CHAPTER NINE

Investigation of Break-Induced
Replication in Yeast
Rajula Elango, Zachary Kockler, Liping Liu, Anna Malkova1
University of Iowa, Iowa City, IA, United States
1
Corresponding author: e-mail address: anna-malkova@[Link]

Contents
1. Introduction 162
2. Calculating BIR Efficiency 165
2.1 Equipment and Materials 168
2.2 Solutions and Reagents 168
2.3 Procedure 168
2.4 Notes 169
3. Analysis of BIR Kinetics 170
3.1 Determining BIR Kinetics in a Time-Course Experiment 170
3.2 Preparing Agarose Plugs With Embedded DNA for CHEF 172
3.3 Analysis of BIR Products Using CHEF Gel Electrophoresis 174
3.4 Capillary Transfer and Hybridization 175
4. Analysis of Mutagenesis Associated With BIR 178
4.1 Analysis of Frameshift Mutagenesis 179
4.2 Analysis of Base-Substitution Mutagenesis Associated With BIR 181
4.3 Analysis of Lys+ and Ura+ Mutations by Sanger Sequencing 183
4.4 Sequencing of URA3 and LYS2 Sequences Located on Either Donor or
Recipient Chromosomes 185
5. Analysis of BIR Structural Intermediates by Two-Dimensional Gel Electrophoresis 186
5.1 Preparation of Yeast Samples 186
5.2 DNA Cross-linking 188
5.3 Extraction of DNA 189
5.4 DNA Separation and Collection 191
5.5 DNA Digestion and Gel Separation in the First Dimension 193
5.6 Two-Dimensional Gel Electrophoresis: Second Dimension 195
5.7 Capillary Transfer and Hybridization 196
6. Alternative Method of Genomic DNA Preparation for 2D Analysis by
Spheroplasting Cells 198
6.1 Plug Preparation 198
6.2 1D Gel Electrophoresis 200

Methods in Enzymology, Volume 601 # 2018 Elsevier Inc. 161

ISSN 0076-6879 All rights reserved.
[Link]
162 Rajula Elango et al.

7. Conclusions 201
Acknowledgment 201
References 201
Further Reading 203

Abstract
Break-induced replication (BIR) is an important mechanism aimed to repair one-ended
double-strand DNA breaks. BIR is initiated by invasion of a broken DNA end into a
homologous template followed by DNA synthesis that can proceed for hundreds of
kilobases to the end of the chromosome. Unlike S-phase replication, BIR is carried
out by a migrating DNA bubble and is associated with conservative inheritance of newly
synthesized DNA. The unusual mode of DNA synthesis during BIR leads to an increased
level of genetic instabilities including increased mutagenesis and chromosomal
rearrangements. Here, we describe our experimental system in yeast Saccharomyces
cerevisiae where BIR is initiated by a site-specific DNA break and where the repair
involves two copies of chromosome III. This system allows investigation of BIR using
genetic and molecular biology approaches, and can be used for characterization of
the BIR mechanism, roles of individual proteins in BIR, and for the analysis of genetic
instabilities associated with BIR.

ABBREVIATIONS
BIR break-induced replication
CHEF contour-clamped homogenous electric field
DSB double-strand break
GC gene conversion
GCRs gross chromosomal rearrangements
HR homologous recombination
NHEJ nonhomologous end joining
ssDNA single-strand DNA

1. INTRODUCTION
Double-strand breaks (DSBs) are a dangerous type of DNA damage
that can result from various sources including exposure of DNA to radiation
or chemicals, from the collapse of DNA replication, or from problems
occurring during chromosome segregation (reviewed in Mehta & Haber,
2014). The repair of DSBs is critical for cell survival and for maintaining
genetic integrity. Two major pathways of DSB repair include non-
homologous end joining (NHEJ) and homologous recombination (HR).
During NHEJ, the broken DNA ends are ligated back together and this
often leads to the loss or alteration of genetic information (reviewed in
Investigation of Break-Induced Replication in Yeast 163

Rodgers & McVey, 2016). In contrast, HR utilizes a homologous DNA

template for repair of DSBs. HR repair can be further divided into several
pathways including: gene conversion (GC), which can occur via gap repair
or by synthesis-dependent strand annealing, single-strand annealing, and
break-induced replication (BIR) (reviewed in Mehta & Haber, 2014).
In this review, we focus on BIR pathway, which is employed only when
one end of the DNA break can find homology in the genome for invasion
and repair (Fig. 1A) (reviewed in Sakofsky & Malkova, 2017). This situation
typically arises at eroded telomeres or at collapsed replication forks. BIR is
initiated by a 50 –30 resection of the broken DNA end (Fig. 1B) followed by
invasion of 30 single-strand end into a homologous DNA template (Fig. 1C)
and copying of DNA template that can proceed for more than 100 kb
through the telomere (Fig. 1D). Unlike S-phase DNA replication, which
is carried out by a replication fork, BIR proceeds via a migrating DNA

Fig. 1 Mechanism of BIR initiation and progression. (A) One-ended DSB. (B) 50 –30 resec-
tion of the break results in exposed 30 ssDNA. (C) The ssDNA end invades into the homol-
ogous template initiating DNA synthesis. (D) Leading strand synthesis progresses by a
migrating bubble. ssDNA accumulates behind the migrating bubble due to uncoupled
leading and lagging strand synthesis. (E) Conservative inheritance of DNA.
164 Rajula Elango et al.

bubble with asynchronous synthesis of the leading and lagging strands, and
leads to conservative inheritance of newly synthesized DNA (Fig. 1E). This
unusual type of DNA synthesis leads to accumulation of large amounts of
single-strand DNA (ssDNA), which promotes mutagenesis associated with
BIR (Saini et al., 2013; Sakofsky et al., 2014). In addition, BIR leads to fre-
quent genomic rearrangements similar to those associated with cancer in
humans (Sakofsky et al., 2015; Smith, Lam, & Symington, 2009; Vasan,
Deem, Ramakrishnan, Argueso, & Malkova, 2014).
For a long time budding yeast remained the only eukaryotic organism
where BIR could be studied, and several different approaches have been
employed to investigate BIR in the budding yeast Saccharomyces cerevisiae.
First, a site-specific HO endonuclease was used to initiate one DSB in chro-
mosome III in a way that only one broken end could invade the homologous
chromosome and initiate BIR that can proceed for approximately 100 kb
through the telomere (Malkova, Ivanov, & Haber, 1996; Malkova,
Naylor, Yamaguchi, Ira, & Haber, 2005). Second, a linearized fragment
was transformed into yeast, where it invaded the homologous template
chromosome and initiated BIR (Davis & Symington, 2004; Morrow,
Connelly, & Hieter, 1997). Third, BIR was initiated in telomerase-deficient
yeast strains to maintain their telomeres by alternative lengthening of telo-
meres (ALT) pathway (Lydeard, Jain, Yamaguchi, & Haber, 2007; Teng,
Chang, McCowan, & Zakian, 2000; Teng & Zakian, 1999).
Recently, development of new experimental approaches has allowed
detection of BIR in higher eukaryotes including human cells (Costantino
et al., 2014; Hashimoto & Costanzo, 2011; Minocherhomji et al.,
2015). It has been demonstrated that at mammalian fragile sites, BIR-like
repair synthesis is initiated following replication collapse (Bhowmick,
Minocherhomji, & Hickson, 2016; Minocherhomji et al., 2015). Also, it
was shown that overexpression of oncogenes in human cells leads to the ini-
tiation of BIR promoting chromosomal rearrangements (Costantino et al.,
2014; Sotiriou et al., 2016). Finally, several important observations have
recently been made investigating ALT (that is carried out by BIR-like path-
way) studied in human telomerase-defective cells (Cho, Dilley, Lampson, &
Greenberg, 2014; Dilley et al., 2016).
In this review we focus on one experimental system in yeast where BIR
is initiated by a site-specific HO endonuclease and involves two copies of
chromosome III (Deem et al., 2008). This system allows for a detailed inves-
tigation of BIR by using genetic and molecular biology techniques. Using
this system, we were able to describe the mechanism of BIR, including its
Investigation of Break-Induced Replication in Yeast 165

kinetics, molecular intermediates, and also to identify proteins that carry out
and regulate BIR (Deem et al., 2008; Saini et al., 2013; Vasan et al., 2014). In
addition, this experimental system allowed us to study mutagenesis and
chromosomal rearrangements associated with BIR (Saini et al., 2013;
Sakofsky et al., 2015, 2014). In this review we describe this BIR system
as well as various genetic and molecular biology techniques that we use
to study BIR in yeast.

2. CALCULATING BIR EFFICIENCY

The efficiency of BIR is determined using our yeast strain disomic for
chromosome III AM1003 and its derivatives (Fig. 2) with the genotype
hmlΔ::ADE1/hmlΔ::ADE3 MATa-LEU2-tel/MATα-inc hmrΔ::HPH
FS2Δ::NAT/FS2 leu2/leu2-3112 thr4 ura3-52 ade3::GAL::HO ade1
met13 (Deem et al., 2008). In this strain, HO endonuclease is under the reg-
ulation of a galactose-inducible promoter and the HO recognition site is at
the MATa locus of the truncated copy of chromosome III acting as the
recipient chromosome. The full-length chromosome III, acting as the donor
copy, contains an uncleavable MATα-inc allele and serves as the template for
DSB repair. The HO endonuclease gene is inserted at ADE3 gene located at
chromosome VII and is placed under the control of GAL1/GAL10
(galactose-inducible) promoter. Almost all sequence homology (except
for 46 bp) between the recipient and donor on centromere-distal side of
the break was eliminated via replacement with LEU2 and telomere
sequences, allowing for efficient DSB repair through BIR. The outcomes
resulting from DSB repair by BIR and alternative repair pathways can be
followed based on maintenance of the markers located at all arms of the
two recombining chromosome IIIs (Deem et al., 2008; Vasan et al., 2014).
The efficiency of DSB repair in AM1003 can be assayed by plating cells
on a YEP-Gal (galactose-containing) medium to induce expression of HO
endonuclease leading to the DSB formation. After plating on YEP-Gal
followed by replica plating of the colonies on a series of dropout media,
approximately 80% of the colonies show the expected BIR phenotype of
Ade+ Leu (Deem et al., 2008; Fig. 3). A small fraction of colonies (approx-
imately 10%) show the Ade+ Leu+ phenotype indicative of GC. Approxi-
mately 2%–5% of the colonies are Ade Leu indicative of failed repair
resulting in the loss of the broken (recipient) chromosome. These chromo-
some loss (CL) events are easily distinguished by accumulation of red pig-
ment on adenine dropout media due to the loss of a functional ADE1
166 Rajula Elango et al.

Fig. 2 Schematic of the AM1003 disome and DSB repair outcomes. (A) Depiction of
the AM1003 experimental disome system that contains two copies of chromosome
III. One copy is truncated and contains a GAL::HO site at the MATa locus. The other, full
length, chromosome III (MATα-inc) serves as a donor template for repair. Rectangles
denote insertions of mutation reporters, including the frameshift (lys2::A4) or base-
substitution (ura3-29) reporters with red letters corresponding to mutant bases in the
reporter. (B) BIR repair outcome (Ade+ Leu). (C) Gene conversion repair outcome
(Ade+ Leu+). (D) Chromosome loss outcome (Adered Leu). (E) Half-crossover outcome
(Adewhite Leu).

gene (this phenotype is indicated as Adered Leu) (Fig. 3; Deem et al.,

2008). Another rare type of aberrant repair, called half-crossovers, consti-
tutes 2%–5% of all events and leads to the Adewhite Leu phenotype where
the colonies fail to grow on adenine dropout media but appear white due to
the absence of any pigment accumulation resulting from the loss of ADE3
gene. These colonies also fail to grow on histidine dropout media because
Investigation of Break-Induced Replication in Yeast 167

Fig. 3 BIR repair plating assay. The cells were pregrown in YEP-lactate liquid media
followed by plating on YEP-galactose plates. The colonies were replica-plated on the
Sc-Ade dropout plates. The left panel shows the replica of AM1003 colonies on
Sc-Ade media. The right panel is the replica of a rad51Δ AM1003 derivative. Arrows point
to different repair outcomes, green arrow indicates a BIR outcome, black arrow indicates
a half-crossover, and the red arrow indicates a chromosome loss outcome.

the protein encoded by ADE3 plays also an important role in the biosynthe-
sis of histidine. Half-crossovers are formed due to interruption of BIR
leading strand synthesis leading to resolution of BIR intermediates produc-
ing a fusion of one arm of the donor chromosome and one arm of the
recipient chromosomes with all other arms lost (Deem et al., 2008).
Because the repair by BIR occurs in G2/M cells (Malkova et al., 2005),
two copies of each chromosome are present. Therefore, each colony grown
on YEP-Gal plate is considered to be a result of two separate repair events
that occurred in two broken chromosomes. Therefore, while calculating the
total number of BIR events, the number of full Ade+ Leu colonies is mul-
tiplied by two, while for the colonies containing one Ade+ Leu sector, the
number of BIR events is counted as one. For example, for the colony where
one half is Ade+ Leu, while another half is Adered Leu, we conclude that
it represents two separate events: the repair of one broken chromatid by
BIR, while another sister chromatid failed to repair and was lost (see
Deem et al., 2008 for details). Also, in some DNA repair mutants, we
observed the formation of sectored colonies that were smaller than a half
of the colony, which resulted from the retention of an unrepaired chromatid
for several cell divisions (Malkova et al., 1996; Vasan et al., 2014). Most of
the cells in such colonies eventually stabilize their broken chromatids by BIR,
GC, or half-crossover, while in others the repair fails, leading to a CL.
168 Rajula Elango et al.

The formation of colonies with small and often multiple repair sectors has
been observed in a number of different mutants, including rad9Δ, rad24Δ,
and rad51Δ (Fig. 3; Malkova et al., 1996; Vasan et al., 2014).

2.1 Equipment and Materials

• Hemocytometer (Hausser scientific, #3100)
• 1.5-mL microcentrifuge tubes
• Tabletop vortex
• Incubator (30°C)
• 30°C incubator with shaking table
• Olympus Microscope (with 40 ocular) (or a similar microscope)
• Petri dishes (100  15 mm)

2.2 Solutions and Reagents

• Sc-Leu dropout liquid: 2% D-glucose (RPI, #50-99-7), 0.67% yeast
nitrogen base without amino acids (US Biological, #Y2025), 0.087%
Leu dropout amino acid mix, pH 5.5
• YEPD plates: 2% D-glucose, 2.5% agar (Difco, #214520), 1% yeast
extract (Difco, #212750), 2% peptone (Difco, #211677), pH 5.5
• YEP-lactate liquid: 3.15% lactic acid (Fisher, #A162-500), 1% yeast
extract, 2% peptone pH 5.5
• YEP-galactose plates: 2% galactose (Sigma-Aldrich, #59-23-4), 2.5%
agar, 1% yeast extract, 2% peptone pH 5.5
• MilliQ water
• Sc-Ade dropout plates: 2% glucose, 2.5% agar, 0.67% yeast nitrogen base
without amino acids, 0.087% Ade dropout amino acid mix pH 5.5
• Sc-His dropout plates: 2% glucose, 2.5% agar, 0.67% yeast nitrogen base
without amino acids, 0.087% His dropout amino acid mix pH 5.5
• Sc-Leu dropout plates: 2% glucose, 2.5% agar, 0.67% yeast nitrogen base
without amino acids, 0.087% Leu dropout amino acid mix pH 5.5
• Yeast strain AM1003 (Deem et al., 2008) or its derivative (available upon
request from the corresponding author)

2.3 Procedure
1. Inoculate a single colony of AM1003 in liquid Sc-Leu dropout media
and grow for 24 h at 30°C to saturation (approximately to
1  108 cells/mL).
2. Transfer 5 mL of this culture (inoculum) to 45 mL of YEP-lactate liquid
media in 250 mL flask and grow for 16 h at 30°C with constant aeration.
Investigation of Break-Induced Replication in Yeast 169

3. Count cells using a hemocytometer to calculate the cell density. The

expected cell density is approximately 2  107 cells/mL.
4. Serially dilute the cell culture to a density that allows plating of approx-
imately 60 cells/plate by initially adding 6  105 cells to 1 mL of sterile
water. Make three serial 1:10 dilutions in sterile water to achieve a final
concentration of 6  102 cells/mL.
5. Plate 0.1 mL of cell suspension (containing approximately 60 cells) onto
1 plate of YEPD and 1 plate of YEP-galactose media. Plate at least 3
YEPD plates and 5–10 YEP-galactose plates.
6. Incubate YEPD and YEP-galactose plates at 30°C for 3–5 days. Typi-
cally, the colonies will form in 3 days on YEPD and in 5 days on
YEP-galactose.
7. Replica plate the colonies onto Sc-Ade and Sc-Leu, dropout media
plates.
8. Score repair outcomes based on auxotrophic marker phenotype (Figs. 2
and 3).
a. BIR: Ade+ Leu
b. GC: Ade+ Leu+
c. Half-crossover: Adewhite Leu
d. CL: Adered Leu
9. Calculate the frequency of BIR outcomes using the following formula:
2  ð# of full BIR coloniesÞ + # of colonies with a BIR sector
BIR frequency ¼
2  ðtotal coloniesÞ

2.4 Notes
1. Initial inoculum can be started from a patched colony, or a single
colony.
2. Sc-Leu dropout media is used for the inoculum to eliminate cells that
underwent spontaneous BIR and therefore became Leu prior to the
beginning of experiment.
3. The lack of glucose in the media enables the efficient galactose uptake
ensuring the maximum HO induction and therefore DSB initiation.
It is expected that more than 90% of cells will undergo HO-induced
DSB in less than 20 min following galactose addition.
4. The comparison of the number of colonies grown on YEP-galactose vs
YEPD allows to determine cell viability following DSB induction. The
cell viability can be calculated using the formula:
c:f :u: per mL on YEP-Gal
Cell viability ð%Þ ¼  100
c:f :u: per mL on YEPD
170 Rajula Elango et al.

where c.f.u. ¼ colony-forming units.

Inability to form colonies, or loss of viability after the DSB, is rare due
to the design of the disomic system that contains two chromosomes III
(Fig. 2A). These cells remain viable even when repair of the one (broken)
chromosome is unsuccessful and it is lost. However, the loss of viability
can occur due to more complex events that alter both recombining
chromosomes.
5. It is important to note that not all Ade+ Leu events have been proven to
be BIR. Some Ade+ Leu outcomes contain gross chromosomal
rearrangements (GCRs). BIR can be distinguished from GCR out-
comes using contour-clamped homogenous electric field (CHEF) gel
electrophoresis (see Section 3). The frequency of GCRs among Ade+
Leu in each strain background should be taken into account when cal-
culating BIR efficiency (see Vasan et al., 2014 for details).

3. ANALYSIS OF BIR KINETICS

BIR kinetics is analyzed by separating BIR products from other chro-
mosomes using CHEF gel electrophoresis. Using this technique, the BIR
product (300 kb) can be distinguished from the original truncated chro-
mosome III that is approximately 200-kb-long before HO-induced DSB
(Fig. 4). BIR repair time-course is performed by collecting 50 mL samples
for CHEF before and after DSB induction. BIR typically takes 4 h for ini-
tiation and 5–10 h for completion. Therefore, time points are collected
every 2 h following BIR induction for the total of 8–10 h. Cells collected
at each time point are spun down and embedded in 1% low melting agarose
plugs after treatment with zymolyase. The resulting DNA plugs are then
used for CHEF gel electrophoresis using the Bio-Rad-CHEF-DRII or
other CHEF machines.

3.1 Determining BIR Kinetics in a Time-Course Experiment

3.1.1 Equipment
• 50-mL Conical tubes
• Tabletop centrifuge for spinning 50-mL conical tubes (Beckman Coul-
ter Allegra 25R or similar model)
• 30°C Incubator with shaking table
• 280°C freezer
Investigation of Break-Induced Replication in Yeast 171

Fig. 4 BIR kinetics assay using CHEF gel electrophoresis. The aliquots were taken from
the liquid cultures undergoing BIR repair before (0 h) and following the addition of galac-
tose to the media every 2 h. The genomic DNA was separated by CHEF. The left panel is
an ethidium bromide-stained gel of the separated chromosomes. The middle panel rep-
resents the results of Southern blot analysis using ADE3-specific probe that highlights
the donor chromosome III. The right panel represents the results of Southern blot analysis
using ADE1-specific probe highlighting the recipient chromosome III. Chr., chromosome.

3.1.2 Solutions and Reagents

• YEP-lactate liquid
• Sc-Leu dropout liquid
• 20% Galactose: 20 g galactose dissolved in 100 mL of MilliQ water and
filter sterilized
• 10% Sodium azide: 10 g sodium azide dissolved in 100 mL MilliQ water
(Sigma-Aldrich, #26628-22-8)
• 50 mM EDTA: 14.6 g dissolved in 1 L MilliQ water pH 7.5 (Fisher,
BP120-1)

3.1.3 Procedure
1. Inoculate AM1003 in 50 mL of Sc-Leu dropout media and incubate at
30°C with shaking for 24 h. Culture should be of a final density of 108
cells/mL.
2. Add 5 mL of inoculum to 500 mL of YEP-lactate to obtain a cell den-
sity of 1  106 cells/mL. The culture was incubated at 30°C with shaking
for approximately 16 h, to reach a cell density of 2  107 cells/mL.
3. Collect 50 mL of culture for a 0-h sample (sample taken before galactose
addition) in a 50-mL conical tube, then add 0.5 mL of 10% sodium azide
to this aliquot. Mix thoroughly.
4. Pellet the sample using a tabletop centrifuge by spinning at 3000 rpm for
5 min at 4°C.
172 Rajula Elango et al.

5. Pour off supernatant into hazardous waste container and resuspend in

50 mL of 50 mM EDTA pH 7.5 solution (chilled).
6. Pellet the sample using a tabletop centrifuge by spinning at 3000 rpm for
5 min. Discard supernatant.
7. Store pellet in 280°C freezer until needed for plug preparation.
8. Induce the DSB by adding 50 mL of 20% galactose to the YEP-lactate
culture.
9. Every hour (or every 2 h) after the galactose addition, collect 50 mL of
culture and repeat steps 3 through 7.

3.1.4 Notes
• DSB formation should occur within the first hour following addition of
galactose.
• 4 h after the break, repair products of BIR usually appear.

3.2 Preparing Agarose Plugs With Embedded DNA for CHEF

3.2.1 Equipment
• 40°C water bath
• 37°C water bath
• 50°C water bath
• 30°C incubator with shaking table
• 4°C refrigerator
• Plug molds (Bio-Rad, #1703713)
• 50-mL conical tubes
• 1.5-mL microcentrifuge tubes
• Serological pipettes (5 and 10 mL)
• Centrifuge for 50-mL conical tubes (Beckman Coulter Allegra 25R cen-
trifuge or a similar brand)
• Scoopula

3.2.2 Solutions and Reagents

• SCE solution: 1 M sorbitol (RPI, #10043-35-3), 0.1 M sodium citrate
dihydrate (RPI, #6132-04-3), 50 mM EDTA pH 7.5
• 50 mM EDTA pH 7.5 solution in water
• LMP agarose (Fisher, 9012-36-6) 1% LMP agarose in a mix of 0.75 mL
water and 0.25 mL 0.5 M EDTA pH 7.5
• Zymolyase buffer: 0.005 g 20T zymolyase (MP#320921), 1 mL SCE
solution, 50 μL 2-mercaptoethanol (AMRESCO, #60-24-2)
Investigation of Break-Induced Replication in Yeast 173

• Incubation buffer 1: 50 mM EDTA pH 7.5, 0.01 M Tris–HCl (RPI,

#77-86-1), pH 7.5
• RNase A 10 mg/mL: (Thermo Fisher Scientific #EN0531)
• Incubation buffer 2: 0.01 M Tris–HCl pH 7.5, 0.25 M EDTA pH 7.5,
1% Sarkosyl (Fisher, #137-16-6)
• Wash buffer: 10 mM Tris pH 7.5, 50 mM EDTA pH 7.5
• Storage buffer: 40 mM EDTA pH 7.5, 55% glycerol (RPI, #56-81-5)
• Proteinase K: (RPI, #39450-01-6)
• MilliQ water

3.2.3 Procedure
1. Thaw pellets from the time-course assay on ice.
2. During thawing, LMP agarose is prepared by adding 0.01 g of LMP
agarose to 0.75 mL of MilliQ water then adding 0.25 mL 0.5 M EDTA
pH 7.5. Heat LMP agarose on heating block to 100°C to dissolve the
LMP agarose. Zymolyase buffer is prepared by adding 50 μL of
2-mercaptoethanol and 0.005 g 20T zymolyase to 1 mL of SCE where
it then stored on ice.
3. Place LMP agarose in 40°C water bath to equilibrate temperature to
40°C.
4. Loosen the pellet using tabletop vortex and equilibrate in a 40°C
water bath.
5. Mix 0.415 mL of agarose with 0.085 mL zymolyase buffer quickly and
then add 0.25 mL cells to this mixture.
6. Quickly transfer the cell agarose mixture into plug molds by pipetting.
7. Chill plug molds containing cell agarose mixture in refrigerator at 4°C
for 5 min.
8. Prepare incubation buffer 1 mix by adding 750 μL mercaptoethanol
and 10 μL 10 mg/mL RNase into 10 mL of incubation buffer 1. Dis-
pense 2.5 mL of incubation buffer 1 mixture into 50-mL conical tubes.
9. Use a toothpick to remove solidified plugs from the plug molds and
into conical tubes with incubation buffer 1 mix from the step 8, and
incubate in 37°C water bath for 1 h.
10. Decant incubation buffer from conical tubes, while using scoopula to
prevent losing the plugs.
11. Rinse plugs in MilliQ water and decant water while using scoopula to
prevent losing the plugs.
12. Prepare incubation buffer 2 mix by adding 0.005 g proteinase K into
10 mL of incubation buffer 2.
174 Rajula Elango et al.

13. Add 2.5 mL of incubation buffer 2 mixture from step 12 into each con-
ical tube with plugs and incubate at 50°C for 16 h.
14. Rinse plugs in MilliQ water and decant water while using scoopula to
prevent losing the plugs.
15. Rinse plugs in 2.5 mL of wash buffer.
16. Plugs can be stored in wash buffer for 2 days at 4°C and used for CHEF
gel electrophoresis. Long-term storage requires decanting wash buffer,
using scoopula to prevent losing the plugs, and adding 50 mL of storage
buffer and keeping at 20°C for up to 6 months.

3.2.4 Notes
1. Colonies for analysis can be patched beforehand to allow for further
studies to be performed.
2. LMP agarose mix should not be hot at the time of mixing with
zymolyase to prevent degradation of zymolyase.
3. When transferring cell mixture, with LMP agarose and zymolyase, into
plug molds avoid air bubbles in the mold so as to have a solid plug.
4. A pressurized stream of air can also be used to remove plugs from the
plug mold as an alternative to a toothpick.
5. For long-term storage of the plugs, the volume of storage solution should
be at least half of the storage tube to prevent freezing of the plugs as well
as to protect against breaking of the plugs.

3.3 Analysis of BIR Products Using CHEF Gel Electrophoresis

3.3.1 Equipment
• CHEF-DRII (Bio-Rad) apparatus or similar brands.
• CHEF gel-casting tray.

3.3.2 Solutions and Reagents

• 5 TBE: 64 g Tris base (RPI, #77-86-1), 31 g boric acid (RPI,
#10043-35-3), 3.35 g EDTA disodium salt dihydrate (Fisher, BP120-1)
for 1 L of water
• 1% Pulse-field certified agarose gel (Bio-Rad, #L004315B): 2 g pulse-
field certified agarose in 200 mL 0.5 TBE
• Wash buffer: 10 mM Tris pH 7.5, 50 mM EDTA pH 7.5

3.3.3 Procedure
1. Make 3 L of 0.5 TBE by adding 300 mL of 5 TBE and 2.7 L of
MilliQ water to a 4-L beaker.
Investigation of Break-Induced Replication in Yeast 175

2. Assemble gel-casting tray (a part of CHEF Bio-Rad equipment) with

black base plate on the bottom and dams tightened on each side.
3. Using a scoopula remove each plug and rinse in wash buffer. Then place
the plug against the long wall of the gel-casting tray.
4. Remove 200 mL of the previously made 0.5 TBE and make a 1%
pulse-field agarose gel, and microwave until it dissolves. Let the 1%
pulse-field agarose gel cool to about 60°C then pour into gel-
casting tray.
5. Transfer the remaining 0.5 TBE buffer into CHEF machine to circu-
late and precool buffer to 14°C.
6. Once the gel solidifies, disassemble the gel-casting tray while leaving the
gel adhered to the black plate.
7. Place the gel and the black base plate into frame in the middle of CHEF
apparatus.
8. Close the lid of the CHEF machine.
9. On the control module enter the parameters of the initial and final switch
times, the duration, and the voltages. For the CHEF-DRII apparatus to
separate the donor chromosome III (356 kb) and the repaired recipient
chromosome III (346 kb) of AM1003, the parameters are: initial switch
time of 10 s, final switch time of 35 s, running time of 40 h, and the volt-
age is 6 V/cm.

3.3.4 Notes
1. When pouring the gel into the casting tray, be sure that the holes in each
corner of the black plate are filled without air bubbles. Failure for the
holes to be filled could lead to the gel breaking away and floating in
the buffer.
2. When removing the gel from the casting tray, remove as much excess gel
from the edges and bottom of the black plate so as to not clog the CHEF
apparatus.
3. For different CHEF machines the optimal running parameters to sepa-
rate the chromosomes of interest vary.

3.4 Capillary Transfer and Hybridization

3.4.1 Equipment
• Glass baking dish 19  28 cm (or similar dish)
• UV Cross-linker wavelength 254 nm (Stratagene Stratalinker UV 2400)
• UV Gel imager (Gel DOC Bio-Rad or similar brand)
• Glass plate 20.5  32 cm
176 Rajula Elango et al.

• Whatman paper 45  45 cm
• Scott C-fold paper towels (Fisher, #06-666-32B)
• Amersham Hybond Membrane-N+ (GE Healthcare, #RPN303B)
• 1.5-mL microcentrifuge tubes
• 1.5-mL microcentrifuge tube locker (VWR, #14229-941)
• 37°C dry bath
• 100°C dry bath
• Hybridization tubes
• Hybridization Oven (BigShot 3 or similar brand)
• Geiger counter (Ludlum 5000)
• Typhoon Phosphorimager FLA7000 (or similar brand)

3.4.2 Solutions and Reagents

• Ethidium bromide (Sigma-Aldrich, #1239-45-8)
• 20 SSC pH 7: 175.3 g NaCl, 88.2 g sodium citrate, 900 mL MilliQ
water
• Alkaline solution: 20 g NaOH (RPI, #1310-73-2), 87.7 g NaCl (RPI,
#7647-14-5) in 1 L of MilliQ water
• Neutralization solution: 87.7 g NaCl (RPI, #7647-14-5), 121.4 g Tris
base (RPI, #77-86-1) in 1 L of MilliQ water, pH 7
• 1 TBE: dilute 5  TBE by adding 200 mL 5  TBE in 1 L of MilliQ
water
• 1% agarose gel: 1 g of agarose (Sigma-Aldrich, #A0169) in 100 mL of
1  TBE
• QIAGEN gel extraction kit: (QIAGEN, #28704)
• P32-dCTP (Perkin Elmer, #BLU513H250)
• RmT Random primer kit (Agilent, #300392)
• Hybridization buffer: 50 mL of 0.5 M sodium phosphate buffer pH 7.2
(Fisher, #S374-1), 25 mL of 20% SDS (Fisher, #BP166-5), 200 μL of
0.5 M EDTA pH 8. Make up to 100 mL using MilliQ water
• Low stringency wash buffer: 2  SSC, 0.1% SDS
• MilliQ water

3.4.3 Procedure
1. Add 300 mL MilliQ water into glass baking dish.
2. Remove the CHEF gel from the CHEF apparatus and place into the
glass baking dish that contains 0.5  TBE.
3. Add 9 μL of ethidium bromide solution into the glass dish containing
the CHEF gel and incubate for 30 min with constant gentle shaking.
4. Carefully remove gel from black base plate.
Investigation of Break-Induced Replication in Yeast 177

5. Using UV gel imager, take a picture of the CHEF gel with separated
chromosomes.
6. Remove buffer with ethidium bromide and place gel into UV cross-
linker and set machine to 600 μJ total exposure at 254 nm wavelength
light.
7. After cross-linking, submerge the gel in alkaline solution and incubate
for 30 min with constant gentle shaking.
8. Decant alkaline solution.
9. Submerge gel in neutralization solution and incubate for 30 min with
constant gentle shaking.
10. Transfer gel via capillary transfer methods described in (Sambrook &
Russell, 2006) using 10 SSC and let the transfer occur for 20 h.
11. Cross-link membrane (DNA side up) in UV cross-linker at 254 nm
wavelength light using the autocrosslink function (1200 μJ).
12. ADE1-specific DNA probe targets not only the recipient copy of chro-
mosome III (before and after the DSB, and also following DSB repair)
but also chromosome I containing a native ADE1 gene. This probe is
prepared by PCR amplification of the ADE1 fragment from the yeast
genome (using genomic DNA prepared from AM1003) and using the
following PCR primers:
FP: 50 -GGTTTGAAACAACCTCAAGGACTT-30
RP: 50 -AAGTCCTTGAGGTTGTTTCAAACC-30 .
13. Run the PCR probe fragment on a 1% agarose 1  TBE gel and excise
the fragment.
14. Using the QIAGEN Gel extraction kit extract the probe PCR probe
fragment.
15. Resuspend and dissolve the contents of one tube of the RmT Random
primer kit in 37 μL of MilliQ water, and transfer to a 1.5-mL
microcentrifuge tube.
16. Add 5 μL (70 ng) of the gel-extracted PCR probe fragment to the
reaction mixture in step 18.
17. Incubate RmT Random primer kit and probe fragment mixture at
100°C for 5 min. Be sure to add a microcentrifuge tube locker to keep
the lid closed.
18. After incubation period, place reaction mixture on ice for 1 min
and briefly spin down (to collect all the liquid on the bottom of the
tube).
19. Add 5 μL of P32-dCTP to the cooled reaction mixture, and add 3 μL of
magenta polymerase from the RmT Random primer kit, and incubate
at 37°C dry bath for 10 min.
178 Rajula Elango et al.

20. Add 2 μL of stop mix from the RmT Random primer kit to stop the
reaction.
21. Add 200 μL of MilliQ water to the reaction mixture and denature at
100°C dry bath for 10 min and snap cool on ice for 1 min.
22. Prehybridize the cross-linked membrane from step 14 in 25 mL of
hybridization buffer at 65°C for at least 10 min.
23. Hybridize the membrane at 65°C using 25 mL of hybridization buffer
for 24 h (Church & Gilbert, 1984).
24. Wash the membrane using 65°C low stringency wash buffer in an open
tray. Gently rock the wash buffer and membrane for 10 min. Discard
waste under the guidelines of your radiation safety officer.
25. Repeat wash two more times or until background is washed away, as
being checked using a Geiger counter.
26. Using 20  25 cm GE Phosphor screen (or similar screen), expose
membrane for 1 day and image on a Typhoon Phosphorimager
FLA7000 (or similar brand). See Fig. 4 for the example of Southern blot
hybridization images.

3.4.4 Notes
1. Follow precautions while using P32. The laboratory staff using P32 has to
undergo radiation safety training prior to usage of these materials.

4. ANALYSIS OF MUTAGENESIS ASSOCIATED WITH BIR

DNA synthesis during BIR is associated with a very high level of
mutagenesis which could be 100- to 1000-fold higher as compared to
S-phase DNA replication (Deem et al., 2011). Both frameshifts and base
substitutions are drastically increased during BIR (Deem et al., 2011;
Saini et al., 2013; Sakofsky et al., 2014). Mutagenesis associated with BIR
can be assayed using our reporter systems. The level of frameshift mutagen-
esis during BIR can be assessed using the lys2::A4 reporter (Fig. 2; Deem
et al., 2011). This reporter can be inserted at several positions on the donor
chromosome, on the track of BIR to determine the level of mutagenesis
during its entire course of synthesis (Fig. 2). The level of base-substitution
mutations formed during BIR could be determined by using the ura3-29
reporter (Saini et al., 2013) inserted at various positions along the track of
BIR (Fig. 2).
Investigation of Break-Induced Replication in Yeast 179

4.1 Analysis of Frameshift Mutagenesis

The reporter lys2::A4 is an allele of the LYS2 gene with an insert of
61 bp resulting in a +1-bp frameshift in the LYS2 reading frame, leading
to a Lys phenotype (Tran, Keen, Kricker, Resnick, & Gordenin, 1997).
A Lys+ phenotype can be restored by a frameshift mutation that can occur
in an approximately 71-bp region of the allele (that includes the inserted
sequence) and restores the reading frame. Several isogenic strains were
created by inserting the lys2::A4 reporter into three positions on the donor
(MATα-inc-containing) copy of chromosome III along the track of BIR
synthesis. In our previous work (Deem et al., 2011) three insertion positions
of lys::A4 reporter were employed: at MATα-inc (“MAT” or “0 kb” posi-
tion), 16 kb centromere-distal from MATα-inc, in the region between
RSC6 and THR4 (“16 kb” position), and 36 kb centromere-distal to
MATα-inc, in the region between SED4 and ATG15 (“36 kb” position).
In all these strains, LYS2 copy was fully deleted from its native location
in chromosome II to prevent recombination with the reporter at
chromosome III.

4.1.1 Equipment
• 30°C incubator
• 30°C incubator with shaker table (New Brunswick Scientific I2500
series or similar model)
• Hemocytometer (Hausser scientific, #3100)
• 50-mL conical tubes
• Tabletop centrifuge for 50-mL conical tubes (Beckman Coulter Allegra
25R centrifuge or similar model)

4.1.2 Solutions and Reagents

• Sc-Leu dropout liquid: 2% D-glucose, 0.67% yeast nitrogen base without
amino acids, 0.087% Sc-Leu dropout amino acid mix
• YEP-lactate liquid media: 3.15% lactic acid, 1% yeast extract, 2% pep-
tone, pH 5.5
• 20% Galactose: 20 g galactose dissolved in 100 mL MilliQ water and fil-
ter sterilized
• Sc-Lys dropout plates: 2% D-glucose, 2.5% agar, 0.67% yeast nitrogen
base without amino acids, 0.087% Sc-Lys dropout amino acid mix
pH 5.5
180 Rajula Elango et al.

• YEPD plates: 2% D-glucose, 2.5% agar, 1% yeast extract, 2% peptone

pH 5.5
• MilliQ water

4.1.3 Procedure
1. Inoculate a single colony of a strain (isogenic to AM1003) containing
lys2::A4 reporter into 50 mL of Sc-Leu dropout media and incubate
with constant shaking at 30°C 24 h.
2. Transfer 10 mL of inoculum into 100 mL YEP-lactate media (in a
500-mL flask), with a starting cell density of 1  106/mL. Incubate
at 30°C for 16–20 h with constant shaking, to a final concentration
of 1–2  107/mL.
3. Collect 50 mL of culture from step 2 into a conical tube. Take this as
“0 h” time point. This sample is used to assess frameshift mutagenesis
associated with S-phase replication (as a control).
4. Serially dilute these samples by transferring 0.1 mL of culture into
0.9 mL water. Typically, four 10-fold dilutions are made to obtain
100 cells/100 μL of water.
5. Plate 0.1 mL per YEPD plate and use three plates in total (approxi-
mately 100 cells per plate). This will allow to measure the concentration
of cells in the culture.
6. To estimate the number of mutations accumulated in the culture at 0 h
(before induction of DSB by HO) collect the “0 h” culture and cen-
trifuge at 5000 rpm for 2 min at 25°C. Decant the supernatant and keep
the pellet. Resuspend the pellet with water to a final volume of 0.5 mL
and spread equal amounts onto Sc-Lys dropout plates (0.1 mL per
plate). Be cautious to not plate >1  108 cells per plate to avoid forma-
tion of a lawn due to residual growth.
7. Add 5.5 mL 20% galactose (filter sterilized) to the remaining 50 mL of
the culture from step 2, to obtain a final concentration of 2% galactose.
Addition of galactose initiates formation of DSB. Incubate with con-
stant aeration at 3000 rpm at 30°C for 7 h.
8. After 7 h of incubation, collect 5 mL of culture from step 7 and cen-
trifuge at 5000 rpm for 2 min at 25°C. Decant the supernatant and
resuspend the pellet with water to a final volume of 0.5 mL and spread
them evenly onto Sc-Lys dropout plates (0.1 mL per plate). Be cautious
to not plate >1  108 cells per plate to avoid formation of a lawn.
9. Incubate all plates at 30°C for 5 days and then count the colonies on
each plate.
Investigation of Break-Induced Replication in Yeast 181

10. Calculate mutation rate during BIR using the following equation (the
example for the volume and dilutions mentioned here):
Mutation Rate associated with BIR ¼ Mutation frequency (7 h)–Mutation
frequency (0 h).
Total number of colonies on all Sc  Lys plates ð0 hÞ
Mutation frequency ð0 hÞ ¼
Average number of colonies on YEPD plates ð0 hÞ  5  106

Total number of colonies on all Sc  Lys plates ð7 hÞ

Mutation frequency ð7 hÞ ¼
Average number of colonies on YEPD plates ð7 hÞ  5  105

4.1.4 Notes
1. The best way to determine mutation rate during S-phase is by using a
no-DSB control strain. A no-DSB strain has a genotype similar to
AM1003 except it contains MATα-inc-LEU2-tel, and in these strains
DSBs cannot be induced by HO endonuclease.
2. Due to the accumulation of mutations over several generations, the rate
of spontaneous mutagenesis (the rate of mutations during S-phase rep-
lication) is calculated using Drake equation: μ ¼ 0.4343 f/log(Nμ),
where μ is the rate of spontaneous mutagenesis, f is mutation frequency,
and N is the number of cells in yeast culture (see Deem et al., 2011 for
details). However, during BIR, the cells are arrested at G2/M by DSB
induction until they are repaired, and then they recover from the arrest.
This means that there are no cell divisions between 0 and 7 h time points
following galactose addition. This is why the mutation rate during BIR
can be calculated using a modified Drake equation. (Mutation Rate
during BIR ¼ Mutation frequency (7 h)–Mutation frequency (0 h)).
3. Typically for wild-type strains, 5 mL samples are collected at 7 h.
However, for certain mutants, more cells need to be collected due to
a lower mutation rate.

4.2 Analysis of Base-Substitution Mutagenesis Associated

With BIR
The ura3-29 reporter (used to assay the frequency of base substitutions asso-
ciated with BIR) is an allele of the URA3 gene containing a mutation at the
position 257 resulting in an amino acid change from phenylalanine to serine
rendering the strain Ura (Shcherbakova & Pavlov, 1996). This reporter can
be inserted in two orientations—Ori1 (AGA in the mutant position in the
leading strand of BIR) and Ori2 (TCT in the leading strand). CG ! AT,
182 Rajula Elango et al.

CG ! TA, and CG ! GC base-substitution events in the mutant position

leads to restoration of a Ura+ phenotype. In two strains isogenic to
AM1003, AM2161, and AM2820, the ura3-29 reporter is inserted in
Ori1 and Ori2 orientations, respectively, at the position located 16 kb
centromere-distal from MATα-inc in the region between RSC6 and
THR4 (16 kb) (Saini et al., 2013). In these strains, URA3 was fully deleted
from its native location in chromosome V.

4.2.1 Equipment
• 30°C incubator
• 30°C incubator with shaker table (New Brunswick Scientific I2500
series or similar model)
• Hemocytometer (Hausser scientific, #3100)
• 50-mL conical tubes
• Tabletop centrifuge for 50-mL conical tubes (Beckman Coulter Allegra
25R centrifuge or similar model)

4.2.2 Solutions and Reagents

• Sc-Leu dropout liquid: 2% D-glucose, 0.67% yeast nitrogen base without
amino acids, 0.087% Sc-Leu dropout amino acid mix
• YEP-lactate liquid: 3.15% lactic acid, 1% yeast extract, 2% peptone,
pH 5.5.
• 20% Galactose: 20 g galactose dissolved in 100 mL of water and filter
sterilized
• Sc-Ura dropout plates: 2% D-glucose, 2.5% agar, 0.67% yeast
nitrogen base without amino acids, 0.087% Sc-Ura dropout amino
acid mix
• YEPD plate media: 2% D-glucose, 2.5% agar, 1% yeast extract, 2%
peptone, pH 5.5
• MilliQ water

4.2.3 Procedure
1. Inoculate a single colony of a strain containing ura3-29 reporter into
10 mL of Sc-Leu liquid dropout media and incubate with constant
shaking at 30°C for 24 h.
2. Transfer 10 mL inoculum into 100 mL YEP-lactate media, with a
starting cell density at 1  106/mL. Incubate at 30°C for 16–20 h
with constant shaking, to a final concentration of 1–2  107/mL.
Investigation of Break-Induced Replication in Yeast 183

3. Collect 50 mL of culture from step 2 into a conical tube. Take this as

“0 h” time point. This sample is used to assess base-substitution muta-
genesis associated with S-phase replication (used as a control).
4. Serially dilute these samples by transferring 0.1 mL of culture into
0.9 mL water. Typically, four 10-fold dilutions are made to obtain
100 cells/100 μL of water.
5. Use 0.1 mL per plate and use 3 YEPD plates in total (approximately 100
cells per plate). This will allow to measure the concentration of cells in
the culture.
6. To estimate the number of mutations accumulated in the culture at 0 h
(before induction of DSB by HO) collect the “0 h” culture and cen-
trifuge at 5000 rpm for 2 min at 25°C. Decant the supernatant and keep
the pellet. Resuspend the pellet with water to a final volume of 0.5 mL
and spread equal amounts onto Sc-Ura dropout plates (0.1 mL per
plate). Be cautious to not plate >1  108 cells per plate.
7. Add 5.5 mL of 20% galactose (filter sterilized) to the remaining 50 mL
of the culture from step 2, to obtain a final concentration of 2% galac-
tose. Incubate with constant aeration at 3000 rpm at 30°C for 7 h.
8. After 7 h, collect 5 mL of culture from step 7 and centrifuge at
5000 rpm for 2 min at 25°C. Decant the supernatant and resuspend
the pellet in water to a final volume of 0.5 mL and spread them evenly
onto Sc-Ura dropout plates (0.1 mL per plate).
9. Incubate all plates at 30°C for 5 days and then count the colonies on
each plate.
10. Calculate mutation rate during BIR using the following equation.
Mutation Rate associated with BIR ¼ Mutation frequency (7 h)–Mutation
frequency (0 h).
See Section 4.1.3 and the notes in Section 4.1.4 for the details of calcu-
lating mutation rates. (Here, use the number of colonies grown on Sc-Ura
dropout plates instead of Sc-Lys dropout plates that are used in the
Section 4.1.3.)

4.2.4 Notes
1. Typically for wild-type strains, 5 mL samples are collected at 7 h. How-
ever, for certain mutants, more cells are collected due to a lower
mutation rate.

4.3 Analysis of Lys+ and Ura+ Mutations by Sanger Sequencing

4.3.1 Equipment
• Thermal cycler (Bio-Rad T100 thermal cycler) or a similar model.
184 Rajula Elango et al.

4.3.2 Solutions and Reagents

• PCR reaction kit containing dNTPs (SibEnzymes, #N026), PCR
buffer and Taq Polymerase (SibEnzymes, #E331).

4.3.3 Procedure
1. Pick Lys+ or Ura+ colonies from Sc-Lys dropout and Sc-Ura dropout
plate, respectively, and streak for singles to isolate single colonies.
2. Take the singles from last step and patch them on YEPD.
3. Replica plate these patches on Sc-Leu and Sc-Ade to confirm that the
colonies have completed BIR repair. (They should be Ade+ Leu).
4. To amplify the mutant Ura+ or Lys+ alleles, use colony PCR.
5. A 50-μL PCR reaction is set up for each isolate and a small amount of
cells are scooped using a sterile toothpick and resuspended in the PCR
reaction mixture.
The primers used to amplify the mutant fragment from Lys+ BIR iso-
lates are:
FP: 50 -GTTCGTACCCCTCTCGAGAATA-30 and
RP: 50 -ATTTGAGGCAAATTTTTCGTTCCAA-30
The primers used to amplify the mutant fragment from Ura+ BIR
isolates are:
FP: 50 -GTGTGCTTCATTGGATGTTCGTAC-30 and
RP: 50 -AAAAGGCCTCTAGGTTCCTTTGTT30
The PCR reaction condition used for colony PCR is:

a. Initial denaturation 95°C–2 min

b. Denaturation 95°C–30 s
c. Annealing 55°C–1 min
d. Extension 72°C–1 min
e. Repeat steps b, c, d 30–40 times
f. Final extension 72°C–10 min

6. Once the PCR fragments are obtained, they are sent to the DNA
sequencing facility along with the following sequencing primers.
Sequencing primer for Lys+ BIR isolates: 50 -GTTCGTACCCCT
CTCGAGAATA-30
Sequencing primer for Ura+ BIR isolates: 50 -GTTCGTACCCCT
CTCGAGAATA-30 .
Investigation of Break-Induced Replication in Yeast 185

4.3.4 Notes
1. Be careful not to scoop out too many cells for colony PCR. The cellular
debris interferes with PCR amplification.

4.4 Sequencing of URA3 and LYS2 Sequences Located on Either

Donor or Recipient Chromosomes
Following BIR, the repair outcomes contain two copies of the reporter gene
(URA3 or LYS2). When mutation occurs during BIR, only one copy of the
reporter contains mutation, while another one does not. Here, we describe
the method allowing to determine which copy of chromosome III (the
donor or the recipient (the BIR product)) contains mutation.

4.4.1 Equipment
• 68°C water bath
• 40°C water bath
• CHEF-DRII apparatus (Bio-Rad)
• Razor blades

4.4.2 Solutions and Reagents

• Beta-Agarase enzyme (Lonza, #BMA58005)
• 50 Lonza Beta-Agarase buffer (Lonza, #BMA58005)
• Ethidium bromide (Sigma, #1239-45-8)
• 5  TBE: 64 g Tris base (RPI, #77-86-1), 31 g boric acid (RPI,
#10043-35-3), 3.35 g EDTA disodium salt dihydrate (Fisher, BP120-1).
Start from the new lane here and insert the new bullet. Wash buffer:
10 mM Tris pH 7.5, 50 mM EDTA pH 7.5
• Low melting agarose (Fisher, #9012-36-6 (BP165-25))
• PCR reaction kit containing dNTPs (SibEnzymes, #N026), PCR
buffer, and Taq Polymerase (SibEnzymes, #E331)

4.4.3 Procedure
1. Take agarose plugs containing DNA isolated from cells undergoing
BIR repair and wash with wash buffer similar to what is described in
Section 3.2.3.
2. Make 3 L of 0.5 TBE by adding 300 mL of 5  TBE and 2.7 L of
MilliQ water to a 4-L beaker.
3. Equilibrate plugs in 0.5 TBE for 1 h.
4. Run CHEF (refer to Section 3.3 for details) using 1% low melting aga-
rose gel and 0.5 TBE.
186 Rajula Elango et al.

5. CHEF conditions: 6 V/cm, 41 h, initial switch time—29.7, final

switch time—30.9, 14°C.
6. After 41 h, stain the gel with 3 μL ethidium bromide per 100 mL of
0.5 TBE buffer for about an hour.
7. Using new razor blade excise donor and recipient chromosome bands
from the gel and incubate it 1 Lonza beta-agarase buffer for 1 h.
8. Remove buffer and add fresh 2 beta-agarase buffer for 1 h.
9. Decant the 2 beta-agarase buffer after the 1 h incubation.
10. Melt the gel at 68°C for 10 min.
11. Equilibrate the melted gel at 40°C for 5 min.
12. Add 1 μL of beta-agarase per 100 μL of gel and incubate at 40°C for
2 h.
13. The gel can be stored at 4°C until further use.
14. To PCR a desired fragment from either the donor chromosome III or
recipient chromosome III, add 2 μL of DNA to 50 μL of PCR reaction
mixture.

5. ANALYSIS OF BIR STRUCTURAL INTERMEDIATES

BY TWO-DIMENSIONAL GEL ELECTROPHORESIS
While previous methods described in this review allowed for the
detection of BIR products, 2D electrophoresis allows for the analysis of
the DNA intermediates as BIR progresses. This method involves two
separate electrophoresis steps described later. The first-dimension (1D) gel
electrophoresis separates samples on the basis of molecular weight, whereas
the second dimension separates DNA on the basis of both size and shape.
Here, we describe a modified procedure for 2D gel electrophoresis
(Brewer & Fangman, 1987) to separate the DNA intermediates formed
during BIR.

5.1 Preparation of Yeast Samples

5.1.1 Equipment
• Shaking incubator 30°C
• 50-mL conical tubes
• 4 L Erlenmeyer flask
• Hemocytometer (Hausser scientific, #3100)
• 500 mL centrifuge bottle
• Ultracentrifuge (Beckman Coulter J2-21 centrifuge or a similar one)
Investigation of Break-Induced Replication in Yeast 187

5.1.2 Solutions and Reagents

• Sc-Leu liquid dropout media: 2% D-glucose (RPI, #50-99-7), 0.67%
yeast nitrogen base without amino acids (US Biological, #Y2025),
0.087% Sc-Leu dropout amino acid mix, pH 5.5.
• YEP-raffinose: 3% raffinose (MP, #4010-022), 1% yeast extract (BD,
#212750), 2% peptone (BD, #211677) pH 5.5.
• 50 mM EDTA (Fisher Bioreagents, #BP120-1).
• 20% Galactose: 20 g of galactose (Sigma-Aldrich, #G0750, 59-23-4) dis-
solved in 100 mL MilliQ water and filter sterilized.
• 0.2 M EDTA pH 7.5: 400 mL of 0.5 M EDTA pH 7.5 in 600 mL of
MilliQ water.
• MilliQ water chilled on ice (4°C).
• 10% Sodium azide (Sigma-Aldrich, #26628-22-8).
• Liquid nitrogen.
• Nocodazole 10 mg/mL (US Biological, #N3000).

5.1.3 Procedure
1. Inoculate 50 mL Sc-Leu dropout media and incubate at 30°C for 24 h.
2. Transfer 20 mL of the inoculum into 850 mL of YEP-raffinose to a
final concentration of 1  106 cells/mL.
3. Incubate YEP-raffinose for 16 h at 30°C with constant shaking.
4. After 16 h calculate cell density. Cell density should reach
1  107 cells/mL.
5. Add 2.5 mL of 10% sodium azide to a 500-mL autoclaved cen-
trifuge bottle containing 81 mL of 0.2 M EDTA pH 7.5 frozen
in a slant.
6. Collect 0 h 2D sample (before addition of galactose) by collecting
250 mL of log-phase culture (a total of 109 cells), into the centrifuge
bottle containing sodium azide and EDTA slants.
7. Shake bottles (like a martini) until the EDTA dislodges from the side of
the bottle.
8. Spin bottles at 6000 rpm for 5 min at 4°C and discard supernatant.
9. Resuspend the pellet in 10 mL of cold sterile H2O and transfer into pre-
chilled 50 mL conical tube.
10. Spin down at 6000 rpm for 5 min.
11. Decant supernatant and freeze pellet in liquid nitrogen for approxi-
mately 2 min and store in 80C.
12. Take 50 mL sample for a 0-h time point in 50 mL conical tube (for
CHEF analysis similar to Section 3.3).
188 Rajula Elango et al.

a. Spin down and discard supernatant.

b. Rinse with 50 mL 50 mM EDTA pH 7.5 containing 0.5 mL 10%
sodium azide.
c. Spin down at 3000 rpm at 4°C. Decant supernatant.
d. Store in 80°C until use.
13. Add 60 mL of 20% galactose to the remaining culture from step 6 (to a
working concentration of 2%).
14. Three hours after addition of galactose, add 1.2 mL nocodazole to the
final concentration of 0.015 mg/mL.
15. At 7 h after addition of galactose, collect 250 mL of culture following
steps 5–11 to collect the 2D samples. These samples can be used for fur-
ther 2D analysis.

5.1.4 Notes
1. In parallel with the samples for 2D, samples are always collected for
CHEF to detect the presence of final BIR product (see Section 3.3).
2. Nocodazole prevents cell progression into the cell cycle after BIR repair.
3. The samples are always placed on ice (until mentioned otherwise).

5.2 DNA Cross-linking

5.2.1 Equipment
• DNA cross-linker wavelength 365 nm (Stratagene Stratalinker UV 2400
or similar model)
• Tabletop centrifuge for spinning 50-mL conical tubes (Beckman Coul-
ter Allegra 25R or similar model)
• Petri dishes (100  15 mm)
• 10-mL serological pipettes
• 50-mL conical tubes
• 15-mL conical tubes

5.2.2 Solutions and Reagents

• Cross-link wash buffer: 50 mM Tris and 50 mM EDTA in ddH2O
• Psoralen solution: 4.8 mg of psoralen (Sigma-Aldrich, #T6137-500MG)
in 9 mL of 200 proof ethanol (Deacon, #64-17-5)
• Cross-link buffer: 2.8 mL of psoralen solution in 11.2 mL of cross-link
wash buffer

5.2.3 Procedure
1. Thaw the frozen pellet on ice for 1 h (keep cold throughout the
protocol).
Investigation of Break-Induced Replication in Yeast 189

2. While thawing the samples make cross-link wash buffer:

Dissolve 4.8 mg of psoralen in 9 mL of 200 proof ethanol by constant
vortexing. Psoralen may take up to 1 h to dissolve.
3. Make fresh cross-link buffer by adding 2.8 mL of psoralen solution to
11.2 mL of cross-link wash buffer and resuspend the pellet by vortexing
with this buffer.
4. Prewarm cross-linking bulbs (monochromatic white light at wavelength
365 nm) for 2 min.
5. Spread a thin layer of the suspended pellet in cross-link buffer onto six
Petri dishes.
6. Place plates in the UV cross-linker at wavelength 365 nm and cross-link
for 4 min. After the 4 min swirl the cell suspension on the plate to expose
more cells. Repeat the cross-linking step twice more at 3 min cycles
(total of 10 min).
7. Wash Petri dishes with chilled cross-linking wash buffer and add to con-
ical tube and keep on ice.
8. Repeat wash two times till the Petri dish is clear of cells.
9. Spin down 6000 rpm for 4 min and discard supernatant.

5.2.4 Notes
1. Psoralen solution (ethanol and psoralen) should be wrapped in a paper
towel/aluminum foil due to psoralen’s light sensitivity and vortexed
for approximately 1 h to allow the psoralen to dissolve into solution.
2. Note, normally cross-link buffer will form a white precipitate instead of
being transparent.
3. Washing of Petri dishes done with serological pipette and automatic
pipetter to collect as many cells as possible.

5.3 Extraction of DNA

5.3.1 Equipment
• Tabletop vortex
• Phase-contrast microscope (Leica DMRBE trinocular research micro-
scope or similar model)
• 37°C water bath
• 0.5 mm diameter acid-washed glass beads (BioSpec products,
#11079105)
• 5.75 in. disposable Pasteur pipettes (Fisher, #13-678-20A)
• Round bottom 50 mL centrifuge tube (Thermo scientific, #3119-0050)
• Opti-seal tubes (Beckman Coulter, #361627)
190 Rajula Elango et al.

• 4°C ultracentrifuge with 50 mL round bottom tube rotor tabletop

(Beckman model J2-21 centrifuge or similar model)

5.3.2 Solutions and Reagents

• NIB solution: 17% glycerol (RPI, #56-81-5), 50 mM MOPS (RPI,
#1132-61-2), 150 mM Potassium acetate (RPI, #127-08-2), 2 mM
Magnesium chloride (Fisher Scientific, #7791-18-6), 500 μM
Spermidine (Sigma-Aldrich, #S2626-5G), 150 μM Spermine (Sigma-
Aldrich, #S3256-1G), filter sterilize
• TEN solution pH 8.0 : 50 mM Tris base, 50 mM EDTA, 100 mM NaCl,
filter sterilize
• 10% Sarkosyl (Fisher, #137-16-6)
• Proteinase K (RPI, #39450-01-6)
• Cesium chloride (Fisher, #7647-17-8)
• Hoechst 33258 dye 5 mg/mL (Life technologies, #23491-44-3)

5.3.3 Procedure
1. Resuspend pellet from Section 5.2.3 (step 9) with 4 mL NIB solution
(keep on ice).
2. Add 0.5 mm acid-washed glass beads (1/3 the volume of the pellet).
3. Vortex for 1 min and then place on ice for 1 min.
4. Repeat for 20 cycles then using phase-contrast microscope look for the
ratio of spheroplasts to unbroken yeast cells.
5. Repeat the vortexing cycles until 80% of cells are spheroplasts (look
like “ghost” cells).
6. Carefully transfer the cells from glass beads to a sterile 50-mL round
bottom centrifugation tube using Pasteur pipettes.
7. Wash beads at least twice with 4 mL NIB solution and transfer into the
round bottom centrifugation tube.
8. Spin cells suspended in NIB at 10,000 rpm for 25 min at 4°C.
9. Save pellet and discard the supernatant.
10. Gently resuspend the pellet using 1.5 mL of TEN solution. Gently stir
pellet with pipette tip.
11. Add 250 μL of 10% Sarkosyl to the suspended cells.
12. Add a scoop of proteinase K (0.005 g/sample).
13. Gently swirl the mixture to ensure Sarkosyl and proteinase K are evenly
mixed.
14. Incubate in 37°C incubator for 1 h (without shaking).
15. Centrifuge at 4°C 5000 rpm for 5 min.
Investigation of Break-Induced Replication in Yeast 191

16. Decant the supernatant into a 15-mL conical tube and make the vol-
ume up to 2.5 mL using TEN solution.
17. For 3.3 mL opti-seal tubes, weigh out 3.7 g of cesium chloride and
gently rock tube back and forth until cesium chloride is dissolved.
18. Add 70 μL of Hoechst 33,258 dye 5 mg/mL and gently rock conical
tube once.

5.3.4 Notes
1. Steps (1–7) should be kept on ice.
2. After step 6 do not vortex any samples. Vortexing can shear the DNA.
3. After second step of bead wash the solution should be clear. If it is not
wash one more time with 4 mL of NIB.
4. Resuspension of pellet into TEN should be done carefully by gently stir-
ring so as not to break the spheroplasts. Do not pipette up and down or
vortex.
5. For opti-seal tube of size 4.5 mL make up volume of TEN in step 16 to
4 mL and add 4.3 g cesium chloride at step 17. Finally, add 125 μL of
Hoechst stain 33258 dye in step 18.

5.4 DNA Separation and Collection

5.4.1 Equipment
• Analytical balance
• 1.5-mL microcentrifuge tubes
• 5.75 in. disposable Pasteur pipettes (Fisher, #13-678-20A)
• 3.5 mL opti-seal tubes (Beckman Coulter, #361627)
• 100,000 rpm ultracentrifuge at 20°C (Beckman Coulter Optima Max-E
tabletop Ultracentrifuge 100,000 rpm) with TLN100 rotor
• Benchtop UV imager
• Ring stand tube clamp
• 18-gauge needle (BD, #305185)
• 3 mL syringe (BD, #309657)
• 4°C refrigerator

5.4.2 Solutions and Reagents

• MilliQ water
• Isopropanol and MilliQ water mixture: 5:1 isopropanol to water mixture
• 70% ethanol in MilliQ water
192 Rajula Elango et al.

5.4.3 Procedure
1. Transfer the TEN and cesium chloride solution to a 3.3-mL opti-seal
tube and seal the tube with the plug.
2. Place balanced tubes in rotor.
3. Place gold cap spacer on top of tubes in rotor.
4. Grease the threads of the rotor screws and tighten the rotor screws to
120 lb–ft using a torque wrench.
5. Spin samples at 100,000 rpm at 20°C for 12 h.
6. Carefully remove the opti-seal tube and carry the tube to the dark room
as smoothly as possible. (Too much shaking will mix DNA layers).
7. Remove opti-seal plug and place the opti-seal tube in a ring stand tube
holder and set up the UV imager to visualize DNA stained by
Hoechst dye.
8. White gooey polysaccharide layer (probably trehalose) should be visible
without UV. Mark the location of this layer with a permanent marker.
9. While using face shield for protection, turn on UV imager where three
bands should fluoresce. The bottom should be the visible gooey poly-
saccharide band, the middle is genomic DNA, and the top band is
mitochondrial DNA.
10. Using an 18-gauge needle and syringe with the needle hole facing
upward, pierce through the side of the tube at the base of the chromo-
somal DNA layer.
11. Carefully remove the chromosomal DNA layer using the needle (do
this quickly and smoothly).
12. Remove needle from the syringe and transfer the DNA to a 15-mL
conical tube.
13. Chill mixture containing 5:1 isopropanol to H2O and 70% ethanol in
ice bucket.
14. Add equal volume of 5:1 isopropanol mixture to the collected DNA
sample. For instance, add 100 μL of 5:1 isopropanol mixture to
100 μL of extracted DNA.
15. Swirl the tube with a flick of the wrist then incubate on ice for 2 min.
16. Remove and discard the top phase of the mixture.
17. Repeat steps 14 through 16 two more times. (On the last wash remove
as much of top layer as possible).
18. Slowly add 2 the volume of chilled 70% ethanol to the tube and mix
carefully by a single quick swirling motion. (DNA will come out of
solution as a white precipitate in a middle phase).
19. Melt the tip of a Pasteur pipette to seal the end into a little ball.
Investigation of Break-Induced Replication in Yeast 193

20. DNA can be removed from the solution by touching the cooled melted
tip to the DNA clot (the DNA will stick to the melted tip), and then
DNA can be spooled on the stick.
21. Dip the DNA clot in 70% ethanol several times and air dry DNA pellet
by placing the Pasteur pipette in a rack DNA end up.
22. Place the tip of Pasteur pipette with the DNA clot attached into a
microcentrifuge tube filled with 100 μL MilliQ water.
23. Keep the tip of Pasteur pipette in the tube for half an hour to allow
DNA to dislodge from the tip.
24. Allow DNA clot to dissolve at 4°C in the microcentrifuge tube
overnight.

5.4.4 Notes
1. Opti-seal tube must be balanced to each other before the centrifugation
as accurately as possible.
2. Before inserting needle, be sure to move plunger up and down for
smoother pulling of the plunger.
3. Needle should be inserted at the base of the chromosomal DNA band
with the needle hole up to prevent the clogging of the needle.
4. Needle is removed, and DNA is transferred into 15-mL conical tube.
Removal of needle is critical to avoid breaking of DNA structures.
5. Residual DNA can be removed from the needle by one full push of the
syringe through the needle.
6. 5:1 isopropanol to MilliQ water is added to DNA to remove the
Hoechst dye.
7. Care should be taken throughout all the steps so as to not break DNA
structures that are formed.

5.5 DNA Digestion and Gel Separation in the First Dimension

5.5.1 Equipment
• 37°C water bath
• 20°C freezer
• 1.5-mL microcentrifuge tubes
• Tabletop microcentrifuge (LabNet International, Inc. #Spectrafuge
24D or similar model)
• Electrophoresis chamber (Thermo Scientific Owl A1 large gel system
#09528100 or similar model)
• Electrophoresis power pack (Bio-Rad PowerPac or similar model)
194 Rajula Elango et al.

5.5.2 Solutions and Reagents

• MilliQ water
• NEBuffer 3.1 (NEB, #B7203S)
• PstI 10 U/μL (NEB, #R0140S)
• Agarose (Sigma-Aldrich, #A0169)
• 70% ethanol in water
• 200 proof ethanol (Deacon, #64-17-5)
• 3 M Sodium acetate pH 5.5 (Fisher, #S210-500): Add 24.6 g sodium
acetate in 100 mL of MilliQ water, and pH using glacial acetic acid
(Fisher, #64-19-7)
• 5 TBE: 0.45 M Tris base, 0.45 M boric acid (RPI, #10043-35-3),
0.02 M EDTA
• Promega blue orange 6  loading dye (Promega, #G1881)

5.5.3 Procedure
1. Prepare enzyme digestion mix using the correct restriction enzyme.
The choice of enzyme depends on specific chromosomal location
where BIR is analyzed. PstI restriction enzyme is used here.
a. 100 μL dissolved DNA
b. 256 μL ddH2O
c. 40 μL NEBuffer 3.1
d. 4 μL PstI
2. Incubate 16 h at 37°C.
3. Add 40 μL of 3 M sodium acetate followed by 1 mL of cold 200 proof
ethanol to the digestion mix and invert once.
4. Incubate on ice for 30 min.
5. Spin in tabletop microcentrifuge at 13,000 rpm for 15 min.
6. Discard supernatant.
7. Wash DNA pellet with 70% EtOH.
8. Spin at 13,000 rpm for 5 min.
9. Carefully discard supernatant.
10. Dry the pellet on the bench with the tubes open until dry.
11. Add 6 μL 1 TBE and 6 μL 6 loading dye to the sample (do not
vortex).
12. Allow the sample to dissolve for at least an hour.
13. In 300 mL of 1 TBE make a 0.4% agarose gel (no ethidium bromide)
and pour with the tiny well comb (<2 mm wide comb).
14. Load samples carefully so as to not overflow the well and run at 55 V for
17 h (for optimal separation of 5 and 10 kb DNA fragments).
Investigation of Break-Induced Replication in Yeast 195

5.5.4 Notes
1. Restriction enzymes were chosen based on the restriction map of the
area where BIR intermediates are expected to form. The fragment size
is usually chosen to be between 5 and 6 kb in size.
2. The well for the first dimension is to be as small as possible. This will
allow the DNA to migrate through the gel in as thin a band as possible.
The well has to be large enough so that the sample will not overflow.
3. The first dimension is run without ethidium bromide. Running with
ethidium bromide may lead to issues in DNA migration.

5.6 Two-Dimensional Gel Electrophoresis: Second Dimension

5.6.1 Equipment
• Large electrophoresis box with buffer pump (Thermo Scientific Owl A3
electrophoresis chamber with Bio-Rad variable speed pump or similar
model)
• Benchtop UV transilluminator
• Razor blades

5.6.2 Solutions and Reagents

• 5 TBE: 0.45 M Tris base, 0.45 M boric acid, 0.02 M EDTA
• Agarose (Sigma-Aldrich, #A0169)
• Ethidium bromide 10 mg/mL (Fisher, #15-585-011)

5.6.3 Procedure
1. After running the first dimension, stain gel with ethidium bromide (3 μL
per 100 mL of buffer).
2. Using a transilluminator to excise the separated DNA from the gel in a
thin strip. Excise the gel containing all DNA molecules larger than 5 kb.
3. Place gel strip with DNA horizontally in the large 2D gel tray.
4. Make 5 L of 1 TBE.
5. Using 1 TBE make 1 L of 1.2% agarose gel.
6. Cool remaining 4 L of 1 TBE to 4°C and add 120 μL of ethidium
bromide.
7. When agarose cools to 65°C, add 30 μL of ethidium bromide, mix, and
pour into gel box around the first-dimension strips of gel.
8. When gel solidifies, place gel into gel box with prechilled 1 TBE.
9. Run gel for 12 h at 340 V while in the cold room while pumping buffer
through the gel box.
196 Rajula Elango et al.

5.6.4 Notes
1. Fragments of size 5 kb (the region flanked by two PstI restriction sites)
and larger fragments (until the well) are excised. Other areas of chromo-
somes can be studied using different restriction enzymes. Excision of this
band would have to be adjusted accordingly for different restriction frag-
ment sizes.
2. Two gel slices will fit at the top of the gel box and two more will fit at
halfway down the gel box for a total of four samples.
3. Do not pour the 1.2% agarose gel until it is 65°C so as to not denature the
DNA structures within the first-dimension gel slice.

5.7 Capillary Transfer and Hybridization

5.7.1 Equipment
• Glass baking dish
• Glass plate
• Whatman paper 45  45 cm
• Scott C-fold paper towels (Fisher, #06-666-32B)
• DNA cross-linker wavelength 254 nm (Stratagene Stratalinker UV 2400
or similar model)
• 20  25 cm GE Phosphor screen (#GE28-9564-75)
• Phosphorimager (GE Typhoon FLA 7000 cat #28983618 or similar
model)
• Geiger counter (Ludlum 5000 or similar model)

5.7.2 Solutions and Reagents

• 0.2 M HCl: 21 mL of concentrated HCl (Fisher, #A144-500) into
979 mL MilliQ water.
• Alkaline solution: 20 g NaOH (RPI, #1310-73-2), 87.7 g NaCl (RPI,
#7647-14-5) in 1 L of MilliQ water.
• Neutralization solution: 87.7 g NaCl (RPI, #7647-14-5), 121.4 g Tris
base (RPI, #77-86-1) in 1 L of MilliQ water, pH 7.
• 20 SSC pH 7: 175.3 g NaCl, 88.2 g sodium citrate, 900 mL MilliQ
water.
• MilliQ water.
• 1 TBE: Dilute 5 TBE by adding 200 mL 5 TBE in 1 L of MilliQ
water.
• 1% agarose gel: 1 g of agarose (Sigma-Aldrich, #A0169) in 100 mL of
1 TBE.
Investigation of Break-Induced Replication in Yeast 197

• QIAGEN gel extraction kit: (QIAGEN, #28704).

• Amersham Hybond-N+ charged transfer membrane (GE Healthcare,
#RPN303B).
• PerfectHyb Plus hybridization buffer (Sigma-Aldrich, #H7033).
• Prime-it RmT Random primer kit (Agilent, #300392).
• P32 radiolabeled dCTP (Perkin Elmer, #BLU513Z250).
• Low stringency wash buffer: 80 mL 20 SSC, 4 mL 20% SDS (Fisher,
#BP166-5) made up to 800 mL MilliQ water.

5.7.3 Procedure
1. Cut the gel in half (separating top from bottom) and image using UV
transilluminator with camera.
2. Submerge gel in 0.25 M HCl for 30 min and keep with constant gentle
shaking.
3. Submerge gel in alkaline solution and keep for 30 min with constant
gentle shaking.
4. Submerge gel in neutralization solution and keep for 30 min with con-
stant gentle shaking.
5. Transfer via capillary transfer methods described in Sambrook and
Russell (2006). Refer to Section 3.4.3 for further details.
6. Cross-link membrane in UV cross-linker at 254 nm wavelength light.
7. DNA probe specific to the chromosome III position located 24 kb
centromere distal from MAT is made using AM1003 genomic DNA
as a template and the following primers:
FP: 50 -AAGGGAGCAAACTGTCTGTC-30
RP: 50 -GGTACGGTCTACAGGCAAGG-30
8. Run the PCR probe fragment on a 1% agarose 1 TBE gel and excise
the fragment.
9. Using the QIAGEN Gel extraction kit extract the DNA for making the
probe.
10. Resuspend and dissolve the contents of one tube of the RmT Random
primer kit in 37 μL of MilliQ water and transfer to a 1.5-mL
microcentrifuge tube.
11. Add 5 μL (70 ng) of the gel-extracted PCR probe fragment.
12. Incubate RmT Random primer kit and probe fragment mixture at
100°C for 5 min. Be sure to add a microcentrifuge tube locker to keep
the lid closed.
13. After incubation period, place reaction mixture on ice for 1 min.
198 Rajula Elango et al.

14. Add 5 μL of P32-dCTP to the cooled reaction mixture, and add 3 μL of

magenta polymerase from the RmT Random primer kit, and incubate
at 37°C dry bath for 10 min.
15. Add 2 μL of stop mix from the RmT Random primer kit to stop the
reaction.
16. Add 200 μL of MilliQ water to the reaction mixture and denature at
100°C dry bath for 10 min and snap cool on ice for 1 min.
17. Prehybridize the cross-linked membrane from step 6 in 25 mL of Per-
fectHyb Plus hybridization buffer at 65°C for at least 10 min.
18. Hybridize the membrane at 65°C using 25 mL of the PerfectHyb Plus
hybridization buffer for 24 h.
19. Wash the membrane using 65°C low stringency wash buffer in an open
tray. Gently rock the wash buffer and membrane for 10 min. Discard
waste under the guidelines of your radiation safety officer.
20. Repeat wash two more times or until background is washed away, as
being checked using a Geiger counter.
21. Using 20  25 cm GE Phosphor screen, expose membrane for 5 days
and image on a Typhoon Phosphorimager FLA7000.

5.7.4 Notes
1. Gel is cut in half to fit in the glass baking dish. If there is a glass baking dish
large enough then cutting may not be necessary.
2. Machine pressure transfer could be an alternative to capillary DNA trans-
fer to membranes.

6. ALTERNATIVE METHOD OF GENOMIC DNA

PREPARATION FOR 2D ANALYSIS BY
SPHEROPLASTING CELLS
6.1 Plug Preparation
6.1.1 Equipment
• Hemocytometer (Hausser scientific, #3100)
• Plug mold: (Bio-Rad, #1703713)
• Phase-contrast microscope (Leica DMRBE trinocular research micro-
scope or similar model)
• 30°C incubator
• Tube mixer (Fisher, #120529060)
Investigation of Break-Induced Replication in Yeast 199

6.1.2 Solutions and Reagents

• 1 TE: 1 M Tris (RPI, #77-86-1) and 0.1 M EDTA (Fisher Bio-
reagents, #BP120-1), pH 7.5
• Digestion buffer: 1 M sorbitol (RPI, #50-70-4) and 0.1 M EDTA pH
7.5
• Lyticase 5 mg/mL (Sigma-Aldrich, #L2524)
• TEN solution pH 8: 50 mM Tris base, 50 mM EDTA, 100 mM NaCl
(RPI, #7647-14-5)
• Low melting agarose (Fisher, #9012-36-6)
• Incubation buffer: 10 mM Tris, 50 mM EDTA, 0.5% Sarkosyl (Fisher
Bioreagents, #137-16-6), 0.005 g/mL proteinase K (RPI, #39450-
01-6) added fresh

6.1.3 Procedure
1. Spin down cells at 3000 rpm for 5 min at 4°C following UV cross-
linking by Psoralen (see Section 5.2 for details) and keep on ice.
2. Resuspend cells in 1 TE and using a hemocytometer count the num-
ber of cells.
3. Determine cell concentration and add 2.5  109 cells per plug.
4. Take cells and spin down to remove excess 1 TE buffer. Resuspend
cells in 500 μL digestion buffer.
5. Add 10 μL of lyticase (5 mg/mL) to the cells and incubate at 37°C for
1 h. Note that lyticase stock is made in digestion buffer.
6. Use phase-contrast microscope to check for spheroplasts. Cells must be
approximately 80% ghosts. If cells remain undisrupted, add another
15 μL of lyticase and incubate for another hour.
7. Following lyticase digestion, spin down the cells at 13,000 rpm for
1 min.
8. Wash cells in 750 μL of TEN solution.
9. Repeat the wash with TEN solution two times.
10. Place cells in a tube mixer to loosen the cells (do not vortex).
11. Take 50 μL of loosened cells and place it in a 50°C water bath to
equilibrate (do not leave it for too long or it will degrade the
spheroplasts).
12. Add 30 μL of 2% molten low melting agarose to the cells and mix the
cells using the pipette approximately three times.
13. Add this mixture (cell + agarose) to the mold and allow it to solidify for
30 min.
200 Rajula Elango et al.

14. After plugs solidify, transfer the plugs into a 50-mL conical tube con-
taining 0.005 mg Proteinase K per 10 mL of incubation buffer. Incu-
bation buffer and incubate at 30°C overnight.

6.2 1D Gel Electrophoresis

6.2.1 Equipment
• 50-mL conical tubes
• Electrophoresis chamber (Thermo Scientific Owl A1 large gel system
#09528100 or similar model)
• Electrophoresis power pack (Bio-Rad PowerPac or similar model)

6.2.2 Solutions and Reagents

• 1 TE: 1 M Tris (RPI, #77-86-1) and 0.1 M EDTA (Fisher Bio-
reagents, #BP120-1), pH 7.5
• Isopropanol (Fisher, #67-63-0)
• 100 μM Phenylmethane sulfonyl fluoride (PMSF): 17.4 g of PMSF
(RPI, #329-98-6) in 100 mL isopropanol
• MilliQ water
• Buffer O restriction buffer: (Thermo Fisher Scientific, #B05)

6.2.3 Procedure
1. Cut the plugs to desired size (approximately 2–3 mm in width).
2. Place the plug in 1 TE (30 mL) and leave it at 25°C for 16 h.
3. Remove 1 TE and replace 30 mL of fresh 1  TE and incubate for 1 h
at 25°C.
4. Discard 1 TE and replace with fresh 1  TE containing100mM
PMSF and incubate for 1 h at 25°C.
5. Wash for 1 h with MilliQ water.
6. Transfer plug from 50-mL conical tube to 2 mL sterile microcentrifuge
tubes.
7. Submerge plugs in 2 restriction buffer (buffer O) and incubate for 1 h
at 25°C.
8. Remove 2 restriction buffer and add 1 restriction buffer with 10 μL
of restriction enzyme. Incubate at 37°C for 16 h.
9. After restriction digestion, equilibrate the plugs in 1 TBE for 1 h at
25°C.
10. Insert the plug into the well of a set 1% agarose gel and seal the well with
the molten agarose to prevent plugs from shifting during
electrophoresis.
Investigation of Break-Induced Replication in Yeast 201

11. Run the 1D and 2D as previously described earlier in Section 5.5.3

through Section 5.6.3.

6.2.4 Notes
• Enzymes using the Orange buffer from Thermo Fisher Scientific are the
most compatible with in-plug digestion.

7. CONCLUSIONS
The experimental system and methods described in this review allow
to study BIR initiated by a site-specific HO endonuclease in yeast. This sys-
tem is convenient for identification of proteins that are involved in BIR and
for investigation of their role in BIR. This system can be used to determine
the frequency and efficiency of BIR by using genetic and physical (molec-
ular biology) methods, for exploring the mechanism of BIR and its kinetics.
In addition, this system allows to investigate mutagenesis and chromosomal
rearrangements associated with BIR.

ACKNOWLEDGMENT
The research in Malkova lab is supported by R01GM084242 grant from NIGMS.

REFERENCES
Bhowmick, R., Minocherhomji, S., & Hickson, I. D. (2016). RAD52 facilitates mitotic
DNA synthesis following replication stress. Molecular Cell, 64(6), 1117–1126. https://
[Link]/10.1016/[Link].2016.10.037.
Brewer, B. J., & Fangman, W. L. (1987). The localization of replication origins on ARS plas-
mids in S. cerevisiae. Cell, 51(3), 463–471.
Cho, N. W., Dilley, R. L., Lampson, M. A., & Greenberg, R. A. (2014). Interchromosomal
homology searches drive directional ALT telomere movement and synapsis. Cell, 159(1),
108–121. [Link]
Church, G. M., & Gilbert, W. (1984). Genomic sequencing. Proceedings of the National Acad-
emy of Sciences of the United States of America, 81(7), 1991–1995.
Costantino, L., Sotiriou, S. K., Rantala, J. K., Magin, S., Mladenov, E., Helleday, T., et al.
(2014). Break-induced replication repair of damaged forks induces genomic duplications
in human cells. Science, 343(6166), 88–91. [Link]
Davis, A. P., & Symington, L. S. (2004). RAD51-dependent break-induced replication in
yeast. Molecular and Cellular Biology, 24(6), 2344–2351.
Deem, A., Barker, K., Vanhulle, K., Downing, B., Vayl, A., & Malkova, A. (2008). Defec-
tive break-induced replication leads to half-crossovers in Saccharomyces cerevisiae. Genetics,
179(4), 1845–1860. [Link]
Deem, A., Keszthelyi, A., Blackgrove, T., Vayl, A., Coffey, B., Mathur, R., et al. (2011).
Break-induced replication is highly inaccurate. PLoS Biology, 9(2), e1000594. https://
[Link]/10.1371/[Link].1000594.
202 Rajula Elango et al.

Dilley, R. L., Verma, P., Cho, N. W., Winters, H. D., Wondisford, A. R., &
Greenberg, R. A. (2016). Break-induced telomere synthesis underlies alternative telo-
mere maintenance. Nature, 539(7627), 54–58. [Link]
Hashimoto, Y., & Costanzo, V. (2011). Studying DNA replication fork stability in Xenopus
egg extract. Methods in Molecular Biology, 745, 437–445. [Link]
61779-129-1_25.
Lydeard, J. R., Jain, S., Yamaguchi, M., & Haber, J. E. (2007). Break-induced replication and
telomerase-independent telomere maintenance require Pol32. Nature, 448(7155),
820–823. [Link]
Malkova, A., Ivanov, E. L., & Haber, J. E. (1996). Double-strand break repair in the absence
of RAD51 in yeast: A possible role for break-induced DNA replication. Proceedings of the
National Academy of Sciences of the United States of America, 93(14), 7131–7136.
Malkova, A., Naylor, M. L., Yamaguchi, M., Ira, G., & Haber, J. E. (2005). RAD51-
dependent break-induced replication differs in kinetics and checkpoint responses from
RAD51-mediated gene conversion. Molecular and Cellular Biology, 25(3), 933–944.
[Link]
Mehta, A., & Haber, J. E. (2014). Sources of DNA double-strand breaks and models of
recombinational DNA repair. Cold Spring Harbor Perspectives in Biology, 6(9). .
a016428[Link]
Minocherhomji, S., Ying, S., Bjerregaard, V. A., Bursomanno, S., Aleliunaite, A., Wu, W.,
et al. (2015). Replication stress activates DNA repair synthesis in mitosis. Nature,
528(7581), 286–290. [Link]
Morrow, D. M., Connelly, C., & Hieter, P. (1997). “Break copy” duplication: A model for
chromosome fragment formation in Saccharomyces cerevisiae. Genetics, 147(2), 371–382.
Rodgers, K., & McVey, M. (2016). Error-prone repair of DNA double-strand breaks. Journal
of Cellular Physiology, 231(1), 15–24. [Link]
Saini, N., Ramakrishnan, S., Elango, R., Ayyar, S., Zhang, Y., Deem, A., et al. (2013).
Migrating bubble during break-induced replication drives conservative DNA synthesis.
Nature, 502(7471), 389–392. [Link]
Sakofsky, C. J., Ayyar, S., Deem, A. K., Chung, W. H., Ira, G., & Malkova, A. (2015).
Translesion polymerases drive microhomology-mediated break-induced replication
leading to complex chromosomal rearrangements. Molecular Cell, 60(6), 860–872.
[Link]
Sakofsky, C. J., & Malkova, A. (2017). Break induced replication in eukaryotes: Mechanisms,
functions, and consequences. Critical Reviews in Biochemistry and Molecular Biology, 52(4),
395–413. [Link]
Sakofsky, C. J., Roberts, S. A., Malc, E., Mieczkowski, P. A., Resnick, M. A.,
Gordenin, D. A., et al. (2014). Break-induced replication is a source of mutation clusters
underlying kataegis. Cell Reports, 7(5), 1640–1648. [Link]
celrep.2014.04.053.
Sambrook, J., & Russell, D. W. (2006). Southern blotting: Capillary transfer of DNA to
membranes. CSH Protocols, 2006(1). [Link]
Shcherbakova, P. V., & Pavlov, Y. I. (1996). 30 !50 Exonucleases of DNA polymerases epsi-
lon and delta correct base analog induced DNA replication errors on opposite DNA
strands in Saccharomyces cerevisiae. Genetics, 142(3), 717–726.
Smith, C. E., Lam, A. F., & Symington, L. S. (2009). Aberrant double-strand break repair
resulting in half crossovers in mutants defective for Rad51 or the DNA polymerase delta
complex. Molecular and Cellular Biology, 29(6), 1432–1441. [Link]
MCB.01469-08.
Sotiriou, S. K., Kamileri, I., Lugli, N., Evangelou, K., Da-Re, C., Huber, F., et al. (2016).
Mammalian RAD52 functions in break-induced replication repair of collapsed DNA
Investigation of Break-Induced Replication in Yeast 203

replication forks. Molecular Cell, 64(6), 1127–1134. [Link]

molcel.2016.10.038.
Teng, S. C., Chang, J., McCowan, B., & Zakian, V. A. (2000). Telomerase-independent
lengthening of yeast telomeres occurs by an abrupt Rad50p-dependent, Rif-inhibited
recombinational process. Molecular Cell, 6(4), 947–952.
Teng, S. C., & Zakian, V. A. (1999). Telomere-telomere recombination is an efficient bypass
pathway for telomere maintenance in Saccharomyces cerevisiae. Molecular and Cellular Biol-
ogy, 19(12), 8083–8093.
Tran, H. T., Keen, J. D., Kricker, M., Resnick, M. A., & Gordenin, D. A. (1997). Hyper-
mutability of homonucleotide runs in mismatch repair and DNA polymerase proofread-
ing yeast mutants. Molecular and Cellular Biology, 17(5), 2859–2865.
Vasan, S., Deem, A., Ramakrishnan, S., Argueso, J. L., & Malkova, A. (2014). Cascades of
genetic instability resulting from compromised break-induced replication. PLoS Genetics,
10(2), e1004119. [Link]

FURTHER READING
Donnianni, R. A., & Symington, L. S. (2013). Break-induced replication occurs by conser-
vative DNA synthesis. Proceedings of the National Academy of Sciences of the United States of
America, 110(33), 13475–13480. [Link]
Wilson, M. A., Kwon, Y., Xu, Y., Chung, W. H., Chi, P., Niu, H., et al. (2013). Pif1 heli-
case and Poldelta promote recombination-coupled DNA synthesis via bubble migration.
Nature, 502(7471), 393–396. [Link]