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J. Biol. Chem., Vol. 277, Issue 40, 37207-37211, October 4, 2002
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andFrom the Institut de Biologia Molecular de Barcelona, Consejo Superior de Investigaciones Científicas, Jordi Girona 18-26, 08034 Barcelona, Spain
Received for publication, July 4, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In the yeast Saccharomyces
cerevisiae, DNA topoisomerases I and II can functionally
substitute for each other in removing positive and negative DNA
supercoils. Yeast The budding yeast Saccharomyces cerevisiae has three
structurally different DNA topoisomerases: topoisomerase I, a type IB enzyme encoded by TOP1; topoisomerase II, a type II enzyme
encoded by TOP2; and topoisomerase III, a type IA enzyme
encoded by TOP3 (For recent reviews see Refs. 1 and 2).
Topoisomerase II is essential for cell viability because it is required
to unlink intertwined pairs of replicated DNA domains at the time of
mitosis (3). Another important role of topoisomerases is to prevent excessive supercoiling of DNA (4, 5). Yeast topoisomerase I efficiently
relaxes the positive and negative DNA supercoils generated,
respectively, in front of and behind the molecular ensembles that track
along the double helix (6-8). Because yeast null top1
mutants are viable (9, 10), the cellular roles of topoisomerase I must
be fulfilled by yeast topoisomerase II, which is also efficient in the
relaxation of positive and negative supercoils (Fig.
1). Conversely, the less abundant
topoisomerase III does not have significant effects on the overall
supercoiling of intracellular DNA (11).
top1 top2(ts) mutants grow
slowly and present structural instability in the genome; over half of
the rDNA repeats are excised in the form of extrachromosomal rings, and
small circular minichromosomes strongly multimerize. Because these
traits can be reverted by the extrachromosomal expression of either
eukaryotic topoisomerase I or II, their origin is attributed to the
persistence of unconstrained DNA supercoiling. Here, we examine whether
the expression of the Escherichia coli topA gene, which
encodes the bacterial topoisomerase I that removes only negative
supercoils, compensates the phenotype of top1
top2(ts) yeast cells. We found that top1
top2(ts) mutants expressing E. coli
topoisomerase I grow faster and do not manifest rDNA excision and
minichromosome multimerization. Furthermore, the recombination frequency in repeated DNA sequences, which is increased by nearly two
orders of magnitude in top1 top2(ts) mutants
relative to the parental TOP+ cells, is restored to normal
levels when the bacterial topoisomerase is expressed. These
results indicate that the suppression of mitotic hyper-recombination
caused by eukaryotic topoisomerases I and II is effected mainly by the
relaxation of negative rather than positive supercoils; they also
highlight the potential of unconstrained negative supercoiling to
promote homologous recombination.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Capacity of different topoisomerases to relax
DNA supercoils in yeast cells. Endogenous topoisomerase I and II
remove both the positive (S (+)) and the negative (S
( 
)) supercoils generated during DNA transcription. E. coli topoisomerase I, which can be expressed in S. cerevisiae from a plasmid-borne top A gene, removes
mainly the negative supercoils.
Genetic and biochemical studies have revealed that the DNA relaxation
activity of yeast topoisomerases I and II has deep implications in
genome stabilization (5, 12). Null top1 mutants have a high
frequency of mitotic recombination in the rDNA cluster relative to
TOP+ cells (13). Single top2(ts)
mutants, in which the weak topoisomerase II activity is abolished at
35 °C, also show increased recombination in the rDNA (13) when grown
at a permissive temperature (26 °C). More striking features of
genome instability are observed in topl
top2(ts) double mutants (Fig.
2). In these cells, over half of the rDNA
genes are excised in the form of extrachromosomal rings, which contain
one or more copies of the 9-kb rDNA unit (14), and small circular
minichromosomes form broad distributions of multimers, which
consist of tandemly repeated copies of their monomeric sequences (15).
These traits, probably mediated by homologous recombination, have been
attributed to excessive supercoiling of DNA (12, 14, 15). Whereas in
topl or top2(ts) single mutants the
presence of either topoisomerase II or I suffices to relax DNA,
supercoils persist longer in the double mutant because of the limited
activity of topoisomerase II available at 26 °C. The formation of
extrachromosomal rDNA rings and the multimerization of circular
minichromosomes can be reverted by the extrachromosomal expression of
yeast topoisomerase I or II (14, 15). Because these enzymes are
structurally different, only their common ability to relax DNA
supercoils can account for the suppression of the instability
traits.
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Here we investigate whether the positive or the negative supercoiling
of DNA differentially determine the genetic instability found in
topoisomerase-deficient cells. We examined the phenotype of
topl top2(ts) mutants supplemented by the
extrachromosomal expression of the S. cerevisiae
topoisomerase I, the S. cerevisiae topoisomerase II, and the
Escherichia coli topoisomerase I. Unlike the yeast
topoisomerases I and II, which relax positive and negative supercoils,
E. coli topoisomerase I is a type IA enzyme that
normally removes only negative supercoils (16) (Fig. 1). Previous
studies have demonstrated that it can be expressed and that it is
active in yeast cells (7, 8). We report that the compensations achieved
by expressing the E. coli DNA topoisomerase I are comparable with those attained with the yeast topoisomerases I and II. These observations reveal that genetic instability in
topoisomerase-deficient cells is mainly a result of the failure to
relax the negative rather than the positive supercoiling of
intracellular DNA.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Strains and Plasmids--
Yeast strains were originally obtained
from the laboratory of James C. Wang (Harvard University). All strains
are derivatives of FY251 (MATa his3-200 leu2-1
trp1-63 ura3-52). JCW26 (top2-4), harboring a
thermosensitive mutation in the TOP2 gene, was derived by
one-step gene replacement (17). JCW27 (top1) and JCW28
(top1 top2-4) were derived from FY251 and JCW26,
respectively, by the hit-and-run system of gene replacement to create a
null mutation in the TOP1 gene (18). Yeast cells were grown
in synthetic selective or rich medium (19), and cell transformations
were done by the lithium acetate method (20).
Plasmids YEptopA-PGAL1, pRK-G1T1, and YEpTOP2GAL1 were provided by James C. Wang (Harvard University). YEptopA-PGAL1 (7) contains the topA gene (coding for E. coli topoisomerase I) downstream of the yeast PGAL1 promoter, the 2µ plasmid origin of replication, and the URA3 marker. pRK-G1T1 (14) derives from the single-copy yeast vector YCp50 and expresses the yeast TOP1 gene from the galactose-inducible yeast promoter pGAL1. YEpTOP2GAL1 (21) derives from the multicopy yeast vector YEp24 (NEB) and expresses the yeast TOP2 gene from the yeast promoter pGAL1. Construction of the yeast minicircle Yp 3.4 (3.4 kb containing the yeast TRP1-ARS1 sequence) has been reported previously (15).
Plasmid pRS314-L was provided by Andrés Aguilera (Universidad de Sevilla). It contains two 598-bp ClaI-EcoRV LEU2 fragments repeated in direct orientation and separated by 31 bp of linker (22).
Plasmid pLeeuu2 was constructed in two steps. First, two overlapping fragments from the LEU2 gene (2225 bp) of YEp13 (23) were inserted sequentially into a regular cloning vector. One XhoI-EcoRI fragment comprised the first 1295 bp of LEU2, and the other ClaI-SalI fragment comprised the last 1416 bp of LEU2. The EcoRI-AccI 628-bp fragment of pBR322 was then inserted between these overlapping LEU2 sequences. From this construct, a 3339-bp XhoI-SalI fragment spanning the overlapping LEU2 sequences plus the linker region was obtained and inserted into the SalI site of Yp 3.4, which provided the yeast ARS1 sequence and the TRP1 marker.
Determination of Recombination Frequencies--
Recombination
frequencies were calculated as the median of three fluctuation tests,
each performed with six independent colonies for each transformant
studied (22). Yeast strains carrying plasmids with the URA3
marker were grown in SC (ura) medium. After transformation with
pRS314-L or pLeeuu2, which carried the TRP1 marker,
independent colonies selected on SC (trp) plates were serially
diluted and plated on SC (leu) to determine the median frequency of
Leu+ recombinants. The viable cell number was
simultaneously determined on SC (trp) plates.
DNA Extraction and Electrophoretic Analysis--
DNA from
transformed yeast cells was prepared from yeast spheroplast (24). DNA
blot-hybridization (25) was done using 32P labeled DNA
probes obtained by random priming of gel-purified DNA fragments.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Expression of the E. coli Topoisomerase I Compensates the Slow
Growth of top1 top2-4 Yeast Mutants--
The yeast strain JCW28
(top1 top2-4) was transformed with pRK-G1T1,
YEpTOP2GAL1, or YEptopA-PGAL1. These plasmids carried under
the yeast GAL1 promoter the genes encoding for
S. cerevisiae topoisomerase I, S. cerevisiae topoisomerase II, and E. coli topoisomerase I, respectively. JCW28 cells were also transformed with YEp24, a
control plasmid. Single transformants, selected from SD (ura) plates,
were grown at 26 °C in liquid selective media containing a
range of glucose or galactose concentrations, to express the topoisomerases by gradual activation of the GAL1 promoter.
Cell duplication times were then measured during exponential growth (Fig. 3). The JCW28 strain harboring the
control plasmid duplicated every 8 h in media containing 2%
glucose, and every 9 h in media containing 2% galactose. Cells
harboring pRK-G1T1, however, improved their growth rate in media
containing 2% glucose (duplication every 6 h) and reached optimal
growth in media containing 1% glucose + 1% galactose (duplication
time about 4 h). In media with 2% galactose, their growth was
impaired because overexpression of eukaryotic topoisomerase I became
toxic. Similarly, cells harboring YEpTOP2GAL1 grew faster with 2%
glucose (duplication every 6 h) and reached optimal growth with
1.4% glucose + 0.6% galactose (duplication time about 4 h).
Overexpression of topoisomerase II by increasing the galactose
concentrations also became toxic. When the cells harbored
YEptopA-PGAL1, the expression of E. coli topoisomerase I also improved the growth rate of the double
mutant, although a higher induction of the GAL1 promoter was
required. These cells duplicated in less than 6 h in media
containing 0.2% glucose + 1.8% galactose; their growth rate did not
improve when the galactose concentration was raised to 2%.
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E. coli Topoisomerase I Reduces the Excision of rDNA Genes in
top1 top2-4 Yeast Cells--
The JCW28 (top1
top2-4) cells, transformed with either pRK-G1T1, YEpTOP2GAL1, or
YEptopA-PGAL1, were grown at 26 °C in selective media SC
(ura) containing amounts of glucose and galactose that yield optimal
growth rate, as inferred above. For each case, JCW28 cells harboring
YEp24 (the control plasmid) were grown in parallel conditions. After a
minimum of 50 generations, DNA of the cells was extracted and analyzed
by electrophoresis to examine the migration of rDNA sequences.
Extrachromosomal rDNA rings (R in Fig. 4) were present in
the cells carrying YEp24 (Fig. 4,
lanes 1, 3, and 5). As expected,
either the expression of yeast topoisomerase I (Fig. 4, lane
2) or yeast topoisomerase II (Fig. 4, lane 4) reduced the formation of extrachromosomal rDNA rings. The expression of E. coli topoisomerase I (Fig. 4, lane 6) also
decreased ring excision, because the rDNA sequences were found
to be integrated mostly in the chromosomal DNA (C in Fig.
4).
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E. coli Topoisomerase I Prevents DNA Ring Multimerization in
top1 top2-4 Yeast Cells--
The same set of cells described above
for the analysis of the rDNA sequences was further transformed with the
Yp 3.4 minicircle, which multimerizes in the top1 top2-4
mutant (15). Single colonies of the double transformants were grown at
26 °C in selective media SC (uratrp) containing optimal amounts
of glucose and galactose. After a minimum of 50 generations, DNA was
extracted and analyzed by electrophoresis to examine the migration of
Yp 3.4. In the cells harboring the control plasmid (Fig.
5, lanes 1, 3, and
5), over half of the Yp 3.4 minicircles (Fig. 5,
M1) were present as multimeric forms (M2, M3, M4,
M5). As expected, in the cells expressing yeast topoisomerase I
(Fig. 5, lane 2) or yeast topoisomerase II (Fig. 5,
lane 4) the Yp 3.4 circles were found stabilized in the
monomeric form (M1). Notably, in the cells that expressed the E. coli topoisomerase I (Fig. 5, lane 6), multimerization of Yp 3.4 was also prevented.
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Expression of E. coli Topoisomerase I Prevents the DNA
Hyper-recombination Found in top1 top2-4 Yeast Mutants--
To
explore whether E. coli topoisomerase I had a more general
effect on genome stabilization in yeast cells defective in
topoisomerase activities, we determined its effect on homologous
recombination frequency. As substrates for DNA recombination, we used
either a yeast multicopy plasmid, pLeeuu2, or a yeast centromeric
plasmid, pRS314-L (Fig. 6A).
In both constructs, homologous recombination between two directly
repeated sequences would reconstitute the yeast LEU2 gene
(Fig. 6B). We determined the recombination frequency of
pLeeuu2 and of pRS314-L within the set of isogenic strains FY251
(TOP+), JCW26 (top2-4), JCW27
(dtop1), and JCW28 (dtop1 top2-4). Each
strain carried YEp24, pRK-G1T1, YEpTOP2GAL1, or YEptopA-PGAL1 at the time of transformation with pLeeuu2 or
pRS314-L. Recombination frequencies were calculated as explained in the experimental section.
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The graphs in Fig. 7 summarize the
averaged values obtained in each of the above described 24 double
transformants after three independent tests. The recombination
frequencies for pLeeuu2 and for pRS314-L were similar and their
variations parallel. When the strains harbored the control plasmid
YEp24, recombination frequency in TOP+ cells was about
3 × 10
4. This value did not increase significantly
in the cells with single mutations in the TOP1 or
TOP2 genes. Conversely, in the top1 top2-4
cells, recombination frequency was nearly two orders of magnitude
higher than the parental TOP+ cells.
Extrachromosomal expression of yeast topoisomerase I or yeast
topoisomerase II did not alter the recombination values in
TOP+ cells or in cells with single mutations in the
TOP1 or TOP2 genes. However, in the double mutant
(top1 top2-4), expression of either yeast topoisomerase I or II prevented hyper-recombination. Comparable effects were found in
cells expressing the E. coli topoisomerase I. Whereas recombination frequencies were not altered in TOP+ or in the
cells with single mutations in the TOP1 or TOP2
genes, the bacterial topoisomerase reduced the hyper-recombination
of the top1 top2-4 double mutants.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The effect of E. coli topoisomerase I on the slow
growth of top1 top2-4 yeast cells indicates that the
bacterial enzyme fulfills a task commonly shared by yeast topoisomerase
I and II. The yeast double mutant duplicates every 8 h, which is
about four times slower than single top1 or
top2-4 mutants and parental TOP+ cells. Slight
expression of either yeast topoisomerase I or II, from plasmid-borne
genes located under the yeast GAL1 promoter, is enough to
improve the growth rate of the double-mutant. Higher expression of
these enzymes was toxic, especially in yeast topoisomerase I, in
which TOP1 gene cannot be placed in multicopy plasmids. The
growth compensation by E. coli topoisomerase was achieved by
placing the topA gene under the GAL1 promoter, in
a multicopy plasmid, under high induction conditions (1.8% galactose,
0.2% glucose). Because higher induction (2% galactose) did not
improved the growth compensation, we consider these conditions close to providing optimal expression of E. coli topoisomerase I for
the compensation of the top1 top2-4 phenotype.
The excision of rDNA genes and the multimerization of small plasmids in
top1 top2-4 cells are dynamic bidirectional processes, probably mediated by homologous recombination, the origin of which is
attributed to the persistence of unconstrained positive and/or negative
supercoils (14, 15). We have corroborated this origin by showing
how both instabilities are reverted by the extrachromosomal expression
of either yeast topoisomerases I or II. The analogous effect of
expressing the E. coli topoisomerase I indicates that relaxation of negative supercoils is sufficient as a substitute for the normal roles of the yeast topoisomerases in suppressing these
forms of instability. Still, we considered the possibility that the
stabilizing effect of the E. coli topoisomerase I
could affect DNA recombination pathways in a different manner. Like other type IA enzymes, namely bacterial topoisomerase III (26) and
yeast topoisomerase III (27), E. coli topoisomerase I could influence recombination by acting on linkages of single strands of DNA
rather than by relaxing supercoiled duplexes. Nevertheless, the
stabilizing effect of E. coli topoisomerase I by its removal of negative supercoils was corroborated by examining the frequency of
homologous recombination in several yeast strains expressing the
topA gene. In agreement with previous reports (13), for top1 or top2-4 single mutants the
recombination frequency in repeated sequences (other than the rDNA) was
similar to TOP+ cells. Because the expression of E. coli topoisomerase I did not alter these values, it is unlikely
that the activity of the bacterial enzyme inhibits or interferes with
mitotic recombination in yeast. In the top1 top2-4
double mutant, however, the frequency of homologous recombination
increased by nearly two orders of magnitude. Extrachromosomal expression of either yeast topoisomerase I or II fully prevented this
increment; and, in this case, expression of E. coli
topoisomerase I also reduced markedly the hyper-recombination. Because
the only common feature shared by the bacterial topoisomerase I and the yeast topoisomerases I and II is the ability to relax negative DNA
supercoils, these are likely the main determinant for the DNA
instabilities occurring in the top1 top2-4 mutants.
The complementation achieved by the DNA relaxation activity of E. coli topoisomerase I in yeast cells is not surprising. First, yeast topoisomerase I and II can replace each other for DNA relaxation, despite their different mechanism and mode of interaction with DNA (4-8). This indicates that most supercoil removal in yeast DNA does not rely on specific molecular interactions. Second, the main role of E. coli topoisomerase I in the bacteria is to relax negative supercoils. Early observations showed that E. coli cells defective in topoisomerase I can survive if they have compensatory mutations affecting DNA gyrase (28), a type II topoisomerase that introduces negative supercoils. Alternatively, E. coli topoisomerase I mutants can be compensated either by the expression of eukaryotic topoisomerase I (29) or by the overexpression of bacterial topoisomerase IV (30). Both enzymes are efficient supercoil removers. More recent evidence confirms the essential function of E. coli topoisomerase I in relaxing unconstrained negative supercoils to avoid R-loop formation (31).
Our results strengthen the notion that DNA supercoiling can be a strong
promoter of genome instability in eukaryotic cells. How unconstrained
supercoils stimulate mitotic recombination remains to be explored.
Supercoiling in general could disrupt chromatin structure, promote DNA
structural transitions, or increase DNA sensitivity to nucleases.
Negative supercoiling, in particular, could facilitate recombination by
favoring DNA strand invasion or branch migration. Further studies with
yeast cells harboring different topoisomerase activities are required.
So far, we conclude that the genetic instability generated by the
combined deficiency of eukaryotic topoisomerases I and II can be
attributed mainly to failure to remove negative supercoils rather than
positive ones.
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ACKNOWLEDGEMENTS |
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We thank J. C. Wang and A. Aguilera for providing yeast strains and plasmids.
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FOOTNOTES |
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* This study was supported by Grants PB95-0131 and PB98-0487 from the Ministry of Science of Spain.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a pre-doctoral training fellowship from the Ministry
of Science of Spain. Current address: Dept. of Molecular and Cellular
Biology, Harvard University, 7 Divinity Ave., Cambridge, MA 02138.
§ To whom correspondence should be addressed: Institut de Biologia Molecular de Barcelona-Consejo Superior de Investigaciones Científicas, Jordi Girona 18-26, E-08034 Barcelona, Spain. Tel.: 34-93-4006178; Fax: 34-93-2045904; E-mail: jrbbmc@cid.csic.es.
Published, JBC Papers in Press, July 31, 2002, DOI 10.1074/jbc.M206663200
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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