Sgs1 and exo1 redundantly inhibit break-induced replication and de novo telomere addition at broken chromosome ends.
ABSTRACT In budding yeast, an HO endonuclease-inducible double-strand break (DSB) is efficiently repaired by several homologous recombination (HR) pathways. In contrast to gene conversion (GC), where both ends of the DSB can recombine with the same template, break-induced replication (BIR) occurs when only the centromere-proximal end of the DSB can locate homologous sequences. Whereas GC results in a small patch of new DNA synthesis, BIR leads to a nonreciprocal translocation. The requirements for completing BIR are significantly different from those of GC, but both processes require 5' to 3' resection of DSB ends to create single-stranded DNA that leads to formation of a Rad51 filament required to initiate HR. Resection proceeds by two pathways dependent on Exo1 or the BLM homolog, Sgs1. We report that Exo1 and Sgs1 each inhibit BIR but have little effect on GC, while overexpression of either protein severely inhibits BIR. In contrast, overexpression of Rad51 markedly increases the efficiency of BIR, again with little effect on GC. In sgs1Delta exo1Delta strains, where there is little 5' to 3' resection, the level of BIR is not different from either single mutant; surprisingly, there is a two-fold increase in cell viability after HO induction whereby 40% of all cells survive by formation of a new telomere within a few kb of the site of DNA cleavage. De novo telomere addition is rare in wild-type, sgs1Delta, or exo1Delta cells. In sgs1Delta exo1Delta, repair by GC is severely inhibited, but cell viability remains high because of new telomere formation. These data suggest that the extensive 5' to 3' resection that occurs before the initiation of new DNA synthesis in BIR may prevent efficient maintenance of a Rad51 filament near the DSB end. The severe constraint on 5' to 3' resection, which also abrogates activation of the Mec1-dependent DNA damage checkpoint, permits an unprecedented level of new telomere addition.
- SourceAvailable from: nyu.edu[show abstract] [hide abstract]
ABSTRACT: Homologous recombination (HR) serves to eliminate deleterious lesions, such as double-stranded breaks and interstrand crosslinks, from chromosomes. HR is also critical for the preservation of replication forks, for telomere maintenance, and chromosome segregation in meiosis I. As such, HR is indispensable for the maintenance of genome integrity and the avoidance of cancers in humans. The HR reaction is mediated by a conserved class of enzymes termed recombinases. Two recombinases, Rad51 and Dmc1, catalyze the pairing and shuffling of homologous DNA sequences in eukaryotic cells via a filamentous intermediate on ssDNA called the presynaptic filament. The assembly of the presynaptic filament is a rate-limiting process that is enhanced by recombination mediators, such as the breast tumor suppressor BRCA2. HR accessory factors that facilitate other stages of the Rad51- and Dmc1-catalyzed homologous DNA pairing and strand exchange reaction have also been identified. Recent progress on elucidating the mechanisms of action of Rad51 and Dmc1 and their cohorts of ancillary factors is reviewed here.Annual Review of Biochemistry 08/2008; 77:229-57. · 27.68 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Double-strand breaks (DSBs) occur frequently during cell growth. Due to the presence of repeated sequences in the genome, repair of a single DSB can result in gene conversion, translocation, deletion or tandem duplication depending on the mechanism and the sequence chosen as partner for the recombinational repair. Here, we study how yeast cells repair a single, inducible DSB when there are several potential donors to choose from, in the same chromosome and elsewhere in the genome. We systematically investigate the parameters that affect the choice of mechanism, as well as its genetic regulation. Our results indicate that intrachromosomal homologous sequences are always preferred as donors for repair. We demonstrate the occurrence of a novel tri-partite repair product that combines ectopic gene conversion and deletion. In addition, we show that increasing the distance between two repeated sequences enhances the dependence on Rad51 for colony formation after DSB repair. This is due to a role of Rad51 in the recovery from the checkpoint signal induced by the DSB. We suggest a model for the competition between the different homologous recombination pathways. Our model explains how different repair mechanisms are able to compensate for each other during DSB repair.Nucleic Acids Research 07/2009; 37(15):5081-92. · 8.28 Impact Factor
Article: Recombination proteins in yeast.[show abstract] [hide abstract]
ABSTRACT: The process of homologous recombination promotes error-free repair of double-strand breaks and is essential for meiosis. Central to the process of homologous recombination are the RAD52 group genes (RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2), most of which were identified by their requirement for the repair of ionizing radiation-induced DNA damage in Saccharomyces cerevisiae. The Rad52 group proteins are highly conserved among eukaryotes. Recent studies showing defects in homologous recombination and double-strand break repair in several human cancer-prone syndromes have emphasized the importance of this repair pathway in maintaining genome integrity. Herein, we review recent genetic, biochemical, and structural analyses of the genes and proteins involved in recombination.Annual Review of Genetics 02/2004; 38:233-71. · 17.44 Impact Factor
Sgs1 and Exo1 Redundantly Inhibit Break-Induced
Replication and De Novo Telomere Addition at Broken
John R. Lydeard¤, Zachary Lipkin-Moore, Suvi Jain, Vinay V. Eapen, James E. Haber*
Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts, United States of America
In budding yeast, an HO endonuclease-inducible double-strand break (DSB) is efficiently repaired by several homologous
recombination (HR) pathways. In contrast to gene conversion (GC), where both ends of the DSB can recombine with the
same template, break-induced replication (BIR) occurs when only the centromere-proximal end of the DSB can locate
homologous sequences. Whereas GC results in a small patch of new DNA synthesis, BIR leads to a nonreciprocal
translocation. The requirements for completing BIR are significantly different from those of GC, but both processes
require 59 to 39 resection of DSB ends to create single-stranded DNA that leads to formation of a Rad51 filament required
to initiate HR. Resection proceeds by two pathways dependent on Exo1 or the BLM homolog, Sgs1. We report that Exo1
and Sgs1 each inhibit BIR but have little effect on GC, while overexpression of either protein severely inhibits BIR. In
contrast, overexpression of Rad51 markedly increases the efficiency of BIR, again with little effect on GC. In sgs1D exo1D
strains, where there is little 59 to 39 resection, the level of BIR is not different from either single mutant; surprisingly, there
is a two-fold increase in cell viability after HO induction whereby 40% of all cells survive by formation of a new telomere
within a few kb of the site of DNA cleavage. De novo telomere addition is rare in wild-type, sgs1D, or exo1D cells. In sgs1D
exo1D, repair by GC is severely inhibited, but cell viaiblity remains high because of new telomere formation. These data
suggest that the extensive 59 to 39 resection that occurs before the initiation of new DNA synthesis in BIR may prevent
efficient maintenance of a Rad51 filament near the DSB end. The severe constraint on 59 to 39 resection, which also
abrogates activation of the Mec1-dependent DNA damage checkpoint, permits an unprecedented level of new telomere
Citation: Lydeard JR, Lipkin-Moore Z, Jain S, Eapen VV, Haber JE (2010) Sgs1 and Exo1 Redundantly Inhibit Break-Induced Replication and De Novo Telomere
Addition at Broken Chromosome Ends. PLoS Genet 6(5): e1000973. doi:10.1371/journal.pgen.1000973
Editor: Gregory P. Copenhaver, The University of North Carolina at Chapel Hill, United States of America
Received February 1, 2010; Accepted April 29, 2010; Published May 27, 2010
Copyright: ? 2010 Lydeard et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: NIH grants GM20056 and GM76020. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
¤ Current address: Department of Pathology, Harvard Medical School, Boston, Massachusetts, United States of America
DNA double-strand breaks (DSBs) are generated by normal
cellular processes including DNA replication or by exposure to
DNA damaging agents or ionizing radiation. To maintain cell
viability and preserve genomic integrity, cells employ multiple
pathways of homologous recombination (HR) to repair DSBs
[1–4]. A key initial step in HR is 59 to 39 resection of DSB ends to
create single-stranded DNA (ssDNA) that recruits formation of a
Rad51 filament, which engages in a search for homologous
sequences. The predominant HR pathway is gene conversion
(GC), a conservative mechanism in which both ends of the DSB
share homologous sequences on a sister chromatid, a homologous
chromosome, or at an ectopic location. Rad51-mediated strand
invasion of the 39-ended ssDNA allows the initiation of new DNA
synthesis to copy a short region of the template and patch up the
DSB. When only one DSB end shares homology to a template
elsewhere in the genome, a less-efficient HR mechanism, break-
induced replication (BIR), can be used to repair the break [5,6].
In BIR, recombination is used to establish an uni-directional
replication fork that can copy the template DNA to the end of the
chromosome. If homologous sequences are located ectopically,
BIR will result in formation of a non-reciprocal translocation with
loss of the distal part of the broken chromosome and may be a
significant source of gross chromosomal rearrangements (GCRs)
and genomic instability . BIR requires the non-essential
subunit of the Pold polymerase, Pol32, and all of the essential
replication machinery except those excluisvely required for
formation of the pre-replicative complex [8,9]. BIR can be used
to restart stalled or collapsed replication forks during DNA
replication  and elongate telomeres in the absence of
telomerase . An alternative way to repair the DSB is through
de novo telomere addition through the action of telomerase
[11–13], although this is a very inefficient process that is
improved by elimination of the Pif1 helicase .
Genetic and in vivo molecular biological experiments indicate
that the early steps of GC and BIR are shared [15–17].
Following the generation of a DSB, the Tel1/ATM kinase is
loaded at sites of DSBs in an Mre11-Rad50-Xrs2 (MRX)-
dependent manner [18,19]. Tel1 in turn phosphorylates MRX
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[20,21]. The Sae2 and MRX proteins mediate the initial
resection [22,23] which is continued via two alternate pathways,
one using the Exo1 nuclease and the other employing the
multifunctional RecQ family helicase Sgs1, in concert with
Top3, Rmi1 and the essential helicase/nuclease Dna2 [22–24].
DNA resection is also essential to activate the Mec1-dependent
DNA damage checkpoint kinase cascade that triggers a cell
cycle arrest, allowing time for the cell to repair the beak prior to
Following resection, Rad51-mediated strand invasion of the
donor template occurs with similar kinetics, but the initiation of
DNA synthesis at the 39-end of the invading strand is greatly
delayed in BIR as compared to GC [16,17]. Recently, Jain et al
 showed that a ‘‘Recombination Execution Checkpoint’’
(REC) delays the initiation of BIR synthesis if a second DSB end
has not become engaged nearby on the same template. It is
unclear if the delay in BIR synthesis is due to a restructuring of the
strand invasion D-loop and/or the recruitment of BIR associated
proteins. The efficiency of BIR is inhibited by Sgs1, as there is an
increase in BIR in sgs1D cells . Sgs1 also has been shown to
disrupt HR intermediates , inhibit homeologous recombina-
tion [27–29], and to dissolve double Holiday Junctions (dHJ) to
yield noncrossovers [30–32].
To better understand the role of Sgs1 in BIR, we examined
mutations of non-essential genes that either cooperate or act
redundantly with Sgs1 in many of its roles in DNA metabolism,
including DNA resection. Here we show that deletion of SGS1 or
EXO1 increases the efficiency of BIR whereas overabundance of
Sgs1 or Exo1 strongly inhibits it. Overexpression of Exo1 also
inhibits GC. Deletion of other non-essential factors responsible for
DNA resection, TEL1 or SAE2, modestly increases the efficiency of
BIR whereas deletion of MRX impairs BIR. Additionally, we find
that overexpression of Rad51 markedly improves the efficiency of
BIR but has little effect on GC. Finally, we show that Sgs1 and
Exo1 redundantly prevent remarkably efficient de novo telomere
addition at broken chromosome ends, a pathway dependent on
both telomerase and Sae2.
Assays to study break-induced replication and gene
conversion in S. cerevisiae
To study BIR we used the haploid Saccharomyces cerevisiae strain
JRL346. A galactose-inducible HO endonuclease is expressed to
induce a DSB at a modified CAN1 locus approximately 30 kb from
the telomere in the non-essential terminal region on Chromosome
V (Ch V) (Figure 1A). The HO endonuclease cut site and an
adjacent hygromycin-resistant marker, HPH-MX, was integrated
into the CAN1 locus, deleting the 39 portion of the gene but
retaining the 59 portion of the gene (denoted as CA). A 39 portion
of the gene (denoted as AN1) with 1,157 base pairs of shared
homology to CA on Ch V was introduced in the same orientation
into Ch XI, 30 kb from its telomere. Prior to HO induction, these
cells are canavanine-resistant (CanR) because CAN1 is disrupted.
Completion of BIR results in a non-reciprocal translocation that
duplicates the donor sequences and the more distal part of the left
arm of Ch XI, thus restoring an intact CAN1 gene. These cells
become canavanine-sensitive (CanS) and hygromycin sensitive
(HphS). About 20% of cells are viable with 99.85% of these cells
repairing by BIR and a small fraction by nonhomologous end-
joining (NHEJ). The efficiency of BIR repair allows us to
physically monitor the kinetics of repair by PCR, Southern blot
and pulse-field gel electrophoresis (PFGE), as described in
Materials and Methods.
To compare the effects of mutations on GC, we used the
isogenic strain JRL475 (Figure 1B). The GC strain was modified
from the BIR strain by introducing 2,404 bp of homology marked
by URA3 to the other end of the break (denoted as 1, for the 39-end
of CAN1). The insertion of the URA3-1 sequences also deleted 376
bp in the middle of the CAN1 so there is a gap between the
homology shared by the two DSB ends created by HO cleavage
(CA-URA3-1) with the donor sequences on Ch XI (AN1). Repair
by GC results in restoration of the CAN1 gene, rendering cells
CanS, but, unlike BIR, the Ch V arm distal to the cut site is
retained. When there is a second end of homology to a DSB break,
the cell strongly favors GC over BIR [16,17,33], so that after
induction of a DSB cell viability increases from 20% in the BIR
strain to nearly 70% when there are two ends of homology and
GC is used to repair the break (Figure 1B and Figure 2B).
Deletion of SGS1 increases the efficiency of BIR
To better understand the role of Sgs1 in BIR, we first measured
the viability of sgs1D cells after inducing a DSB (Figure 1A). As
previously shown , sgs1D cells are 1.5 times more efficient in
BIR compared to wild type cells (Figure 2A), repairing the break
with 33% efficiency (p,0.001). To confirm that the increase in
viability directly correlates with an increase in repair product, we
monitored the kinetics of repair using the PCR assay that detects
the first 242 bp of new DNA synthesis. The maximum amount of
product detected by PCR (18% at 12 hours) in wild type cells
(Figure 2D) is comparative to the viability of cells (21%) following
induction of the DSB (Figure 2A). As expected, deletion of SGS1
increased the efficiency of product formation compared to wild
type cells (Figure 2D). Using the previously described BIR system
involving the LEU2 sequences  we also showed that a helicase-
dead allele of Sgs1  behaves like the complete deletion of Sgs1
(Figure S1). We have previously shown that deletion of sgs1D does
not increase the efficiency of GC events in which there is perfect
homology or when there is a small gap in homology of 1.2 kb or
less [16,35]. We confirmed that sgs1D does not affect the efficiency
of GC in the ectopic assay used here (Figure 1B and Figure 2B).
A chromosomal double-strand break (DSB) poses a severe
threat to genome integrity, and budding yeast cells use
several homologous recombination mechanisms to repair
the break. In gene conversion (GC), both ends of the DSB
share homology to an intact donor locus, and the break is
repaired by copying the donor to create a small patch of
new DNA synthesis. In break-induced replication (BIR),
only one side of the DSB shares homology to a donor, and
repair involves assembly of a recombination-dependent
replication fork that copies sequences to the end of the
template chromosome, yielding a nonreciprocal translo-
cation. Both processes require that the DSB ends be
resected by 59 to 39 exonucleases, involving several
proteins or protein complexes, including Exo1 and Sgs1-
Rmi1-Top3-Dna2. We report that ectopic BIR is inhibited
independently by Sgs1 and Exo1 and that overexpression
of Rad51 recombinase further improves BIR, while GC is
largely unaffected. Surprisingly, when both Sgs1 and Exo1
are deleted, and resection is severely impaired, half of the
cells acquire new telomeres rather than completing BIR or
GC. New telomere addition appears to result from the lack
of resection itself and from the fact that, without
resection, the Mec1 (ATR) DNA damage checkpoint fails
to inactivate the Pif1 helicase that discourages new
Sgs1 and ExoI Impair BIR and New Telomere Addition
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Figure 1. Experimental systems of break-induced replication (BIR) and gene conversion (GC). (A) In the experimental system to study BIR,
an HPHMX marked HO cut site (gray bar) is integrated into the CAN1 gene on Ch V, deleting the 39 end portion of the gene, the remaining sequences
are represented as CA. The AN1 donor sharing 1,157 bp homology with CAN1 is integrated into Ch XI. PCR with primers P1 and P2 monitors the
Sgs1 and ExoI Impair BIR and New Telomere Addition
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The non-essential genes required for DNA resection
affect the efficiency of BIR
To better understand the role of Sgs1 in BIR, we investigated a
number genes that have previously been shown to interact
MUS81, YEN1, RAD27, ESC2, DIA2, YBR094w, or RNH202 did
not have a statistically significant effect on BIR when tested for
viability after inducing a DSB that can only be repaired by BIR
(Table S1). However, we found that the other non-essential genes
required for 59 to 39 resection of DSB ends all affect the efficiency
BIR. A deletion of SAE2 resulted in a slight, but statistically
significant, increase in viability (p=0.02). In contrast, deleting
subunits of the MRX complex, mre11D or rad50 D, decreased
viability nearly 2 fold (both p=0.003) (Figure 2A). The effect of
deleting mre11D or rad50D is consistent with results previously seen
in a diploid BIR assay in which a DSB is induced at the MAT locus
on Ch III [17,42], but differs from a transformation-based BIR
assay that saw no requirement for MRX in BIR .
Because Tel1 plays a role in suppressing gross chromosomal
rearrangements and enhances Sae2 and MRX activity in DNA
resection  we asked if deletion of TEL1 would affect BIR.
Similar to sae2D, deletion of TEL1 resulted in a small but
statistically significant increase in viability (p=0.008) (Figure 2A).
Complementation of a tel1D strain with the kinase-dead allele 
partially restored viability to wild type levels (Figure 2A).
The Exo1 nuclease acts redundantly with Sgs1 in DNA
resection after the initial trimming of the ends by Sae2 and
MRX, although by itself exo1D has a minimal impact on 59 to 39
resection [22–24]. Similar to sgs1D, deletion of EXO1 (p=0.001)
increased viability nearly 1.5 times compared to wild type
(Figure 2A). Also like sgs1D, deletion of EXO1 increased the
efficency of BIR when measured by PCR (Figure 2D) and does not
affect the efficiency of GC (Figure 2B).
Overexpression of both SGS1 and EXO1 inhibit BIR
Plamids overexpressing Sgs1 pYES2-SGS1  or Exo1
(pSL44)  were expressed under the control of a galactose-
inducible promoter on a high copy plasmid. These overexpression
plasmids are denoted as pGAL::SGS1 and pGAL::EXO1, respec-
tively. Expression is induced concomitantly with HO induction. In
cells carrying pGAL::SGS1, the efficiency of BIR decreased 5 fold
(p,0.001) whereas in pGAL::EXO1 the efficiency of BIR decreased
10 fold (p,0.001) (Figure 2A). Overexpression of these genes did
not affect cell viability in cells that lacked an HO cleavage site
(data not shown). Furthermore, we found that Exo1 overexpres-
sion inhibited BIR prior to inhibition of new DNA synthesis, by
monitoring the kinetics of repair by PCR (Figure S2). The strong
inhibition of BIR by overexpressing Exo1 depends on the nuclease
activity of this protein, as there is no such inhibition when we
overexpressed plasmids carrying exo1 mutations that are required
for exonuclease activity (Figure 2C). As shown previously ,
increasing the homology in our BIR assay more than two fold to
2,977 bp increases the efficiency of BIR (Figure 3C). The increase
in homology results in slightly higher viability but does not
significantly suppress the effects of overexpressing SGS1 or EXO1
(Figure 3C). When tested in the GC assay, overexpressing Sgs1
had no effect on viability but overproduction of Exo1 decreased
viability by half (Figure 2B).
Overexpression of Rad51 increases the efficiency and
kinetics of BIR
The initiation of BIR is delayed several hours after the ends of
the DSB begin to be resected at a wild type rate of about 4 kb/hr
[22,46]. We have also previously shown that the abundance of
Rad51 is sufficient to continuously coat only about 10 kb of
ssDNA on either side of the break ; consequently it is possible
that excess ssDNA would interfere with forming or maintaining a
stable and efficient Rad51 filament that is needed to promote
strand invasion and initiation of new DNA synthesis. Excess
ssDNA has been previously shown to interfere with recombination
in meiotic cells . We therefore asked if overexpression of
Rad51 would also increase the efficiency of BIR, using well-
characterized high-copy plasmids in which RAD51 was expressed
under the ADH1 promoter (pDBL(RAD51))  or under the PGK
promoter (pSJ5). Strikingly, overexpressing RAD51 in wild type
cells caused a 2.5-fold increase in viability (p,0.001) when
expressed under control of either promoter (Figure 3D). When we
tested the same plasmids in the GC assay we found that there was
a slight but not statistically significant decrease in viability
(Figure 3E). These results clearly indicate that Rad51 overexpres-
sion preferentially stimulates BIR. Overexpression of RAD51 in
the BIR assay with longer homology further increased the
efficiency of BIR (Figure 3C). We also find that the efficiency of
BIR is increased when we tested the kinetics of repair by Southern
blot (Figure 3A) and PCR (Figure 3B). However, when normalized
to the percent of final product the kinetics of repair are not
different from wild type cells (data not shown).
An elevated level of Rad51 increased the viability of sgs1D,
exo1D or tel1D cells to the level seen for overexpressed RAD51
alone (Figure 3D), so the effects of RAD51 expression and deleting
SGS1 or EXO1 are not additive. However, overexpressing RAD51
in cells also overexpressing SGS1 or EXO1 did not significantly
suppress the inhibition of BIR that is seen with overexpressing
SGS1 or EXO1 alone (Figure 3D). These results could suggest that
Sgs1 and Exo1 act prior to the rate-limiting step carried out by
Rad51. In the case of Sgs1, it could be in dismantling transient
strand invasion encounters; for Exo1, there is no evident
mechanism at this point unless a modest increase in resection
 would overwhelm excess Rad51.
Sgs1 and Exo1 redundantly inhibit new telomere
addition at DSBs
We examined a a dramatic 2-fold increase in viability in an
sgs1D exo1D double mutant compared to sgs1D or exo1D alone
when tested in the BIR assay (Figure 4A); however this increase is
not in the level of BIR. Instead, it is due to a dramatic increase in
new telomere addition, as described below. There is in fact no
increase in BIR events compared to the single mutants and repair
appears to be no better than wild type cells when repair was
monitored by PCR (Figure S3). As has previoulsy been reported
initiation of new DNA synthesis while PCR with primers P1 and P4 detects synthesis past the AN1 sequences, specifc to the donor sequences on Ch XI.
Southern blot analysis of AvaI-digested (marked by ‘‘A’’) DNA probed with CAN1 sequences monitors extension of the BIR fork. Completion of BIR is
monitored by Pulse-field gel electrophoresis (PFGE) followed by Southern blot analysis using the MCH2 sequences that are duplicated when the
entire donor chromosome arm is copied. (B) In the experimental system to study ectopic GC. A galactose inducible HO endonuclease generates a DSB
within the CAN1 locus (disrupted by URA3 creating a 376 bp gap) on Ch V. An additional 2,404 bp of homologous sequences to the gene conversion
donor sequences found on Ch XI are distal to the cut site and are denoted as ‘‘1.’’ PCR with primers P1 and P2 monitors both the starting strain and
repair into the CAN1 sequences. PCR with primers P1 and P3 monitors repair by GC in which the distal end of the break is retained.
Sgs1 and ExoI Impair BIR and New Telomere Addition
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[22–24], we found that resection is severely impaired in sgs1D
exo1D cells as evident by the persistence of the cut chromosome
band seen by Southern blot (data not shown). Although TEL1 and
SAE2 moderately inhibit BIR and are involved in DNA resection
like SGS1 and EXO1 , deleting TEL1 did not cause new
telomere additions at the DSB when ablated in combination with
Figure 2. Sgs1 and Exo1 negatively regulate BIR. (A) Efficiency of BIR in cells as measured by viability following a DSB. (B) Efficiency of GC in cells as
measured by viability following a DSB. (C) Efficiency of BIR in wild type (WT), exo1D, overexpression of EXO1 and overexpression of EXO1 nuclease-dead
alleles measured by viability following a DSB. For (A–C), data are the mean 6standard error of the mean. Values marked with asterics are statistically
significant (*represents p,0.05, ** represents p,0.01 compared to wild type). (D) The kinetics of repair are shown for PCR of BIR induced in cycling WT,
sgs1D and exo1D cells amplified with P1 and P2 primer set labeled as ‘‘CAN1’’ and the standard FLO9 locus of. Data are the mean 6standard deviation.
Sgs1 and ExoI Impair BIR and New Telomere Addition
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Sgs1 and ExoI Impair BIR and New Telomere Addition
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sgs1D or exo1D nor did deletion of SAE2 in combination with exo1D
DSBS are frequently repaired by telomere addition in
sgs1D exo1D cells
As mentioned above, when we analyzed the viablity of sgs1D
exo1D cells, we found that half of the survivors did not have the
CanSHphSphenotype indicative of repair by BIR (Figure 4A).
Instead, the new survivors were HphSbut CanR, suggesting that
they might have lost the terminal non-essential portion of Ch V
distal to the cut site but failed to restore a functional CAN1 locus.
Sgs1 has previously been shown to inhibit homeologous
recombination [27,29], specifically the formation of translocations
between CAN1 and two highly diverged CAN1 homologs, LYP1
and ALP1, on Ch XIV ; these rearrangements might be
further elevated by the absence of Exo1. Alternatively, given that
sgs1D exo1D severely retards 59 to 39 resection, the chromosome
end could be stabilized, allowing new telomere addition. To
distinguish between these possibilities, we performed pulse field gel
electrophoresis (PFGE) on 12 independent CanRHphScolonies,
comparing them to the starting strain and a survivor that repaired
by BIR (CanSHphS)(Figure 5). The ethidium bromide-stained
agarose gel (Figure 5A) shows that the majority of the CanRHphS
survivors (lanes 1–11) have a smaller chromosome than the
starting (ST) strain or one repaired by BIR (B). (There is no size
difference in Ch V size prior to DSB induction and after BIR
because the 30 kb of non-essential region distal to the cut site on
Ch V is replaced by a duplication of 30 kb from Ch XI.) We
confirmed by Southern blot that the band remaining at the
original position of Ch V is Ch VIII, which is approximately the
same size as Ch V in this strain background (data not shown). One
CanRHphScolony (lane 12) increased in size from the original
strain. These data indicate the CanRcolonies are not due to
mutations in a restored CAN1 gene, and are therefore not repaired
by BIR nor by NHEJ that could have deleted a small region
including HPH. To confirm that none of the CanRHphScolonies
were repaired by BIR, we probed with the MCH2 probe that
hybridizes proximal to the telomere on Ch XI (Figure 5B). The
MCH2 probe hybridized to sequences on Ch XI in every sample,
but only to Ch V in the CanSHphScolony that repaired by BIR.
To determine what sequences of Ch V were retained in the
CanRHphScolonies, we next probed the blot with a CAN1 probe
that hybridizes to the donor sequences on Ch XI and just proximal
(1 kb) to the cut site on Ch V (Figure 1A, Figure 5C). The CAN1
probe hybridized to sequences on Ch XI in all samples and to Ch
V in the starting and BIR strains, but only to three CanRHphS
colonies (1, 9 and 12). This result indicates that at least 1 kb of
sequence was deleted in the 9 other CanRHphSsurvivors. To
determine approximately how much sequence was deleted in the
other CanRHphScolonies we probed the Southern blot with a
NPR2 probe that specifically hybridizes to Ch V 4 kb proximal to
the cut site (Figure 1A and Figure 5D). In this case, the NPR2
probe hybridized to all CanSsamples except lanes 3, 5, 6, and 7.
When we probed with PRB1 that hybridizes approximately 9 kb
proximal to the cut site on Ch V, the probe hybridized to Ch V in
all CanSsurvivors (Figure 1A and Figure 5E). We also probed the
blot with the highly diverged ALP1 and LYP1 sequences on Ch
XIV with which CAN1 forms translocations in sgs1D cells , but
these sequences did not hybridize to the novel chromosome in lane
12 (data not shown). We have not explored further the structure of
Based on our PFGE and Southern blot analysis we conclude
that the great majority of the CanRHphSsurvivors result in a
truncation of Ch V after limited resection. To show if the
sequences at the terminus of the truncations are indeed new
telomeres, we determined the breakpoint of five independent sgs1D
exo1D CanRHphSrepaired colonies by PCR, using a Ch V-
specific primer and a telomere-specific primer as previously
described [52,53]. As shown in Figure 6, the presence of a new
telomere is indicated by a laddered PCR product. We then
sequenced the PCR product using the Ch V-specific primer. As
shown in Table 1, all five sgs1D exo1D CanRHphScolonies have
new telomere sequences directly added to the Ch V sequences.
Consistent with the PFGE and Southern blot analysis, the
breakpoints were not at a uniform location. Based on our results,
we hypothesize that in the absence of both Sgs1 and Exo1, a DSB
frequently results in a truncated chromosome with newly added
telomeres and that these additions can occur at several different
sites, often as far as between 1 and 4 kb away from the DSB end.
To confirm that these events are telomerase-dependent, we
deleted EST2, an essential components of telomerase. As shown
in Figure 4A, deletion of EST2 does not affect repair by BIR but
eliminates recovery of CanRcolonies.
We next asked if NHEJ or HR pathways contributed to de novo
telomere formation (Figure 4A). Telomere addition was not
dependent on NEJ1, which is required for NHEJ. We next deleted
RAD51, which is required for both BIR and GC. We confirmed
that nearly all BIR is eliminated in sgs1D exo1D rad51D cells but
also found a 20% increase in the number of cells with new
telomeres. Although overexpression of RAD51 increased the
efficiency of BIR it did not suppress new telomere addition
(Figure 4A). We then tested if the MRX-associated exonuclease
Sae2 plays a role in new telomere addition. Recently, Sae2 and
Sgs1 have also been shown to act in parallel telomere processing
pathways . Interestingly, when resection is nearly eliminated
by deletion of sae2D in combination with sgs1D exo1D, new
telomere addition is eliminated and BIR is significantly reduced
(Figure 4A). When TEL1 was deleted in combination with sgs1D
exo1D there was no change in levels of BIR or de novo telomeres
compared to sgs1D exo1D cells.
It has previously been seen that sgs1D exo1D cells are defective in
GC when tested for the ability to successfully complete MAT
switching . When we tested the viability of sgs1D exo1D cells in
our GC assay there was no discenrable effect on viability.
However, when the phenotypes of the viable colonies were
examined only 5% were CanS, which is indicative of repair by GC,
while the remaining viabile colonies were CanR, consistent with a
truncated chromosome (Figure 4B). The drastic decrease in GC is
Figure 3. Overexpression of RAD51 increases the kinetics and efficiency of BIR. (A) Southern blot analysis of the kinetics of repair product in
wild type and pPGK::RAD51 cycling cells as indicated in Figure 1A. Lane S contains DNA from a colony where BIR occurred. (B) Kinetics of repair are
shown for PCR of BIR induced in cycling wild type (WT) and pPGK::RAD51 cells. Data are the mean 6data range. (C) Efficiency of BIR in cells as
measured by viability following a DSB in a BIR assay with increased homology (2,977 bp homology). Data from Figure 2A and Figure 3D (1,157 bp
homology strain) are shown for comparison. Data are the mean 6s.e.m. Values marked with asterics are statistically significant (*represents p , 0.05,
** represents p , 0.01 compared to wild type). (D) Efficiency of BIR in strains graphed in Figure 2 also carrying either pPGK::RAD51 or pADH::RAD51 as
measured by viability following a DSB. Data are the mean 6s.e.m. Values marked with asterics or number sign are statistically significant (*represents
p , 0.05, ** represents p , 0.01 compared to wild type. # represents p , 0.05 to the corresponding single mutant). (E) Efficiency of GC in WT and
pPGK::RAD51 as measured by viability following a DSB.
Sgs1 and ExoI Impair BIR and New Telomere Addition
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Figure 4. The effect of sgs1D exo1D on the viability and repair product in BIR and GC. (A) The viability and phenotypic characterization of
wild type (WT), sgs1D, exo1D, tel1D, sgs1Dsgs1D exo1D, pPGK::RAD51 and indicated double and triple mutant combination cells following a DSB in the
BIR assay. BIR colonies (CanSHphS) represent those that have repaired the DSB by BIR while CanRHphScolonies represent those that have a truncated
Sgs1 and ExoI Impair BIR and New Telomere Addition
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consistent with previously published defects seen in sgs1D exo1D
cells. We analyzed 10 independent CanScolonies by PCR to
ascertain if the break was repaired by GC (Figure 4C). In fact, only
5 of the 10 colonies analyzed (samples S2, S3, S4, S5, S8) repaired
by GC whereas 4 of the colonies repaired the break by BIR (S1,
S6, S7, S10). One colony (S9) had PCR products consistent with
repair by both GC and PCR. The use of BIR to repair half of the
sgs1D exo1D colonies is consistent with the failure of these cells to
activate the DNA damage checkpoint and thus to enter mitosis in
the absence of DSB repair.
To verify that that the DNA damage checkpoint was impaired
by the lack of normal 59 to 39 resection of the DSB ends we
microscopically monitored the length of the cell cycle of individual
cells plated on YEP-Gal to induce HO endonuclease, from the
time that an unbudded G1 cell formed a bud until the dumbbell-
shaped mother-daughter pair formed the next bud . Wild type
cells in which the DSB cannot be repaired remain arrested prior to
anaphase for approximately 6 cell division times relative to an
isogenic strain lacking the HO cleavage site . In contrast, cells
of the BIR strain lacking SGS1, EXO1 and RAD51, so that they
could not repair the DSB by homologous recombination, show a
brief, but significant arrest. These cells extend the cell cycle 1.8
times the length of time of a derivative that lacks the cut site (6.2 h
versus 3.5 h). Thus, there is still a brief activation of DSB-induced
cell cycle arrest but much shorter than when extensive resection
As was the case with CanRsgs1D exo1D colonies found in the
BIR assay, the CanRcolonies in the GC assay appear to be
chromosome truncations with de novo telomere formation. PCR
analysis showed that the broken chromosomes were truncated at
different points proximal of the DSB (Figure S4). When
representative isolates were tested by PCR as mentioned above
we found that consistent with new telomere addition there was a
laddered PCR product as seen in sgs1D exo1D cells in the BIR assay
We conclude that eliminating both Sgs1 and Exo1, by markedly
reducing 59 to 39 resection and most likely by preventing full
activation of the Mec1-dependent DNA damage checkpoint (see
Discussion), allows a dramatic increase in new telomere formation,
rescuing almost half of all cells suffering a DSB.
In this work we show that the RecQ family helicase, Sgs1, and
the Exo1 exonuclease negatively regulate BIR to maintain
genomic integrity. From the observation that the efficiency of
BIR was no greater in sgs1D exo1D than in a single mutant one
might conclude that the helicase/endonuclease (Sgs1-Rmi1-
Top3/Dna2) and Exo1 act in the same pathway, but since the
sgs1D exo1D double mutant has such distinctly different phenotypes
from sgs1D or exo1D it is difficult to know precisely why the double
mutant does not show an increase in BIR similar to that seen when
Rad51 is overexpressed in sgs1D or exo1D alone. We note also that
other proteins responsible for 59 to 39 DNA resection, Sae2 and
MRX, do not inhibit BIR in the same fashion; but the behavior of
sae2D or mre11D may be explained by their other important roles
in other steps in HR [1,3,4].
Sgs1 and Exo1 likely do not act in precisely the same way in
inhibiting BIR. Sgs1-mediated inhibition of BIR may involve
unwinding of a nascent strand invasion D-loop, as demonstrated in
vitro for the human Sgs1 homolog, BLM [56,57]. In vivo it is clear
that the Sgs1 helicase can dismantle strand annealings and strand
invasions if the heteroduplex DNA contains mismatches [27–29].
In meiotic recombination, Sgs1 prevents independent strand
invasions of alternative templates [58,59]. If Sgs1 dismantles
heteroduplex DNA, we might expect that increased homology
between the DSB end and the donor template would lead to a
more stable D-loop that would counteract Sgs1. Increasing the
extent of homology from 1.1 kb to ,3 kb did not significantly
change the response of cells to overexpression of Sgs1. It is also
possible that Sgs1 inhibits the recruitment of some of the BIR-
associated proteins. We note that the effect of deleting Sgs1 or
Exo1 is not apparent in a different BIR assay system in a diploid in
which nearly all homologous sequences distal to the DSB are
deleted [17,60]; and where there are 100 kb of homologous
sequences centromere-proximal to the DSB that can be used to
initiate BIR. However, even in this case, many BIR events fail to
retain a marker 3 kb proximal to the DSB, suggesting either that
more extensive homology increases BIR or that some more
proximal sequences are especially favored in initiating BIR .
Rather than acting on D-loop stability, Exo1 may act on the
assembly of the BIR replication fork. In response to DNA damage
or defective checkpoint activation, Exo1 has also been shown to
process stalled replication forks and resect nascent strands [62,63].
The mechanism by which Exo1 interferes with fork integrity is
unclear; it may be possible that the intermediate steps at which the
BIR replication fork is assembled are an Exo1 substrate. We have
previously shown that overexpression of Exo1 increases the rate of
resection ; this has not been tested for Sgs1 overexpression.
A unifying hypothesis would be that BIR is severely limited if
resection of the DSB ends is too extensive. There is a limited
amount of Rad51 in the cell (about 3,500 molecules), enough to
cover continuously about 10 kb of ssDNA . Although Rad51
will initially form a filament with sequences close to the DSB
(including the relevant ‘‘CA’’ sequences that engage in BIR), as
resection proceeds the continuous polymerization and depolymer-
ization of Rad51 may leave patches of Rad51 along much of the
ssDNA so that by the time BIR is seen, many DSBs will not have a
continuous Rad51 filament near the 39 end to promote the
completion of recombination. Thus, even in wild type cells,
overexpressing Rad51 would ensure that there would be a
functional filament over the CA sequences and BIR would
consequently be more efficient. Deletions of Sgs1 or Exo1 would
partially suppress the problem by slowing down resection (hence
BIR is increased 1.5 times wild type), although we again note that
exo1D by itself has little visible effect on resection. Overexpression
of Rad51 is apparently unable to suppress the consequences of
overexpressing Exo1 or Sgs1. It is important to note that Exo1
overexpression is only effective if nuclease activity is preserved; at
least some of Exo1’s functions in meiosis are independent of
chromosome. Data are the mean 6s.e.m. Values marked with asterics or number sign are statistically significant (*represents p , 0.05, ** represents
p , 0.01 compared to wild type BIR. # represents p , 0.05 to the sgs1D exo1D CanRHphScolonies). (B) The viability and phenotypic characterization
of cells following a DSB in the GC assay. HR colonies (CanS) represent those that have repaired by Homologus Recombination (either BIR or GC) while
CanRcolonies represent those that have a truncated chromosome. Data are the mean 6s.e.m. ** represents p , 0.01 compared to wild type. (C)
Repair of CanScolonies in the GC assay as monitored by PCR. Included are the starting GC strain (ST), ten CanScolonies (S1–S10) and a colony that has
repaired by BIR (B). PCR with primers P1 and P2 detects the starting band and shift to smaller size upon repair into the CAN1 sequences if repair
occurs either by GC or BIR. PCR of primers P1 and P3 monitors retention of the distal end of the DSB and is indicative of repair by GC. PCR with
primers P1 and P4 monitors repair specific to BIR (see Figure 1).
Sgs1 and ExoI Impair BIR and New Telomere Addition
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Figure 5. Characterization of CanRHPHSsgs1D exo1D colonies in the BIR strain by PFGE. (A) Ethidium bromide-stained agarose gel PFGE
gel of sgs1D exo1D colonies that have repaired the DSB. Included are the ladder (L), starting strain prior to DSB induction (ST), CanSHPHScolony that
has repaired by BIR (B), and twelve CanRHPHScolonies (1–12). Arrows indicate additional uncharacterized chromosomal fragments. (B) Southern blot
analysis of (5A) by hybridization with a probe for MCH2 that normally lies 6 kb from the telomere on Ch XI (See Figure 1). (C) The blot was stripped
and Southern blot analysis was performed by hybridization with a probe for CAN1 that normally lies 33 kb from the telomere on Ch V and is 1 kb
proximal to the HO cut site (See Figure 1). (D) The blot was stripped and Southern blot analysis was performed by hybridization with a probe for NPR2
that normally lies 36 kb from the telomere on Ch V and is 4 kb proximal to the HO cut site (See Figure 1). (E) Southern blot analysis was performed on
(5D) by hybridization with a probe for PRB1 that normally lies 40 kb from the telomere on Ch V and is 8 kb proximal to the HO cut site (See Figure 1).
Sgs1 and ExoI Impair BIR and New Telomere Addition
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nuclease activity (N. Hunter, personal communication; L.
Symington, personal communication). Increasing homology in
our assay does not suppress these effects but further increases in
homology may do so, as noted above.
It is possible that overexpressing Rad51 could ensure that the
39-ended single-stranded DNA was better protected against
degradation over the long time required to enact BIR, as
previously suggested . However, we have previously shown
that in single-strand annealing where one of the flanking 1-kb
homologies is very close to the DSB and the other is exposed only
after 6 hr of 59 to 39 resection, at least 85% of cells are able to
accomplish SSA, which would be impossible if even 1 kb of the 39-
end were degraded in the 6-hr period. Moreover, SSA was equally
possible with and without Rad51 , arguing that Rad51 did not
provide end-protection to the 39-ended single-strand.
Eliminating both Sgs1 and Exo1 had a marked defect in
completing GC but did not impair BIR so severely. Because
resection is severely impaired in the sgs1D exo1D double mutant, it
is possible that the more severe defect in GC is attributable to the
need to resect more than 1 kb of intervening sequence before the
‘‘1’’ end of homology would be single-stranded (see Figure 1B).
However, it is also possible that the difference reflects still another
defect in sgs1D exo1D strains, a failure to activate the DNA damage
checkpoint because of a lack of sufficient ssDNA [25,65]. If mitosis
is not arrested, then cells that have an unrepaired DSB will
proceed through mitosis. This may lead to the loss of the acentric
fragment, as we have shown in other assays , so that only the
centromere-proximal DSB end will be inherited. This situation is
not fatal for BIR, which only uses homology on that side of the
DSB; indeed previous studies [17,67] have shown that BIR may
actually increase in a checkpoint-deficient situation whereas GC
will be defective. Thus, even when GC should be possible, half of
the HR outcomes of the sgs1D exo1D GC assay proved to be BIR
Strikingly, Sgs1and Exo1 also redundantly inhibit new telomere
formation. In a previous study , when an HO-induced DSB
was generated in a rad52D strain that could not carry out
recombination but had apparently normal 59 to 39 resection, only
about 1% of cells created new telomeres, and this was only in a
situation where a ‘‘seed’’ of T2G4telomere sequences was located
Figure 6. Marking of the breakpoint and detection of de novo telomere formation by PCR in sgs1D exo1D CanRHphScells. From the
BIR assay. (A) PCR analysis of a starting strain prior to DSB induction (ST), CanSHphScolony that has repaired by BIR (B), and five CanRHPHScolonies
(1–5) with primers that amplify sequences (Ch V 32,763–34,020) approximately 750 bp proximal to the break. (B) PCR with primers that amplify
sequences (Ch V 32,265–34,020) approximately 250 bp proximal to the break. (C) PCR with a Ch V-specific primer that amplifies all colonies indicated
and primer CA16, a telomere-specific primer. (D) PCR product from 6C ran longer an agarose gel to better display the laddered PCR product indicative
of de novo telomere formation in samples 1–5.
Table 1. Sequenced breakpoints in sgs1D exo1D CANRHPHSrepaired colonies.
CANRSample Ch V Breakpoint Sequence
The breakpoint in five independent sgs1D exo1D CanRHphSrepaired colonies were determined by PCR, amplified with Ch V-specific and telomere-specific primers
(Figure 6), and sequenced as described [52,53].
Sgs1 and ExoI Impair BIR and New Telomere Addition
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centromere-proximal to the DSB. In the absence of the T2G4
repeats, new telomeres arose less than 0.1% of the time. The
remarkably high level of new telomere formation (up to 50% of all
cells) must be attributable to the elimination of vigorous resection
in the double mutant strain, but it is also likely that the failure to
activate the Mec1 DNA damage checkpoint also plays a key role.
Recently, Makovets and Blackburn  have shown that the Pif1
helicase, which antagonizes new telomere formation , is
phosphorylated in a Mec1-dependent fashion; hence if sgs1D exo1D
block resection and that prevents Mec1 activation, new telomeres
should increase. However, in the assay used by Makovets and
Blackburn  the level of new telomeres added near an HO
endonuclease-induced DSB was only about 2%. Moreover, Chung
et al  also find that new telomere addition is much less efficient
in cells lacking MEC1 compared to sgs1D exo1D cells. Hence, it is
likely that the 40–50% level of de novo telomere formation we find
reflects both the failure to activate Pif1 when the checkpoint is not
strongly activated and the severe block on resection itself.
Apparently de novo telomere formation does not require the
recruitment of the MRX-Tel1 complex, as a tel1D mutant does not
affect the formation of new telomeres in an sgs1D exo1D strain.
When resection is blocked by deletion of SAE2 in sgs1D exo1D cells,
new telomeres are absent. The fact that new telomeres were added
as far as 4 kb from the DSB site indicates that there is a residual
resection activity that–over a period of perhaps many hours–can
chew away the chromosome end and expose sites suitable for new
telomere addition. However, we show that the MRX-asociated
endonuclease SAE2 is required for de novo telomere formation.
In this work we have expanded our understanding of the genetic
relationships of factors that negatively regulate BIR. Furthermore,
we have provided evidence for a novel repair pathway that is
redundantly impaired by Sgs1 and Exo1. Understanding the
interplay of these factors in response to DNA damage and
uncovering the molecular details of signaling between them to
maintain genomic integrity will be an area of much future
Materials and Methods
Strains and plasmids
The wild type JRL346 was derived from JRL092  by first
disrupting the LEU2 marker with a leu2::hisG construct from
pNKY85  to generate strain JRL187. The HMRa-stk gene
was then knocked out with an hmr::ADE3 fragment generated by
PCR with mixed oligos to generate JRL346. All strains used to
study BIR are isogenic to JRL346 and were created by standard
gene disruption methods and confirmed by PCR unless otherwise
stated . In order to generate an assay to study GC that is
isogenic with JRL346, an HOcs-HPH cassette  was integrated
into Ch V between nucleotides 31,644 and 32,020, resulting in a
truncation of the CAN1 ORF at nucleotide 1,146 to create strain
JRL017 (CL11-7 can1,1-1446::HOcs::HPH). JRL017 was then
modified by transforming in a hphmx::URA3 ‘‘marker swap’’
cassette  to generate JRL472 (CL11-7 can1,1-1446::HOcs::
URA3::AVT2). To introduce another 2,404 bp of homology to the
donor, the can1,1-1446::HOcs::URA3::AVT2 region with Ch V
sequences 29,146 to 32,976 was amplified from JRL472 and
integrated distal to the HO cut site into Ch V in strain JRL346 to
generate JRL475 (can1,1-1446::HOcs::URA3::AVT2 ykl215c::leu2::
hisG::can1DEL1-289::AVT2). As a result, there are Ch V
sequences 33,177–32,020 shared between the donor and
sequences proximal to the break, Ch V sequences 31,644–
29,240 shared between the donor and sequences distal to the
break and a 376 bp gap of homology. All mutant strains were
created by standard gene disruption methods and confirmed by
PCR. Plasmid pSJ5 was constructed by subcloning a XhoI-NotI
fragment containing the RAD51 ORF under the PGK promoter
form pNSU256  into pRS314 .
Logarithmically growing cells grown in YEP+2% Raffinose, or
the appropriate drop-out media +2% Raffinose, were plated on
either YEPD or YEP-Gal, and grown into colonies. Colonies were
counted and were then replica plated onto plates containing either
canavanine or hygromycin to confirm repair occurred by BIR.
Experiments were performed at least 5 times for each strain unless
otherwise indicated. To determine the statistical significance
between strains the student’s t-test was used (paired, two-tailed,
n$4 for all strains).
HO induction and measurement of kinetics of DSB repair
Strains were grown in YEP+2% Raffinose to a cell density of
3610e6 to 1610e7 cells/mL. A 50 mL aliquot of cells was
removed for the zero time point. Freshly made galactose was
added to final concentration of 2% to induce HO expression. Cell
aliquots were taken at the indicated time points throughout the
PCR analysis of BIR was performed as previously described .
Briefly, we monitor the initiation of new BIR DNA synthesis using
a PCR assay in which one primer is specific to Ch V and the other
primer is specific to the donor sequence on Ch XI. Once a
covalent molecule is formed, corresponding to the first 242 bp of
new DNA synthesis, we see PCR product. At least three PCR
reactions from three different experiments were performed for
wild type, sgs1D and exo1D strains. For all other strains tested, at
least three PCR reactions from two experiments were performed.
The technical replicates from each biological experiment was first
averaged and then the technical averages were averaged among
the two experiments to obtain a biological average. Data were
graphed as the biological averages normalized to the maximum
product obtained by amplifying DNA from a strain that has
repaired the DSB by BIR. Error bars represent the data range
between the biological averages.
Repair is also measured by Southern blot that detects
approximately the first 3 kb of new DNA synthesis was performed
as previously described . The analysis by Southern blot or
pulse-field (CHEF) gel electrophoresis followed by Southern blot
was performed as described  using the probes indicated in
Figure 1. The breakpoints and sequences of sgs1D exo1D CanR
HphSrepaired colonies were performed as described [52,53].
BIR. (A) In this assay to study BIR, an HO cut site is integrated
into an ectopically located LEU2 gene on Chromosome V (Ch V)
in which the 39 end portion of the gene is deleted, the remaining
sequences are represented as LE. The donor sequences are the
endogenous LEU2 gene on Ch III. Repair of the DSB only occurs
by BIR resulting in duplication of the LEU2 gene and the distal
sequences on Ch III. (B) Efficiency of BIR as measured by viability
following a DSB in wild type (WT), sgs1D, or sgs1D cells
complemented with a plasmid expressing the sgs1-hd allele
Found at: doi:10.1371/journal.pgen.1000973.s001 (0.21 MB TIF)
The helicase-domain of Sgs1 is required to inhibit
Sgs1 and ExoI Impair BIR and New Telomere Addition
PLoS Genetics | www.plosgenetics.org 12May 2010 | Volume 6 | Issue 5 | e1000973
repair are shown for PCR assays of BIR induced in cycling wild
type (WT) and GAL::EXO1 cells. Data are the mean 6data range.
Found at: doi:10.1371/journal.pgen.1000973.s002 (0.13 MB TIF)
Overexpression of EXO1 inhibits BIR. Kinetics of
cells. Kinetics of repair are shown for PCR assays of BIR induced
in cycling wild type (WT) and sgs1D exo1D cells. Data are the
mean 6 data range for two experiments.
Found at: doi:10.1371/journal.pgen.1000973.s003 (0.16 MB TIF)
The efficiency of BIR is not increased in sgs1D exo1D
telomere formation by PCR in sgs1D exo1D CANRsurvivors from
the GC assay. (A) PCR analysis of a starting strain prior to DSB
induction (ST), CanScolony that has repaired by HR (S), and ten
CanRcolonies (R1–R10) with primers that amplify sequences (Ch
V 39,744–42,157) approximately 7.7 kb proximal to the break. (B)
PCR with primers that amplify sequences (Ch V 34,271–37,985)
approximately 2.2 kb proximal to the break. (C) PCR with
primers that amplify sequences (Ch V 33,007–35,272) approxi-
mately 1 kb proximal to the break. (D) PCR with primers that
amplify sequences (Ch V 32,265–34,020) approximately 250 bp
proximal to the break. (E) PCR with a Ch V-specific primer that
Marking of the breakpoint and detection of de novo
amplifies all colonies indicated and primer CA16, a telomere-
Found at: doi:10.1371/journal.pgen.1000973.s004 (1.41 MB TIF)
The viability of cells that could repair a DSB by BIR as shown in
Figure 1A was compared by plating cells on YEP-galactose to
induce expression of HO endonuclease and on YEPD, as
described in Materials and Methods.
Found at: doi:10.1371/journal.pgen.1000973.s005 (0.07 MB
The effect of varied mutants on the efficiency of BIR.
We are grateful to Ian Hickson, Loraine Symington, and David T. Weaver
for the gift of plasmids and to Grzegorz Ira and Anna Malkova for sharing
results prior to publication.
Conceived and designed the experiments: JRL SJ JEH. Performed the
experiments: JRL ZLM SJ VVE. Analyzed the data: JRL ZLM SJ VVE
JEH. Contributed reagents/materials/analysis tools: JRL. Wrote the
paper: JRL JEH.
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