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433
Lotte Bjergbæk (ed.), DNA Repair Protocols, Methods in Molecular Biology, vol. 920,
DOI 10.1007/978-1-61779-998-3_30, © Springer Science+Business Media New York 2012
Chapter 30
Live Cell Microscopy of DNA Damage Response
in Saccharomyces cerevisiae
Sonia Silva , Irene Gallina , Nadine Eckert-Boulet , and Michael Lisby
Abstract
Fluorescence microscopy of the DNA damage response in living cells stands out from many other DNA
repair assays by its ability to monitor the response to individual DNA lesions in single cells. This is particu-
larly true in yeast, where the frequency of spontaneous DNA lesions is relatively low compared to organ-
isms with much larger genomes such as mammalian cells. Single cell analysis of individual DNA lesions
allows speci fi c events in the DNA damage response to be correlated with cell morphology, cell cycle phase,
and other speci fi c characteristics of a particular cell. Moreover, fl uorescence live cell imaging allows for
multiple cellular markers to be monitored over several hours. This chapter reviews useful fl uorescent mark-
ers and genotoxic agents for studying the DNA damage response in living cells and provides protocols for
live cell imaging, time-lapse microscopy, and for induction of site-speci fi c DNA lesions.
Key words: Homologous recombination , Checkpoint , Fluorescence microscopy
A broad palette of genetically encoded fl uorophores have been
developed for fl uorescence microscopy with the most commonly
used being derivatives of the green and red fl uorescent proteins
(GFP and RFP) from Aequoria victoria and Discosoma sp., respec-
tively (
1 ) . The available variants of these molecules vary with respect
to excitation and emission spectra, brightness, photostability, pKa,
and oligomeric state in vitro. However, in vivo additional factors
may affect the fl uorescence capabilities of fl uorophores. For example,
both GFP and RFP require molecular oxygen for the maturation
of the nascent polypeptide into a chromophore by cyclization and
oxidation of central amino acids before the protein becomes
fl uorescent (
2, 3 ) . Codon usage is also of great importance for the
proper expression and folding of the fl uorophore, and notably the
optimal codon usage in Saccharomyces cerevisiae is different from
1. Introduction
434 S. Silva et al.
that of human cells, for which many GFP and RFP variants have
been optimized. Finally, it is advisable to avoid fl uorophores that
oligomerize because these can compromise biological function of
the fusion protein. We have good experience with three speci fi c
variants of CFP (clone W7) (
4 ) , YFP (clone 1OC) ( 3 ) and RFP
(clone yEmRFP) (
5 ) . To facilitate PCR-based cloning-free tagging
of proteins at their endogenous chromosomal loci with these
fl uorophores, we have engineered PCR template vectors that use
the Kluyveromyces lactis URA3 for selection of transformants. The
K. lactis URA3 marker is subsequently popped out to leave a clean
integration of the fl uorophore encoding open reading frame. The
relevant plasmids (pWJ1162, pWJ1163, pWJ1164, pWJ1165,
pNEB30, and pNEB31) are listed in Table
1 and the tagging pro-
cedure described elsewhere (
13 ) .
The detection of fl uorescent fusion proteins expressed from
their endogenous promoter depends on multiple factors besides
the expression level including protein turnover, incubation tem-
perature, and localization of the protein. Speci fi cally, the number
of fl uorescent molecules available for detection is de fi ned by the
balance between maturation and protein turnover, which may vary
with experimental conditions. For the CFP, YFP, and RFP variants
used in this study, the maturation half-time was previously esti-
mated to 49, 39, and 15 min, respectively (
14, 15 ) . Moreover,
high temperature negatively affects the folding of some GFP and
RFP variants (
1, 16 ) . In this chapter, we present a selection of
DNA repair and checkpoint markers that are easily detected with
most wide- fi eld microscopes equipped with a CCD camera
(Subheading
2 , Table 2 and Fig. 1 ). The selected proteins are pri-
marily involved in recombinational DNA repair and checkpoint
activation.
1. SC (synthetic complete) powder: 100 g yeast nitrogen base
without amino acids and without NH
4 SO
4 , 293.11 g NH
4 SO
4 ,
1.2 g adenine sulfate, 1.2 g
L -arginine sulfate, 1.2 g L -histidine-
HCl, 1.8 g
L -isoleucine, 3.6 g L -leucine, 1.8 g L -lysine-HCl,
1.2 g
L -methionine, 3 g L -phenylalanine, 1.2 g L -tryptophan,
1.8 g
L -tyrosine, 1.2 g uracil, 9 g L -valine ( 18 ) . Grind in ball
mill overnight.
2. SC + Ade liquid medium: 7.25 g SC powder, 0.1 g adenine
sulfate, and 20 g glucose in 1,000 mL. Adjust pH to 5.8 with
NaOH/HCl.
3. Galactose: Filter-sterilize 30 % (w/v) stock solution in H
2 O.
2. Materials
2.1. Media and Sugars
43530 Live Cell Microscopy of DNA Damage Response in Saccharomyces cerevisiae
Table 1
Yeast strains and plasmids
Marker Strain/plasmid name Genotype a References
Rad52-G-CFP ML187-2D MAT a TRP1 lys2 D ( 6 )
Rad52-G-CFP pWJ1218 pRS413-based HIS3/
CEN This study
Rad52-G-YFP W5094-1C MAT a trp1-1 LYS2 (
6 )
Rad52-G-YFP pWJ1213 pRS413-based HIS3/
CEN ( 7 )
Rad52-G-YFP pWJ1344 pRS415-based LEU2/
CEN (
7 )
Rad52-G-yEmRFP NEB322 MAT a trp1-1 LYS2 This study
Mec1
1–64 -YFP-AA-Mec1
65–2,369 ML41-4B MAT a trp1-1 LYS2 This study
YFP-GDPGG-Tel1 ML129-6B MAT a trp1-1 LYS2 This study
Ddc2-AAAA-YFP W5088-4A MAT a trp1-1 LYS2 ( 8 )
Ddc2-AAAA-CFP ML311-18C MAT a TRP1 lys2 D This study
Mre11-LLAKKRKG-YFP ML104-6B MAT a trp1-1 LYS2 ( 8 )
Rfa1-AAAAAAAAG-YFP ML147-2B MAT a trp1-1 LYS2 ( 8 )
Rfa1-AAAAAAAAG-CFP ML67-4B MAT a TRP1 lys2 D This study
YFP-AAAAAAAA-Sml1 W4622-14D MAT a trp1-1 LYS2
bar1::LEU2 ( 9 )
Ddc1-AAAA-YFP W5059-8A MAT a trp1-1 LYS2 This study
YFP-GGPGG-Rad9 SMG242 MAT a trp1-1 LYS2 (
10 )
Mec1
1-64 -YFP-AA-Mec1
65-2,369 ML292-6C MAT a trp1-1 LYS2 This study
Ddc2-AAAA-CFP
YFP-AAAAAAAA-Sml1 IG101-12D MAT a trp1-1 LYS2 This study
Rad52-G-yEmRFP
Mre11-LLAKKRKG-YFP SS54-9B MAT a trp1-1 LYS2
RAD52-RFP This study
Rfa1-AAAAAAAAG-CFP
CFP-3 ¢ -K.l.URA3 pWJ1162 pRS423-based HIS3/2 m (
11 )
CFP-5 ¢ -K.l.URA3 pWJ1163 pRS423-based HIS3/2 m (
11 )
YFP-3 ¢ -K.l.URA3 pWJ1164 pRS423-based HIS3/2 m (
11 )
YFP-5 ¢ -K.l.URA3 pWJ1165 pRS423-based HIS3/2 m (
11 )
yEmRFP-3 ¢ -K.l.URA3 pNEB31 pRS423-based HIS3/2 m This study
yEmRFP-5 ¢ -K.l.URA3 pNEB30 pRS423-based HIS3/2 m This study
a All strains used in this study are ADE2 RAD5 derivatives of W303-1A ( BAR1 ade2-1 can1-100 ura3-1 his3-11,15 leu2-
3,112 trp1-1 LYS2 rad5-535 ) ( 12 )
436 S. Silva et al.
Table 2
DNA damage response markers, exposure time, and biological function
Protein Construct a Biological function Localization
Exposure
time
b References
Rad52 Rad52-G-CFP, Rad52-G-
YFP, Rad52-G-yEmRFP Early marker of HR Diffuse nuclear, partially excluded from the
nucleolus. Relocalizes to foci during HR 1 s ( 5, 6 )
Mec1 Mec1
1–64 -YFP-AA-
Mec1
65–2,369 Single-stranded DNA
damage checkpoint Localizes from diffuse nuclear to foci in
response to DNA damage 3 s (
8 )
Tel1 YFP-GDPGG-Tel1 Double-strand break
checkpoint Localizes from diffuse nuclear to foci in
response to DSBs 7 s (
17 )
Ddc2 Ddc2-AAAA-YFP Single-stranded DNA
damage checkpoint Localizes from diffuse nuclear to foci in
response to DNA damage 3 s (
17 )
Ddc2 Ddc2-AAAA-CFP Single-stranded DNA
damage checkpoint Localizes from diffuse nuclear to foci in
response to DNA damage 1 s (
17 )
Mre11 Mre11-LLAKKRKG-YFP Double-strand break
recognition and processing Localizes from diffuse nuclear to foci in
response to DSBs 3 s (
17 )
Rfa1 Rfa1-AAAAAAAAG-CFP Single-stranded DNA
recognition Multiple faint replication foci during S phase.
Localizes to bright foci in response to DSBs 0.5 s (
17 )
Sml1 YFP-AAAAAAAA-Sml1 Downstream target of
Rad53-dependent DNA
damage checkpoint
Cytoplasmic degraded upon checkpoint
activation and during S phase 3 s (
8, 9 )
Ddc1 Ddc1-AAAA-YFP Single-stranded DNA
damage checkpoint Localizes from diffuse nuclear to faint foci in
response to DNA damage 3 s (
17 )
Rad9 YFP-GGPGG-Rad9 DNA damage checkpoint Localizes from diffuse nuclear to transient
foci in response to DNA damage 3 s (
10 )
a For microscopy of a single protein, YFP is the preferred fl uorophore for tagging, because it gives the best signal-to-noise ratio. For imaging three proteins in the same cell, it is advis-
able to tag the protein of the lowest abundance with YFP to maximize sensitivity, the most abundant protein with CFP to reduce phototoxicity, and the third protein with RFP
b The optimal exposure time depends on the fl uorophore, the abundance of the protein, the sensitivity of the camera and the intensity of the excitation light, which should be
adjusted so that no signi fi cant photobleaching occurs during acquisition
43730 Live Cell Microscopy of DNA Damage Response in Saccharomyces cerevisiae
1. Melt and mix 1 volume of petroleum jelly (vaseline), 1 volume
of beeswax, and 1 volume of lanolin at 80 °C.
2. For sealing the edges of the microscope cover glass for time-
lapse microscopy, the wax mixture can be kept at 65 °C. The
molten wax is applied along the edges of the cover glass with a
fl at metal spatula pre-heated over a Bunsen burner or similar.
The following chemicals were used: zeocin, camptothecin (CPT),
methyl methanesulfonate (MMS), raf fi nose, galactose, petroleum
jelly, beeswax, lanolin, 4-nitroquinoline-1-oxide (4-NQO), and
hydroxyurea (HU).
The essential elements of the microscope hardware are a high sen-
sitivity cooled CCD or EM-CCD camera and a high magni fi cation
objective (100×) with high numerical aperture ( ³ 1.4).
2.2. Wax for Sealing
Microscope Slides
2.3. Reagents
and Chemicals
2.4. Microscope
Hardware
Fig. 1. Representative images of selected DNA repair and checkpoint proteins. Strains
were imaged as described in Subheading
3 after treatment with 100 m g/mL zeocin for
1 h. Strains are ( a ) ML292-6C, ( b ) IG101-12D, ( c ) ML129-6B, ( d ) W5059-8A, ( e ) SMG242,
and ( f ) SS54-9B. Scale bar, 3 µm.
438 S. Silva et al.
For yeast live-cell imaging, the best result is obtained by culturing
and mounting cells in fi lter-sterilized minimal (SC) medium. Live
cell imaging is preferred over fi xed cells, because fi xation may gen-
erate artifacts or otherwise decrease the quality of the obtained
data. A protocol for imaging live yeast cells is described here:
1. Inoculate from a single colony 3 mL of SC medium containing
100 m g/mL adenine (see Note 1).
2. Grow shaking overnight at 25 °C (see Note 2).
3. Dilute culture to OD
600 = 0.2 and grow for at least one cell
cycle (2.5 h for wild type) prior to microscopy.
4. Expose the culture to DNA damage using one of the agents in
Table
3 .
5. Harvest 1.5 mL of cells at OD
600 = 0.4–0.6 by centrifugation at
1,500 × g (see Note 3).
6. Resuspend the pellet of cells in 50 m L of medium by
vortexing.
7. Cells are mounted on standard glass slides and covered by
cover glass appropriate to the optics of the microscope (see
Note 4). For most strains, the cells are immobilized on the
slide simply by adjusting the volume applied (usually 2–3 m L),
so that the cells settle in a monolayer with the cells touching
both the slide and the cover glass.
8. Capture 11 optical sections separated by 0.4 m m around the
focal plane of the cell (see Note 5).
Imaging of cells over time requires that phototoxicity is minimized
and that favorable growth conditions can be maintained (see Note
6). Phototoxicity can be reduced by decreasing the fl uorescence
exposure time or intensity of the excitation light, and by reducing
the number of optical sections acquired. The easiest way to main-
tain favorable growth conditions is to reduce the cell density on
the slide, so that only a single cell is present in the fi eld of view,
thereby prolonging the time that the cell can grow before nutrients
are locally exhausted. For this reason it is not recommended to
concentrate the cells by centrifugation before mounting. For long-
term imaging (>30 min), the edges of the cover glass are sealed
with wax to prevent evaporation (see Subheading
2.2 and Note 7).
The protocol is:
1. Turn on the microscope 1–2 h before the time-lapse is launched
(see Note 8).
2. Cells are cultured and mounted essentially as described above
(see Subheading
3.2 ).
3. Methods
3.1. Basic Imaging
Protocol
for Fluorescence
Microscopy
3.2. Time-Lapse
Microscopy
43930 Live Cell Microscopy of DNA Damage Response in Saccharomyces cerevisiae
3. The appropriate acquisition parameters for time-lapse
microscopy should be determined empirically. With our
microscopy setup, we are able to image Rad52-YFP with
minimal phototoxicity for 20 time points over 4 h by reduc-
ing the number of optical sections from 11 to 9 (0.5 m m
between sections), by reducing the intensity of the excita-
tion light by inserting a neutral density fi lter in the
fl uorescence path that allows only 10 % transmission, and by
reducing the exposure time from 1 to 0.5 s.
Table 3
Compounds for DNA damage induction
Genotoxic
agent
a Dosage
Time of
treatment Targets/lesions
Primary repair
pathway(s) References
HU 100–200 mM 30 min Inhibits ribonucle-
otide reductase
leading to depletion
of dNTP pools and
replication fork
stalling
Replication restart
by homologous
recombination
(
19, 20 )
MMS 0.03 % 1 h Methylates DNA on
N
7 -deoxyguanine
and N
3 -
deoxyadenine,
which leads to
replication fork
stalling and collapse
Replication restart
by homologous
recombination,
DSB repair
(
21, 22 )
IR 1 DSB per
haploid
genome per
2 krad (20 Gy)
Seconds–
minutes In addition to DSBs,
approximately 17
SSBs are generated
per DSB
Homologous
recombination (
23 )
4-NQO 10 ng/mL 1 h Guanine adducts at
positions C
8 and N
2 Nucleotide
excision repair,
translesion
synthesis, HR
(
24, 25 )
CPT 5 m g/mL 1 h Stabilizes covalent
topoisomerase
I-DNA intermedi-
ates, which are
converted to DSBs
during DNA
replication
Excision repair and
homologous
recombination
(
26, 27 )
Zeocin 100 m g/mL 1 h DSBs Homologous
recombination (
28 )
a HU hydroxyurea, MMS methyl methanesulfonate, IR ionizing radiation, 4-NQO 4-nitroquinoline-1-oxide, CPT
camptothecin, SSB single-strand break, DSB double-strand break, HR homologous recombination
440 S. Silva et al.
The advantage of site-speci fi c, inducible DNA lesions is that the
location of the lesion can be marked fl uorescently and therefore
followed during repair. Fluorescence marking of the DNA lesion is
achieved by inserting a tandem array of DNA binding sites for a
fl uorescently tagged protein, e.g., the Lac or Tet repressor, adja-
cent to the DNA lesion as described (
13 ) . Different types of site-
speci fi c DNA lesions can be introduced. For example, the HO and
I-SceI meganucleases are used to induce double-strand breaks
(DSBs) (
21 ) ; a mutant of the Flp recombinase (Flp-H305L) irre-
versibly cleaves one DNA strand forming a covalent Flp-DNA
intermediate (
29 ) , which causes replication fork stalling and col-
lapse; and a mutant of the I-AniI homing endonuclease (I-AniI-
K227M) exhibits site-speci fi c nicking (
30 ) . Strains in which a single
site-speci fi c endonuclease restriction site in the genome has been
marked fl uorescently, such as the HO or the I-SceI sites, are trans-
formed with a plasmid expressing the appropriate endonuclease
from an inducible promoter, e.g., pJH132 (pGAL-HO) or
pWJ1108 (pGAL-I-SceI), respectively (
8, 31 ) .
1. Transform the appropriate plasmid (expressing HO or I-SceI)
into the assay strain by the LiAc method (
32 ) . Select trans-
formants on the appropriate glucose-containing minimal
medium.
2. Inoculate fresh transformants in 2 mL of the appropriate mini-
mal, raf fi nose-based medium (2 %, w/v, raf fi nose, autoclaved)
(see Note 9).
3. Grow shaking at 25 °C for 24 h.
4. Dilute culture to OD
600 = 0.2 and grow for one cell cycle (3.5 h
for wild type) prior to endonuclease induction.
5. Add galactose (sterile- fi ltered, stock solution 30 %, w/v) to a
fi nal concentration of 2–3 % (w/v). This induces expression of
the endonuclease.
6. Grow shaking at 25 °C for another 90 min prior to microscopy.
1. The supplementation of SC medium with 100 m g/mL adenine
is necessary only for ade2 mutant strains, which will otherwise
accumulate a red pigment that is strongly auto fl uorescent.
However, this medium can also be used for ADE2 wild-type
cells.
2. It is important that the culture is well aerated, because matura-
tion of GFP and RFP requires molecular oxygen (
2, 3 ) .
3.3. Site-Speci fi c
DNA Lesions
4. Notes
44130 Live Cell Microscopy of DNA Damage Response in Saccharomyces cerevisiae
3. At this point it is not necessary to use autoclaved microcentrifuge
tubes. In fact, yeast cells tend to adhere to the side of some
microcentrifuge tubes, if they have been autoclaved, whereas
cells will pellet to the bottom of untreated tubes.
4. A high cell density in the fi eld of view is desired to maximize
data acquisition. However, at high cell density and growth rate
(glucose), the medium quickly becomes saturated with carbon
dioxide, which precipitates as gas bubbles that may displace
cells. Therefore, the optimal cell density must often be deter-
mined empirically.
5. The yeast nucleus has a diameter of approximately 2 m m and
the cell 5 m m. The number of optical sections required to sam-
ple the entire volume of the cell is determined by the Nyquist
theorem. Ideally, the sampling distance should be half the
depth of focus, but in our experience a sampling distance of
0.3–0.4 m m is suf fi cient for most applications. However, under-
sampling may cause some structures to be overlooked.
6. Phototoxicity is of particular importance when studying DNA
damage response, because photolesions can be introduced
into the DNA especially when imaging CFP, which has the
excitation wavelength with the highest energy compared to
YFP and RFP.
7. Nail polish or other solutions containing organic solvents
should be avoided when sealing slides for live cell imaging,
because the solvents may harm the cells.
8. Most microscopes will heat up and expand during use, which
will cause the focal plane to change. The temperature of the
microscope will usually reach equilibrium within 1–2 h of use.
9. It is important to use fresh transformants in order to avoid
selecting cells in which mutations have accumulated in the
endonuclease recognition site. Such mutations tend to
appear in old transformants due to leakage of the GAL1-10
promoter.
Acknowledgment
We thank Dr. Neta Dean, Stony Brook University, for sharing the
yEmRFP construct. This work was supported by Fundação para a
Ciência e a Tecnologia (SS), The Danish Agency for Science,
Technology and Innovation (ML, NEB), the Villum Kann Rasmussen
Foundation (ML), and the European Research Council (ML).
442 S. Silva et al.
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