Copyright © 2005 by the Genetics Society of America
Multiple Endonucleases Function to Repair Covalent Topoisomerase I
Complexes in Saccharomyces cerevisiae
Changchun Deng, James A. Brown, Dongqing You1and J. Martin Brown2
Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California 94305-5152
Manuscript received March 15, 2004
Accepted for publication March 4, 2005
Topoisomerase I plays a vital role in relieving tension on DNA strands generated during replication.
However if trapped by camptothecin or other DNA damage, topoisomerase protein complexes may stall
replication forks producing DNA double-strand breaks (DSBs). Previous work has demonstrated that two
structure-specific nucleases, Rad1 and Mus81, protect cells from camptothecin toxicity. In this study, we
used a yeast deletion pool to identify genes that are important for growth in the presence of camptothecin.
In addition to genes involved in DSB repair and recombination, we identified four genes with known or
implicated nuclease activity, SLX1, SLX4, SAE2, and RAD27, that were also important for protection against
camptothecin. Genetic analysis revealed that the flap endonucleases Slx4 and Sae2 represent new pathways
parallel to Tdp1, Rad1, and Mus81 that protect cells from camptothecin toxicity. We show further that
the function of Sae2 is likely due to its interaction with the endonuclease Mre11 and that the latter acts
on an independent branch to repair camptothecin-induced damage. These results suggest that Mre11
(with Sae2) and Slx4 represent two new structure-specific endonucleases that protect cells from trapped
topoisomerase by removing topoisomerase-DNA adducts.
cation fork by transiently cutting and religating a single
strand of the DNA double helix. This reaction involves
forming a covalent (3?-phosphotyrosyl)-enzyme-DNA
complex.These Top1-DNAcovalentcomplexes arenor-
mally transient, but the anticancer drug camptothecin
stabilized complex can then lead to a DNA double-
strand break and cytotoxicity if it blocks a replication
fork (Hsiang et al. 1989). In addition, various other
types of DNA damage, such as base mismatches and
abasic lesions, have been shown to stabilize the Top1-
DNA complex (Pourquier et al. 1999).
The lesion created by the collision of the replication
fork with the Top1-DNA complex requires specialized
enzymes for its repair since the enzyme is covalently
bound to the 3? end of the break and must be removed
before the DNA can be religated. In the budding yeast
Saccharomycescerevisiae, anenzyme, tyrosyl-DNAphopho-
diesterase (Tdp1), has been found that can specifically
hydrolyze this lesion (Yang et al. 1996; Pouliot et al.
NA topoisomerase I (Top1) is an essential enzyme
that relaxes DNA supercoiling ahead of the repli-
1999). However, yeast mutants deleted in TDP1 show little
or no sensitivity to camptothecin (Vance and Wilson
2002) and it has been shown that the structure-specific
heterodimer endonuclease Rad1 Rad10 functions as a
redundant pathway for removing the Top1-DNA lesion
(Vance and Wilson 2002). Thus, whereas the tdp1 and
rad1 deletion mutants have little or no sensitivity to
camptothecin, the double mutant is hypersensitive to
the drug (Vance and Wilson 2002). In addition, dele-
sensitivity to camptothecin apparently through a path-
way parallel to Tdp1 and Rad1, as the triple mutant tdp1
rad1 mus81 is more sensitive than the double-mutant
tdp1 rad1 (Liu et al. 2002; Vance and Wilson 2002).
It would appear, however, that still more enzymes
repair, are considerably more sensitive to camptothecin
than are the double mutation tdp1 rad1 (Vance and
Wilson 2002) and the triple mutant mus81 rad1 tdp1
(Liu et al. 2002). To identify any such alternative path-
ways, we performed a genome-wide screen to detect all
nonessential genes that are important for protection
from growth inhibition or killing produced by continu-
ous exposure to camptothecin. The recent completion
of a systematic deletion of all open reading frames in
yeast (Giaever et al. 2002) has provided a powerful new
tool for screening for genes whose deletion produces
sensitivity to cytotoxic drugs. We and others have used
this collection of mutants to identify novel genes whose
deletion confers sensitivity to ultraviolet radiation (Bir-
1Present address: Department of Radiation Medicine, Second Military
Medical University, 800 Xiangyin Rd., Shanghai, 200433, People’s
Republic of China.
2Corresponding author: Division of Radiation and Cancer Biology,
Department of Radiation Oncology, Stanford University School of
Medicine, CCSR South, Room 1255, 269 Campus Dr., Stanford, CA
Genetics 170: 591–600 (June 2005)
592 C. Deng et al.
strains that were called in at least two of the three replicate
experiments were included in further analysis to determine
of these quality control criteria eliminated 6.3% of all the
strains in the pool (i.e., we are working with 4424 of the total
work that were “not called” in the hybridization experiments
because of their low abundance in the pool was the mre11
Testing of individual strains for sensitivity to camptothecin:
All individual strains tested were haploids in the BY4741 and
BY4742 backgrounds and were generated by the Saccharomyces
ble- and triple-deletion strains were the products of tetrad
dissection. All the deletions were confirmed by genomic PCR
using primer sets flanking the ORF as well as primers within
the deleted coding region or the Kanamycin replacement
marker. In the case of the slx1 tdp1 double deletion we trans-
by PCR because SLX1 and TDP1 are tightly linked and ex-
was then backcrossed with a wild-type strain to minimize influ-
ence of random mutations introduced during transformation.
Spores were selected and confirmed by PCR to be deleted in
both SLX1 and TDP1. The mre11-H125N mutants were kindly
provided by Lorraine S. Symington (Moreau et al. 1999).
Table 1 shows the genotypes of all the strains used.
Measurement of sensitivity to camptothecin was performed
according to the published protocol of Vance and Wilson
(2002). Briefly, cells were grown to midlog phase in the pres-
ence of 1% DMSO and then diluted 1000- to 2000-fold and
treated with camptothecin at various concentrations. OD600
was measured for all testing concentrations when the un-
treated culture of a test strain had proliferated 10 cycles. We
vs. the number of the doubling of the untreated culture. The
sensitivity was determined as the ratio of slope of the treated
sample divided by that of the untreated sample.
rell et al. 2001), ionizing radiation (Bennett et al. 2001;
Game et al. 2003), and other DNA-damaging agents
(Begley et al. 2002; Chang et al. 2002; Wu et al. 2004).
One of the advantages of this resource is that the gene
replacement cassette contains two molecular “bar code”
tags or unique 20-base oligonucleotide sequences, which
allow for unique identification of the strain in a pool
of all deletion mutants by PCR amplification of the tags
and subsequent hybridization to a high-density oligo-
nucleotide array containing the corresponding comple-
mentary sequences (Giaever et al. 2002).
From this screen we identified two further pathways
involved in repairing the Top1-DNA complex, one in-
volving Slx4 (but not its partner Slx1), which acts in a
parallel pathway to Tdp1 and Rad1/Rad10, and Sae2,
which appears to act by stimulating the endonuclease
activity of Mre11.
MATERIALS AND METHODS
Yeast strains, deletion pool, and drug treatment: Genotypes
of the parental diploid yeast strain BY4743, construction of
the homozygous diploid deletion strains, and construction of
the homozygous diploid deletion pool have been described
are available through Open Biosystems (Huntsville, AL) or
EUROSCARF (Frankfurt, Germany). We used a mutant pool
of the diploid pool of deletion strains minimizes the possible
tion of the haploid deletion stains.
An aliquot of the pool containing ?107cells was diluted
10-fold in YPD and then grown for 6–7 hr. Aliquots were then
either mock treated or treated with 50 ?m camptothecin for
in logarithmic growth. Following the 16-hr exposure the cells
were harvested, genomic DNA extracted, PCR amplified, and
hybridized to custom-made Tag3 gene chips (Affymetrix,
Santa Clara, CA) as described previously (Birrell et al. 2002).
Each deletion strain is associated with four hybridization sig-
nals on the high-density oligonucleotide array generated in
two separate PCR labeling reactions: UPTAG (sense and anti-
sense) and DNTAG (sense and antisense). Equal numbers of
cells were harvested in both the control and the treated pools
to produce equal pool label intensities. We calculated the
background intensity of each array and normalized the data
to eliminate any bias created during the PCR amplification
reaction as described earlier (Wu et al. 2004). An experimen-
tal/control intensity ratio was included in the data analysis
only if the signal generated in the untreated control array was
at least twice the background signal. This limit was chosen so
that a maximum value of 0.5 for the ratio of treated/control
hybridizations would be obtained if the treated signal was at
the background level. In addition, each treated tag that fell
within two standard deviations of the background was flagged,
indicating that the measured value may be an overestimation
of the true ratio (i.e., the true value would likely be lower,
indicating greater sensitivity). To yield a more stable estimate
of the average treated/control ratio, we averaged the logs of
the treated/control ratios for each of the four tags assigned
to an individual strain. Those strains that failed to have at
least two of the tags significantly above background were not
called for an individual experiment. In addition, only those
Genomic screen for genes whose deletion produces
sensitivity to camptothecin: To determine which genes
when deleted cause sensitivity to continuous exposure
to camptothecin, we treated the pool of 4728 deletion
strains composing the yeast deletion pool with 50 ?m
camptothecin for 16 hr and then compared the abun-
dance of the strains in the treated pool with those in a
mock-treated pool. This assay measures overall growth
inhibition, which integrates slowing of growth and cell
killing. Table 2 lists the top 30 sensitive deletion strains
and supplemental Table 3 (see supplemental data at
all 4728 strains to the camptothecin exposure). For con-
venience we have also listed in Table 2 the rankings
of the deletion strains used in this investigation. Not
surprisingly, the most sensitive strains were deleted for
genes required for recombinational repair, consistent
with the fact that camptothecin causes DNA double-
strand breaks. In addition to these highly sensitive strains
ity to camptothecin with a ranking of 149 among the
593Endonucleases Resolving Top1-DNA Adducts
Strains used in this study
FigureStrain Relevant genotype
MAT? his3?1 leu2?0 met15?0 slx4?::KanMX
MATa his3?1 leu2?0 slx4?::KanMX tdp1?::KanMX
MATa his3?1 leu2?0 met15?0
MAT? his3?1 leu2?0 tdp1?::KanMX
MATa his3?1 leu2?0 tdp1?::KanMX?::LEU2 slx1?::KanMX
MAT? his3?1 leu2?0
MAT? his3?1 leu2?0 lys2?0 tdp1?::KanMX?::LEU2
MATa his3?1 leu2?0 met15?0 slx1?::KanMX
MAT? his3?1 leu2?0 tdp1?::KanMX
MATa his3?1 leu2?0 slx4?::KanMX tdp1?::KanMX
MAT? his3?1 leu2?0 met15?0 slx4?::KanMX rad1?::KanMX tdp1?::KanMX
MATa his3?1 leu2?0 met15?0 rad1?::KanMX tdp1?::KanMX
MATa his3?1 leu2?0 met15?0 slx4?::KanMX rad1?::KanMX
MATa his3?1 leu2?0 slx4?::KanMX
MAT? his3?1 leu2?0 met15?0 lys2?0
MAT? his3?1 leu2?0 lys2?0 rad1?::KanMX
MATa his3?1 leu2?0 slx4?::KanMX mus81?::KanMX tdp1?::KanMX
MAT? his3?1 leu2?0 slx4?::KanMX tdp1?::KanMX
MATa his3?1 leu2?0 mus81?::KanMX tdp1?::KanMX
MAT? his3?1 leu2?0 tdp1?::KanMX
MATa his3?1 leu2?0 lys2?0 sae2?::KanMX tdp1?::kanmx?::LEU2
MAT? his3?1 leu2?0 met15?0 sae2?::KanMX
MATa his3?1 leu2?0 lys2?0 met15?0 tdp1?::kanmx?::LEU2
MAT? his3?1 leu2?0
MAT? his3?1 leu2?0 lys2?0 met15?0 tdp1?::kanmx?::LEU2 rad1?::KanMX sae2?::KanMX
MAT? his3?1 leu2?0 lys2?0 tdp1?::KanMX rad1?::KanMX
MATa his3?1 leu2?0 tdp1?::KanMX
MATa his3?1 leu2?0 met15?0 tdp1?::kanmx?::LEU2 sae2?::KanMX
MAT? his3?1 leu2?0 tdp1?::KanMX sae2?::KanMX
MATa his3?1 leu2?0 tdp1?::KanMX mus81?::KanMX sae2?::KanMX
MATa his3?1 leu2?0 lys2?0 tdp1?::kanmx?::LEU2
MATa his3?1 leu2?0 lys2?0 tdp1?::kanmx?::LEU2 mus81?::KanMX
MAT? his3?1 lys2?0 leu2?0
MATa his3?1 leu2?0 mre11-H125N
MATa his3?1 leu2?0 lys2?0 met15?0 tdp1?::kanmx?::LEU2 rad1?::KanMX mre11-H125N
MATa his3?1 leu2?0 met15?0 can1 tdp1?::kanmx?::LEU2 rad1?::KanMX
MAT? his3?1 leu2?0 lys2?0 met15?0 tdp1?::KanMX mre11-H125N
MAT? his3?1 leu2?0 met15?0 can1 tdp1?::KanMX
MATa his3?1 leu2?0 met15?0 sae2?::KanMX mre11-H125N
MAT? his3?1 leu2?0 met15?0 mre11-H125N
MATa his3?1 leu2?0 lys2?0 sae2?::KanMX
MAT? his3?1 leu2?0 lys2?0
MATa his3?1 leu2?0 met15?0 lys2?0 sae2?::KanMX
MAT? his3?1 leu2?0 met15?0
MATa his3?1 leu2?0 top1?::KanMX
MAT? his3?1 leu2?0 lys2?0 top1?::KanMX sae2?::KanMX
MAT? his3?1 leu2?0 met15?0
MATa his3?1 leu2?0 sae2?::KanMX mre11?::KanMX
MATa his3?1 leu2?0 met15?0 lys2?0 mre11?::KanMX
MAT? his3?1 leu2?0 lys2?0 sae2?::KanMX
MAT? his3?1 leu2?0 sae2?::KanMX
MATa his3?1 leu2?0 met15?0 rad9?::KanMX
MATa his3?1 leu2?0 met15?0 lys2?0 sae2?::KanMX rad9?::KanMX
MAT? his3?1 leu2?0 lys2?0
MATa his3?1 leu2?0 met15?0 LYS2 rad52?::KanMX
594C. Deng et al.
The most sensitive strains to camptothecin and others used in this study
Gene Protein functionT/CRank
ORF deletes MMS4
DNA helicase involved in DNA repair
Meiotic and mitotic recombination
Ubiquitin-protein ligase, chromosome stability
Top 1 interacting factor
B type cyclin in G1/S transition
Involved in rDNA transcription
ORF deletes MMS4
Protein serine/threonine phosphatase
Regulation of transcription from Pol II promoter
Controls mRNA decay
Biological process unknown
DNA-dependent ATPase, chromosome stability
Controls mRNA decay
Meiotic chromosome segregation
Regulation of transcription from Pol II promoter
Biological process unknown
Ubiquitin-conjugating enzyme in stress response
Endonuclease in nucleotide excision repair.
“Gene” indicates the gene deleted in the test strain. The gene MMS4 is listed three times, because deletions
at three close ORFs are all believed to result in the deletion of MMS4. “T/C” denotes the ratio of abundance
of that strain in the treated group divided by that in the control group. “Rank” denotes the ranking of the
sensitivity of each strain relative to the whole 4728 strains in the deletion pool. Parentheses indicate that
deletion of the ORF deletes MMS4 rather than a new gene.
the existence of other repair mechanisms for removing
Top1-DNA complexes in the absence of Tdp1 (Liu et
al.2002; Vanceand Wilson2002). Totestour hypothe-
sis that there may be other endonucleases capable of
removing the Top1-DNA complex in the absence of
Tdp1, we examined the results of the deletion pool
screen for other endonucleases that also cause a slight
to moderate sensitivity to camptothecin when deleted
(defined as in the top 150 sensitive strains to prolonged
camptothecin exposure). Four such genes were found:
SLX1, SLX4, RAD27, and SAE2. Experiments to deter-
mine their phenotype in relation to sensitivity to camp-
tothecin are described below.
Slx4 but not Slx1 is involved in repairing campto-
thecin-induced damage in the absence of Tdp1: SLX1
and SLX4 were originally identified as genes syntheti-
cally lethal with mutations in SGS1 or TOP3 (Mullen
et al. 2001). Slx1 and Slx4 coimmunoprecipitate, and
in vitro evidence shows that the proteins form a hetero-
dimeric complex that has strong endonuclease activity
(Fricke and Brill 2003). The complex is active in vitro
on branchedDNA substrates includingsimple Y,5? flap,
3? flap, replication forks, and Holliday junction sub-
strates, with a preference for the 5? flap, simple Y, and
replication fork structures. Since Rad1/Rad10 and
a simple Y and 3? flap structures, respectively (Bastin-
Shanower et al. 2003), have both been shown to be
required to remove Top1 complexes from DNA, we
reasoned that Slx1/Slx4 may have a similar function in
595Endonucleases Resolving Top1-DNA Adducts
Figure 1.—Loss of Slx4 but not Slx1 sensitizes tdp1mutants
to camptothecin. (a) Isogenic wild-type, tdp1, slx4, and slx4
tothecin at various concentrations. Their sensitivities were as-
sessed by the ratio of slope of the growth curve of the treated
(c) vs. the untreated (c ? 0) (see materials and methods).
(b) Isogenic wild-type, tdp1, slx1, and slx1 tdp1 single- and
double-deletion strains were tested as in a.
removing covalently complexed Top1. We employed
an assay developed by Vance and Wilson (2002) that
measures growth inhibition during continuous drug ex-
posure to measure the sensitivity of yeast to campto-
thecin. While the tdp1 and slx4 single-mutant strains
were only slightly inhibited by camptothecin, the tdp1
slx4 double mutant was substantially more sensitive to
This synergistic inhibition by camptothecin is similar to
that previously observed for the rad1 and tdp1 deletion
may represent another pathway to remove Top1 com-
plexes stabilized by camptothecin in the absence of
Tdp1. Surprisingly, Slx1 appears not to have the same
function as Slx4, as neither slx1 nor the slx1 tdp1 double
mutant was more sensitive to camptothecin than was the
wild type (Figure 1b). This result provides evidence that
Slx1 and Slx4 have separate functions in addition to their
shared endonuclease activities (Fricke and Brill 2003).
We next examined the relationship between Slx4,
Rad1, and Mus81. The rad1 tdp1 double mutant was
Figure 2.—Epistasis study of slx4, rad1, and mus81. Experi-
ments were carried out as in Figure 1. (a) The slx4 rad1 tdp1
triple-deletion strain is more sensitive to camptothecin than
either the slx4 tdp1 or the rad1 tdp1double-deletion strain. (b)
The slx4 rad1 double-deletion strain is not more sensitive to
camptothecin than either single-deletion strain. (c) The slx4
mus81 tdp1 triple-deletion strain is more sensitive than the slx4
tdp1 and mus81 tdp1 double-deletion strains. Standard errors
for three replicate experiments are shown but are smaller
than the points.
more sensitive to camptothecin than was the slx4 tdp1
mutant (Figure 2a), suggesting that Rad1 plays a more
active role than Slx4 in the removal of Top1 complexes.
As expected, both pathways are engaged only in the
absence of Tdp1, as rad1 and slx4 single and double
596 C. Deng et al.
mutants had sensitivities very similar to that of wild-type
cells (Figure 2b). However, deletion of SLX4 in the rad1
tdp1 or mus81 tdp1 strains caused additional sensitivity
epistasis group as either MUS81 or RAD1 (Figure 2, a
and c). This provides evidence that Slx4 is in another
pathway for repair of Top1-DNA complexes.
Sae2 and Mre11 participate in repairing campto-
thecin-induced damage: Sae2 was originally identified
by its requirement in meiotic recombination, a require-
ment that can be bypassed by a mutation in SPO11
(McKee and Kleckner 1997; Prinz et al. 1997). In the
sae2 mutant, meiotic recombination is blocked at an
intermediate stage, and covalent protein complexes are
found at the broken ends of double-strand breaks
(Keeney and Kleckner 1995). This and other pheno-
types of sae2, including repair of mitotic double-strand
breaks, are also observed in the separation-of-function
mutant mre11s-H125N (McKee and Kleckner 1997;
Prinz et al. 1997; Rattray et al. 2001). Mre11 is an
endonuclease that forms a complex with Rad50 and
Xrs2 to process double-strand breaks during meiosis,
tion of function mutant, rad50s-K81I, the covalent pro-
II-like protein that initiates meiotic recombination by
creating double-strand breaks (Keeney et al. 1997). On
the basis of the similarity of the phenotypes of sae2 and
rad50s-K81I and mre11s-H125N, it has been suggested
that Sae2 is required for the endonuclease activity of
chemical evidence has been reported (Rattray et al.
2001). We therefore asked whether Sae2 is involved in
repairing camptothecin-induced DNA damage. As shown
in Table 2, the sae2 deletion strain is among the strains
most sensitive to camptothecin in the deletion pool.
To examine the relationship between Sae2 and that
of the other proteins involved in repairing Top1-DNA
complexes we determined the sensitivity of strains with
in the pathway. Figure 3a shows that the sae2 deletion
strain was indeed very sensitive to camptothecin. Sur-
prisingly, deletion of TDP1 in this strain did not cause
significantly higher sensitivity. This pattern is similar to
that observed for mus81 (Liu et al. 2002; Vance and
Wilson 2002). To determine whether Sae2 constitutes
an additional pathway to remove Top1 complexes, we
studied the genetic relationship between Sae2, Rad1,
and Mus81. Figure 3, b and c, shows that deletion of
SAE2 in the rad1tdp1 and mus81tdp1 backgrounds caused
greater sensitivity to camptothecin than any double-dele-
tion combinations. These results suggest that Sae2 acts in
a redundant pathway to protect camptothecin toxicity in
the absence of repair by Tdp1, Mus81, or Rad1.
Because of the close similarity of sae2 and mre11s-
H125N mutants, we next examined whether Mre11 en-
Figure 3.—Sae2 is required for protection from campto-
ity compared with wild type or the tdp1 strain. Deletion of
TDP1 in the sae2 strain caused slight additional sensitivity. (b)
The tdp1 rad1 sae2 triple-deletion strain is more sensitive than
either the tdp1 rad1 or the tdp1 sae2 double-deletion strain.
(c) Similarly, the tdp1 sae2 mus81 triple-deletion strain showed
more sensitivity than the double-deletion strain of tdp1 sae2
or tdp1 mus81.
donucleaseactivity isinvolvedinremoving Top1.Figure
4a shows that the H125N mutation of MRE11 alone
caused a mild sensitivity to camptothecin. Moreover,
deletion of TDP1 and the mre11-H125N mutation were
synergistic in their sensitivity to camptothecin. This re-
sult suggests that Mre11 endonuclease activity is in-
597Endonucleases Resolving Top1-DNA Adducts
Figure 4.—Mre11 endonuclease domain is required for
protection from camptothecin, and its function is in the same
epistasis group as Sae2. (a) The mre11-H125N mutation strain
caused mild sensitivity to camptothecin, which is increased by
further deletion of TDP1. The triple-deletion strain mre11-
H125N tdp1 rad1 is more sensitive than either of the double-
deletion strains tdp1 mre11-H125N or tdp1 rad1. (b) The sae2
mre11-H125N double-mutant strain shows essentially the same
sensitivity as the sae2 deletion strain, indicating that they are
in the same epistatic pathway.
volved specifically in removing Top1 protein complex
stabilized by camptothecin at the DNA ends. Deletion
of RAD1 in the mre11-H125N tdp1 double mutant caused
further sensitivity to camptothecin (Figure 4a), suggesting
The suggestion that Sae2 is required for the endonu-
clease activity of the Mre11-Rad50-Xrs2 complex (Rat-
tray et al. 2001) predicts that these two mutants would
be epistatic. We therefore tested the double mutant sae2
mre11-H125N and, in agreement with this prediction,
strain (Figure 4b). This result is confirmed by our find-
ing that the double-deletion mutant sae2 mre11 has the
same sensitivity as the single-deletion mutant mre11
strain (Figure 5b). To test the specificity of the Sae2 for
lesions created by Top1, we tested the effect of deletion
of SAE2 in a strain deleted in TOP1. As expected, dele-
tion of TOP1 abrogated the sensitivity of wild-type cells
to camptothecin, but, more importantly, reversed the
Figure 5.—Sae2 acts on Top1 lesions and is epistatic with
Mre11 and not with Rad9. (a) The sensitivity of the sae2 dele-
tion strain to camptothecin is abrogated by deletion of TOP1,
showing that Sae2 is acting on lesions produced by Top1. (b)
The sae2 mre11 double-deletion mutant is no more sensitive
than the mre11 deletion strain, demonstrating that the two
operate in the same repair pathway. Also shown are three
replicate experiments (with standard errors) for a rad52 dele-
zation to camptothecin.
sensitivity of the sae2 deletion strain to the drug (Figure
5a). This result demonstrates that Sae2 acts on lesions
created by topoisomerase I. As a further test of the
repair pathway of Sae2, we crossed strains with deletions
in SAE2 and RAD9 and found that their sensitivities to
camptothecin were approximately additive (Figure 5c),
598 C. Deng et al.
thecin. This drug provides a useful way of “freezing”
the covalent linkage of Top1 to DNA, thereby revealing
the enzymes involved in normal resolution of the cova-
lent complex on single-stranded DNA. Previous work of
Tdp1 and the structure-specific endonuclease Rad1-
complex (Liu et al. 2002; Vance and Wilson 2002).
The Mus81-Mms4 endonuclease complex has also been
identified as playing a role in protection of cells against
camptothecin, either by restarting replication forks that
have stalled at Top1 complexes or by directly resecting
the DNA ends with covalently attached Top1 from a
duplex flap (Vance and Wilson 2002). Our genetic
evidence suggests that Mre11 and Slx4 also protect cells
against camptothecin and indicates that they are in dif-
ferent branches than the Rad1 and Mus81 pathways. It
therefore seems likely that there are three mechanisms
to protect cells from Top1-DNA complexes stabilized by
camptothecin (Figure 6). First, Top1 can spontaneously
cut and religate the DNA. The stalled replication forks
raman et al. 2001). If spontaneous reversion of Top1
does not occur, then Tdp1 can free Top1, thereby leav-
ing a 3? end phosphate group on the DNA that can
then be removed by Apn1, Apn2, and Tpp1 (Vance
of structure-specific endonucleases, including Rad1,
Slx4, Mre11, and Mus81, may be employed to remove
the DNA ends covalently bound to the Top1 protein
complex (Figure 6).
As these endonucleases have different substrate pref-
erences,on thebasis ofbiochemical evidenceit suggests
that a variety of DNA structures may arise from stalled
replication forks caused by camptothecin. Rad1, Slx4,
and Mus81 all act on branched DNA structures, but also
have their unique substrate specificities. While Rad1
prefers the simple Y flap, Mus81 preferentially cuts the
duplex flap and replication fork (de Laat et al. 1998;
Kaliraman et al. 2001; Bastin-Shanower et al. 2003).
We initially hypothesized that the involvement of Slx4
in Top1 removal would demonstrate the importance of
the Slx1-Slx4 complex, as it has been shown that Slx4
stimulates Slx1 5? flap nuclease activity (Fricke and
Brill 2003). However, the resistance of the slx1 tdp1
double null mutant to camptothecin argues against this
possibility (Figure 1b). It therefore seems likely that the
observed stimulation of Slx1 5? flap nuclease activity is
only one function of Slx4 in vitro. In vivo, Slx4 may be
responsible for a nuclease activity either by itself or
with another protein. The finding that Slx4 has weak
endonuclease activity on several sites of a simple Y sub-
strate appears to support this (Fricke and Brill 2003).
However, this activity is unlikely to be the mechanism
for its in vivo role in Top1 removal as suggested by our
result, because the cleavage sites by Slx4 alone in vitro
are all distal to the branch point; i.e., they are on the
Figure 6.—Diagram of pathways repairing camptothecin-
stabilized Top1 complex. Various lines of evidence indicate
that the substrate for repair is a double-strand break arising
Top1 (Pouliot et al. 1999). (Top) Spontaneous cutting and
camptothecin inhibits the ligation step. Stalled replication
forks can then be restarted possibly by Mus81. (Middle) Alter-
natively, Tdp1 can resect Top1 off the DNA backbone, leaving
a phosphate group at the 3? end of DNA. The phosphate
group can then be removed by the redundant phosphatases
Apn1, Apn2, and Tpp1. (Bottom) Several endonucleases can
directly remove the covalently bound Top1 together with a
DNA fragment. These endonucleases have different site selec-
tivities, implying that different DNA intermediates exist with
stalling of replication forks caused by camptothecin.
demonstrating that the protein products of these two
genesare actingin differentrepair pathways(as demon-
We noted that deletion of SAE2 was previously re-
ported to produce little sensitivity to camptothecin, ei-
ther by itself or in combination with tdp1 (Liu et al.
2002). We suggest that this discrepancy may be due to
the higher sensitivity in the assay we used, which in-
cludes growth inhibition in addition to cell killing.
also a flap endonuclease and is required for processing
tion of RAD27 causes mild sensitivity to camptothecin,
with a sensitivity ranking of 82 among the 4742 deletion
strains. However, deletion of TDP1 in the rad27 strain
did not cause significantly higher sensitivity, consistent
with a previous report (Vance and Wilson 2002). There-
fore Rad27 isnot specifically involved inremoving Top1
complexes from DNA ends.
We report in this work the identification of two new
pathways, involving the proteins Mre11 and Slx4, for
the removal of Top1 from DNA ends. The data have
been obtained using genetic analysis of the response of
mutant strains of yeast to the Top1 poison campto-
599Endonucleases Resolving Top1-DNA Adducts
overhanging single strand. Since Top1 is attached to
the 3? end of DNA, one would expect the cleavage sites
proximal (5?) to the branching point on the duplex to
be more effective in removing Top1, as seen with Rad1
and Mus81 (de Laat et al. 1998; Kaliraman et al. 2001;
Bastin-Shanower et al. 2003). An exception to this
scenario is that the presence of Top1 on the 3? DNA
ends causes significant melting of the duplex DNA ei-
ther through physical displacement caused by the bulky
Top1 protein complex or by a helicase or through a
5?-3? exonuclease on the other strand.
In contrast, Mre11 has single-strand DNA (ssDNA)
endonuclease and dsDNA and ssDNA 3?-5? exonuclease
activities (Usui et al. 1998). The mre11-H125N mutant
lacks endonuclease activity but keeps weak 3?-5? exo-
nuclease activity (Moreau et al. 1999). The resulting
mutant cannot process double-strand breaks (DSBs) in
meiosis, where the DNA 5? ends are covalently attached
to Spo11, but can still process the “clean” DSBs gener-
ated by HO endonuclease (Moreau et al. 1999). This
suggests that Mre11 endonuclease activity is essential to
process the meiotic DSBs that have a protein complex
attached at the 5? end. In meiosis, the ssDNA substrate
for Mre11 endonuclease activity is thought to be gener-
of unwinding duplex DNA, thereby allowing the endo-
exonucleaseactivity ofMre11 islikely tobeimportant in
meiosis but it would not be effective in the case of Top1
attached to the 3? DNA end. A 5?-3? exonuclease is
probably more appropriate to produce the ssDNA sub-
protein could generate the ssDNA substrate for Mre11.
In fact, all the endonucleases implicated in resecting
the Top1 attached DNA strand would be more efficient
with such unwinding activity. Although its nature is not
known, Srs2 is a reasonable candidate for this activity.
The srs2 deletion mutant is sensitive to camptothecin
(Table 1 and Vance and Wilson 2002), and there is a
synergism of camptothecin sensitivity between the srs2
and tdp1 mutants (Vance and Wilson 2002).
to its interaction with Mre11, as in other cases where Sae2
functions have been examined (McKee and Kleckner
1997; Prinz et al. 1997; Moreau et al. 1999; Mullen et
al. 2001), and our data showing that sae2 is epistatic to
the exonuclease-defective mre11-H125N mutant (Figure
ase activity. Thesae2 and mre11-H125N mutantsare simi-
lar to mus81 in that all are sensitive to camptothecin
(Figures 3 and 4). They are also unable to process mei-
otic DSBs, and the mutants are sporulation deficient
(McKee and Kleckner 1997; Prinz et al. 1997; Moreau
et al. 1999; Mullen et al. 2001).
In summary, we have identified Slx4 and Mre11 as
two endonucleases that protect against camptothecin
toxicity, probably by removing Top1 from 3? DNA ends.
Thus, they constitute two more branches of repair by
nucleases, in addition to Mus81 and Rad1. The function
of Slx4 in repairing camptothecin damage is distinct
from its known role as a partner and stimulator for the
ase domain in removing Spo11 in meiosis and Top1 in
both 5? and 3? DNA ends that are covalently attached
to protein complexes.
We thank Lorraine S. Symington (Columbia University) for provid-
ing the mre11-H125N strain and Angela Chu (Stanford University)
for providing the sae2, slx2, and slx4 deletion strains. This work was
supported by National Institutes of Health grant P01 CA67166.
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Communicating editor: L. S. Symington