Excel (Microsoft) for plotting.28
tures (0.8 107, 0.8 106, 0.8 105, 0.8 104, and 0.8 103 cells/
ml) are prepared in sterile water and 5 l of each dilution (corre- MEASURING YEAST SENSITIVITY TO GENOTOXIC
sponding, respectively, to 4 104, 4 103, 4 102, 4 101, and 4 AGENTS BY THE REDUCTION OF 2,3,5-TRIPHENYLTETRA-
100 cells) is spotted on YEPD plates containing different con- ZOLIUM CHLORIDE The water-soluble and colorless
centrations of DNA-damaging agents. The plates are incubated tetrazolium salts are reduced in a variety of cells to red, water-
at 30C for 48 h, and cell survival is scored by visual inspection insoluble formazan compounds. The reaction occurs in the mito-
such as comparing the size of the grown spots of yeast colonies. chondria where these compounds are reduced at different sites
2b. For UV irradiation 5 l of 10-fold serial dilutions of irradi- along the electron-transport chain, with the exact site of reduction
ated and nonirradiated cells (0.8 107, 0.8 106, 0.8 105, 0.8 depending on their chemical substitution. Among these, TTC is
104, and 0.8 103 cells/ml) are spotted on YEPD plates. After one of the most commonly used. Its reduction to red formazan
UV irradiation, the plates are incubated at 30C for 48 h in the occurs at the site of the cytochrome oxidase complex in place of
dark (to avoid photo-reversal), and cell survival is scored by molecular oxygen reduction.29 Consequently, measurements of
visual inspection of the plates. cell metabolism by TTC reduction can be used to determine cell
survival.
SURVIVAL CURVES OF ULTRAVIOLET-IRRADIATED
CELLS BY COLONY COUNTING 1. Yeast cultures are grown to mid-log phase, treated with
DNA-damaging agents, and incubated for different times to allow
1. Yeast stock cultures are grown overnight in 5 ml YEPD at
cells to repair the DNA.
30C and under continuous rotation to early stationary phase.
2. For each time point, 35 ml of cell culture (7 108 cells)
2. Serial dilutions are prepared as follows:
is pelleted, resuspended in 1 ml sodium phosphate buffer (0.05 M,
104 to 106 dilutions of the stock cultures are made for pH 7.5), transferred to an Eppendorf tube containing 350 l of
the controls (no UV) and the WT (or RAD+) strains irradi- TTC stock solution (0.5% TTC in 0.05 M phosphate buffer,
ated at UV doses up to 150 J/m2. Sigma), and mixed well.
103 to 105 dilutions of the stock cultures are made for 3. Incubation is done for 20 h at RT in the dark (without
class I of rad mutant strains (see Introduction) when irra- stirring).
diated at doses between 5 J/m2 and 10 J/m2. For higher 4. Cells are pelleted by centrifugation in a microcentrifuge for
irradiations (up to 30 J/m2) prepare 102 to 100 dilutions. 2 min, washed in sterile water, and pelleted again.
CHAPTER 47 / YEAST AS A MODEL SYSTEM TO STUDY DNA DAMAGE AND DNA REPAIR 449

5. An equal volume of glass beads (425600 m, Sigma) is 2. The cells are vortexed vigorously 1015 times for 30 sec
added to the cell pellet, together with 500 l ethanol : acetone each time with 30-sec gaps on ice.
(1 : 1). The suspension is vortexed vigorously to break open the 3. The cells lysates are transferred to new 15-ml tubes on ice
cells. and the glass beads are rinsed twice with 1 ml NIB. The combined
6. Cell debris and beads are pelleted in a microfuge for 2 min washing extractions are spun in a benchtop centrifuge at 3500 rpm
and the supernatant is transferred into a 15-ml tube. (2000 g) for 2.5 min.
7. Steps 5 and 6 are repeated four times, followed by two 4. The supernatants (nuclei suspensions) are divided into two
extractions with 500 l of 100% acetone. 2-ml Eppendorf tubes and spun in a microcentrifuge for 5 min at
8. For each DNA repair time point, all the supernatants are 4C.
collected and adjusted to 3.5 ml nal volumes with ethanol : 5. The pellets are resuspended in 500 l TE total volume and
acetone (1 : 1), followed by 5 min centrifugation at maximum collected into one 2-ml Eppendorf tube. After addition of 225 l 3 M
speed in a benchtop centrifuge to pellet cell debris. Na-acetate (pH 6.5), mixing, and 35 l 10% sodium dodecyl sulfate
9. Absorbance of the combined washing extractions is read at (SDS), mixing (nal volume 770 l), the samples are extracted twice
485 nm and measurements are conveniently taken in 1-ml plastic with phenol : chloroform : isoamyl alcohol (25 : 24 : 1) (equilibrated
cuvettes. [Note: Dilute the sample if OD485 is above 2.] with Tris to pH neutral) and once with chloroform (avoid taking
10. OD485 measurements of mock-treated cells are considered the interphase). Finally, the nucleic acids are precipitated with one
as 100%, and the percents of OD485 are plotted versus the doses volume isopropanol at 20C from 1 h to overnight.
of DNA-damaging agents employed. 6. After centrifugation for 30 min at 4C, the white pellets are
resuspended (without drying) in 500 l TE; 10 l RNase A (10 mg/
MEASUREMENTS OF DNA DAMAGE AND DNA ml) is added to each tube and the samples are incubated at 37C
REPAIR RATES for 30 min. Thereafter, 225 l 3 M Na-acetate (pH 6.5) and 35 l
10% SDS are added as described in step 5, followed by one
The quantication of the formation and removal of lesions is
extraction with phenol : chloroform : isoamyl alcohol (25 : 24 : 1)
crucial to understanding the processes of DNA damage and repair,
and one extraction with chloroform.
and the preparation of good-quality, high-molecular-weight DNA
8. The samples are precipitated in isopropanol as described
is an essential step of these analyses. Several methods are used to
above. After centrifugation, the pellets are rinsed twice in 70%
break the yeast wall, which are either based on mechanical or
(v/v) ethanol, dried, resuspended in 200 l TE, and stored at
enzymatic assays (Zymolyase, Lyticase, Glusulase).13 Because
20C.
cells could rapidly remove some DNA lesions, it is important that
the initial steps of the DNA isolation be carried out rapidly. In IMMUNOASSAY FOR THE DETECTION OF
our laboratory, the method of choice is to break the cells by vigor- ULTRAVIOLET-INDUCED PHOTOPRODUCTS IN
ous shaking in the presence of glass beads. TOTAL GENOMIC DNA
Currently, the most common techniques that are used to quan-
1. Total genomic DNA is digested with two restriction
tify the number of photoproducts in total genomic DNA are either
enzymes to reduce its average size and resuspended in TE buffer.
immunological methods30 or are based on the T4endoV assay.
The samples are denatured at 95C for 15 min and chilled on ice.
Monoclonal antibodies specic to CPDs and to (6.4)PDs [or to
[Note: the amounts of DNA must be determined empirically,
other types of DNA lesions that are not induced by UV light, such
e.g., 50500 ng per slot.]
as benzo[a]pyrenediol (BPDE)] are commercially available, and
2. The nitrocellulose membrane (Protran, Schleicher &
antibody binding can be measured even with small quantities of
Schuell) is prewetted in ddH2O and then equilibrated in 20
antigens. Alternatively, the T4endoV assay developed in the
SSPE (174 g NaCl, 27.6 g NaH2PO4H2O, 7.4 g EDTA, pH 7.4 per
Hanawalt laboratory31 combines T4 enzymatic activity with
liter) for 30 min. Using a slot-blot apparatus, the DNA samples
Southern blotting. Namely, CPD formation and CPD removal are
are spotted in triplicate onto the membrane as follows:
detected with CPD-specic endonuclease T4endoV, which makes
single-DNA strand cuts at CPD sites. Southern blotting of dena- While applying a constant vacuum add 500 l 20 SSPE
turing (alkaline) agarose gels and hybridization with specic to each well.
radioactive probes are used to quantify the frequency of CPDs in When all the liquid is adsorbed add the DNA samples in
DNA. This technique also makes it possible to determine levels 250 l.
of DNA repair in small fractions of the genome such as single When all the liquid is adsorbed, wait an additional 5 min,
copy genes. keeping the vacuum constant. Then rinse the slots twice
ISOLATION OF GENOMIC DNA FROM YEAST CELLS To with 250 l 20 SSPE.
follow NER of UV-induced photoproducts, the following steps When all the liquid is adsorbed, wait an additional 5 min,
are carried out under yellow lamps (peak at 550650 nm): dismantle the slot-blotter, and place the membrane (DNA-
side up) on 3MM papers and let it dry for 30 min.
1. The cells are irradiated and 50-ml aliquots are collected by
centrifugation as described in the section on Ultraviolet Light 3. After baking the membrane for 2 h at 80C to x the DNA,
Irradiation of Large Cell Cultures. The cell pellets are resus- the membrane is rehydrated in PBS.
pended in ice-cold 1.5 ml NIB (nuclei isolation buffer: 17% glyc- 4. Blocking of the membrane is done in 5% nonfat dry milk
erol, 50 mM MOPS, 150 mM K-acetate, 2 mM MgCl2, 0.5 mM in PBS-T (0.05% Tween-20 in PBS), at RT for 1 h.
spermine, and 0.15 mM spermidine, pH 8.0) and transferred to 5. The blocking solution is discarded and the membrane is
15-ml tubes containing 1.5-ml glass beads (Sigma; 425600 m) briey rinsed twice with PBS-T followed by four washes in PBS-
prewashed twice in NIB and prepared on ice. T, 10 min each.
450 SECTION V / MODELS OF BEHAVIOR

6. Incubation with antibodies for CPDs and (6,4)PDs (e.g., random priming (Amersham). Prehybridization, hybridization,
from Abcam, Aftech, Sigma-Aldrich, Trevigen) is done in 1% and washing are done as described in standard protocols, and the
milk in PBS (0.15 ml/cm2 of membrane surface) for 3 h at RT or membranes are exposed to PhosphorImager screens.
overnight at 4C. [Note: The concentration of the antibodies is 4. Determination of the average DNA lengths (Lav) is done as
predetermined empirically.] follows:
7. Washing of the membrane is done as described in step 5.
The image of one lane is overlaid with an array of dupli-
8. Secondary antibodies (Amersham) are prepared in 1% milk
cated rectangles, with the height that corresponds to 1 mm
in PBS and the incubation is done for 1 h at RT,
distance on the gel [Figure 471B; ()T4endoV]. This
followed by the washing step as described above. [Note: The
array (1 to n) is copied onto the other lanes [Figure 471B;
concentration of antibodies is predetermined empirically.]
(+)T4endoV] and onto an empty lane that is used for
9. Detection is done with chemiluminescence reagents (ECL;
background correction (Figure 471B; N).
Amersham) according to the manufacturers instruction, and the
The image density (or volume) within each rectangle (1
membranes are exposed to X-ray lm or a PhosphorImager.
to n) is measured with ImageQuant and the data are trans-
10. To normalize the amount of DNA in each sample and slot,
ferred to an Excel spreadsheet (Microsoft). A chart is
DNA is spotted onto a parallel membrane (steps 13). Prehybrid-
generated showing the DNA prole of each lane (Figure
ization and hybridization with 32P-labeled DNA are done follow-
471C). [Note: Figure 471C is a schematic representa-
ing standard protocols.
tion of a typical DNA prole of UV-irradiated genomic
11. To determine the number of photoproducts and the DNA DNA, treated or mock treated with T4endoV.]
repair rates, normalize the signals obtained with the antibodies to Three backgrounds are subtracted from the image density
the respective amounts of DNA in each slot. Subtract the signals data shown in Figure 471C. (a) The background gener-
generated from nonradiated samples (UV, controls), which ated from nonspecic binding of radiolabeled probe to the
correspond to the nonspecic binding of the antibodies to nylon membrane is removed by subtracting the density
DNA, and determine the percent repair (0 time repair = 100% values obtained from an empty lane (Figure 471B, N) to
photoproducts remaining). [Note: The same technique can be the corresponding rectangles (1 to n) in the () and (+)
used to detect BPDE, the ultimate carcinogenic metabolite of T4endoV lanes and (b) the background due to random
benzo(a)pyrene.] nicks generated during DNA isolation [visible in the lanes
THE ALKALINE AGAROSE GEL ELECTROPHORESIS AND ()T4endoV] is removed. (b1) First, the signal (smear)
SOUTHERN BLOT ASSAY FOR THE DETECTION OF DNA below the broad band corresponding to high-molecular-
STRAND BREAKS AND ULTRAVIOLET-INDUCED PHOTO- weight DNA [Figure 471B; ()T4endoV, from 5 to n) or
PRODUCTS IN TOTAL GENOMIC DNA The strand breaks, region b1 (Figure 471D): each position in the
for example, induced by T4endoV cleavage at CPD sites, cause ()T4endoV lane is subtracted from itself and from its
a shift of the migration of DNA in denaturing agarose gel elec- corresponding position in the (+)T4endoV lane. (b2)
trophoresis (Figure 471A; compare and + T4endoV, and + Second, region b2 (Figure 471D), which corresponds
UV). The shift can be measured by separation of the DNA in to nicked high-molecular-weight DNA and is described as
alkaline agarose gels, transfer to nylon membranes, hybridization the area under the straight line (y = ax), which is generated
with 32P-labeled genomic DNA (e.g., by random priming), and from the origin of the high-molecular-weight DNA and
detection with a PhosphorImager.32 The storage phosphor technol- the rst data point of the smear (rectangle 5); the y-values
ogy is very sensitive and has a linear response range over ve are subtracted from each corresponding data point in the
orders of magnitude. () and (+)T4endoV lanes. The results obtained after sub-
Discrete-step scans of images (Figure 471B) are recorded traction of the three backgrounds are shown in Figure
using ImageQuant software (Molecular Dynamics, Sunnyvale, 471E.
CA). The half-area DNA migration points, or average length of The total areas encompassed by the corrected scans are
the population of DNA molecules, are calculated on an Excel calculated, and the data points corresponding to half of
spreadsheet (Microsoft, Redmond, WA) from the migration these areas are determined (Figure 471E; Xmed). By using
prole of the population of DNA molecules, relative to DNA size the standard curve generated from the migration of the
standards. Finally, the number of CPDs/kb (or DNA strand DNA size markers (Figure 471A; M), Xmed is converted
breaks/kb) is calculated according to Freeman et al.33 to the median length Lmed (in bases). The average molecu-
lar length (Lav) is then calculated using the following
1. The isolated DNA is treated with T4endoV (Figure 471A) equation35:
as described in the section on The T4endoV Assay.
Lav = 0.6 Lmed
2. Alkaline agarose gels (1%) are prepared as previously
described34 and electrophoresis is run for about 13 h at 25 V assuming a Poisson distribution of DNA fragments.
(0.7 V/cm) with buffer circulation. Thereafter, gels are rst neu- 5a. For UV-irradiated DNA, the number of CPDs/kb is calcu-
tralized for 1 h in 1.5 M NaCl, 1 M TrisHCl (pH 7.5), then soaked lated using the following equation33:
in 0.25 N HCl for 30 min, and nally treated in 0.4 N NaOH for
CPDs/kb = (Lav)1(+)T4endoV (Lav)1()T4endoV
15 min. The DNA is transferred in 0.4 M NaOH to a Hybond-XL
membrane (Amersham) following the alkaline Southern blotting where (Lav)(+)T4endoV and (Lav)()T4endoV are the Lav values for samples
protocol. treated or mock treated with T4endoV, respectively.
3. The radioactive probe is generated from yeast total genomic 5b. For DNA SSBs, for example, induced by BLEO, the
DNA digested with EcoRI (to obtain shorter fragments) using number of SSBs/kb is calculated using the following equation32:
CHAPTER 47 / YEAST AS A MODEL SYSTEM TO STUDY DNA DAMAGE AND DNA REPAIR 451

Figure 471. Measurements of DNA strand breaks (induced by T4endoV cleavage at CPD sites) by alkaline agarose gel electrophoresis and
Southern blot.

SSB/kb = (Lav)1 EcoRI, which releases an 2.9-kb DNA fragment when blots are
hybridized with a probe corresponding to the central portion of
6. For repair experiments, CPDs/kb (or SSB/kb) at various
ribosomal genes (rDNA).]
repair times are expressed as percent of the damage measured at
2. After extraction with phenol:chloroform:isoamyl alcohol
time of 0 repair (see the section on The T4endoV Assay).
and chloroform, the DNA is precipitated in ethanol and resus-
pended in 32 l of T4endoV buffer (50 mM TrisHCl, pH 7.5, and
THE T4ENDOV ASSAY TO MEASURE NUCLEOTIDE
5 mM EDTA).
EXCISION REPAIR OF ULTRAVIOLET-INDUCED PHOTO-
3. On ice, dilute 1 l of T4endoV enzyme (e.g., from Epicen-
PRODUCTS IN SPECIFIC DNA SEQUENCES
tre) 1 : 50 in T4endoV buffer, and prepare the reactions in 0.5-ml
Eppendorf tubes as follows:
1. DNA is isolated before UV irradiation (UV), soon after
UV irradiation (+UV; 0 repair), and after various repair times To the mock-treated samples [()T4endoV] add 2 l of
(+UV; 1/2, 1, 2, and 4 h) (Figure 472A). About 6 g of DNA for T4endoV buffer.
each time point is digested with the suitable restriction enzyme. To the test samples (+)T4endoV] add 2 l of the diluted
[For example, in Figure 472A the DNA was digested with T4endoV enzyme.
452 SECTION V / MODELS OF BEHAVIOR

UV + UV The volume (V) of each band is corrected by subtracting


its average background, which is generated from the non-
0 1/2 1 2 4 Repair (hours) specic binding of radiolabeled probe to the membrane
+ + + + + + T4endoV (rectangle above the band) and from random nicks gener-
WT
ated during DNA isolation (rectangle below the band), and
is called V [e.g., V19 = V19 (V20 + V21)/2].
The number of CPDs per DNA fragment (in Figure 47
+ + + + + +
2A; the 2.9-kb EcoRI rDNA fragment) is calculated with
rad14D the following equation:
A CPDs/DNA fragment = ln(V()T4endoV /V(+)T4endoV)
+ + + + + +
2 5 8 11 14 17 20 23 26
6. The resistance of DNA cutting by T4endoV is monitored
29 32 35
1 4 7 10 13 16 19 22 25 28 31 34 WT as a measure of repair, and the percent of CPD removal is plotted
3 6 9 12 15 18 21 24 27 30 33 36
over the repair incubation time (Figure 472C). (In the rDNA of
1 2 3 4 5 6 group WT cells 50% of CPDs is removed after 2 h and 100% repair
B is achieved after 4 h. No repair is measured for the same DNA
fragment in rad14D cells.)
100
% CPDs removal

WT ACKNOWLEDGMENTS
I would like to thank Dr. K. Kobryn at the Universit de Sher-
50
booke and D. Fahy at Washington State University for critical
reading of the manuscript. Also, I would like to thank the students
rad14D
in my laboratory, M. Toussaint, M. Tremblay, G. Levasseur and
0
0 1/2 1 2 4 J. Doyon for providing me with the most up to date protocols.
C The Natural Sciences and Engineering Research Council of
Time repair (hours)
Canada (NSERC) supported this project.
Figure 472. Measurements of repair of CPDs in dened DNA
sequences by the T4endoV assay.
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