From the Institut de Biologia Molecular de Barcelona, Consejo Superior de Investigaciones Científicas, Jordi Girona 18-26, 08034 Barcelona, Spain
Received for publication, September 29, 2000
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ABSTRACT |
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In topoisomerase-deficient yeast cells, we have
found that circular minichromosomes are present as broad distributions
of multimeric forms, which consist of tandemly repeated copies of their
monomeric sequences. This phenomenon selectively occurs in
Three distinct families of DNA topoisomerases disentangle DNA
strands and modulate DNA supercoiling inside the cell. Type IA
topoisomerases transiently cleave single-stranded DNA to allow the
passage of other DNA segments. Type IB topoisomerases cleave one DNA
strand in a double-helix and allow the rotation of the duplex by the
uncleaved strand before resealing the DNA. Type II topoisomerases
transiently cleave both strands in a double-stranded DNA segment and
transport another double-stranded DNA segment across the gated duplex
(for a review, see Ref. 1).
The main biological roles of eukaryotic DNA topoisomerases has been
inferred from studies in yeast cells. Saccharomyces
cerevisiae has three topoisomerases: topoisomerase I, a type IB
enzyme encoded by the TOP1 gene (2, 3); topoisomerase II, a
type II enzyme encoded by the TOP2 gene (4); and
topoisomerase III, a type IA enzyme encoded by the TOP3 gene
(5). One function of these enzymes is to remove DNA supercoiling.
During replication, DNA ahead of the replication fork becomes
positively supercoiled as the parental DNA strands come apart behind
it. During DNA transcription, the hindrance to the rotation of the
transcription ensemble drives positive supercoiling of DNA ahead of the
polymerase and negative supercoiling behind it (6). Yeast topoisomerase
I serves as the major DNA swivel during replication and transcription,
but this role can be fulfilled by topoisomerase II (7, 8); thus, both
enzymes are capable of relaxing positively and negatively supercoiled
DNA in vivo (9, 10). Because untwining of parental DNA
strands is often incomplete at the end of DNA replication, the other
main role of topoisomerases is to unlink the new pairs of replicated
DNA molecules. Decatenation activity of topoisomerase II is essential
for proper chromosome segregation during mitosis (11, 12) and meiosis
(13). The less abundant topoisomerase III has weak DNA relaxation
activity and does not contribute to supercoil removal in
vivo (14); it is, however, required to complete the final stages
of meiotic recombination (15).
Whereas TOP2 is essential for cell viability, cell growth is
not blocked by null mutations of the yeast TOP1 or
TOP3 genes. However, deficiency of any of the three yeast
topoisomerases is found to affect genome stability (for a review, see
Refs. 16 and 17). A null top1 mutant and a top2
thermo-sensible (ts)1 mutant,
grown at a semipermissive temperature, were shown to have a high
frequency of mitotic recombination in the rDNA cluster (18). In double
mutant yeast cells The mechanisms by which topoisomerases apparently suppress mitotic
recombination remain to be explored. While studying the biological
functions of DNA topoisomerases, using various mutants of the yeast
S. cerevisiae, we found evidence of the involvement of DNA
topoisomerases in genome stability. We observed that in the
topoisomerase double mutant Strains--
The yeast strains used in this study were kindly
provided by R. Kim and J. C. Wang (Harvard University). All
strains are derivatives of FY251 (MATa
his3- Plasmid and DNA Constructions--
Circular constructs Yp 0.8, Yp 1.4, Yp 1.5, and Yp 2, which did not contain DNA sequences to allow
bacterial amplification, were constructed by self-ligation of linear
DNA fragments obtained by the polymerase chain reaction or from
bacterial plasmids. Yp 0.8 (855 bp) was constructed from the
EcoRI yeast chromosomal fragment (1453 bp) containing
TRP1 and ARS1. The following primer sequences:
5'-GCCGGAATTCATTGAGCACGTGAGTATACG-3' and
5'-GCCGGAATTCTTTAGCATTATCTTTACATCTTG-3' were used to
amplify a segment of 869 bp having TRP1 and ARS1, which was then digested with EcoRI (primer-engineered
EcoRI sites are shown in boldface) and self-ligated to
obtain the 855-bp DNA ring. Yp 1.4 (1453 bp) was constructed by
circularizing the 1453-bp EcoRI yeast chromosomal fragment
containing TRP1 and ARS1. Yp 1.5 (1487 bp) was
constructed by circularizing a DNA fragment, which was composed of a
SacI-HindIII segment (1211 bp) containing yeast
URA3, followed by a HindIII-SacI
segment (276 bp) containing yeast ARS-HO. Yp 2 (2055 bp) was
constructed by circularizing a DNA fragment, which was composed of an
EcoRI-HindIII segment (1217 bp) containing yeast
URA3, followed by a HindIII-EcoRI (838 bp) segment containing yeast ARS1. Yp 3.4 (3468 bp) was
constructed by inserting the 1453-bp EcoRI yeast chromosomal
fragment containing TRP1 and ARS1 into pHC624 (a
2015-bp plasmid derivative of pBR322). Yp 4.6 (4414 bp) was constructed
by inserting the 1453-bp EcoRI yeast chromosomal fragment
containing TRP1 and ARS1 into pBluescript-KSII (a
2961-bp plasmid from Stratagene). Dimeric and trimeric constructs of Yp
1.4 were constructed by self-ligation of the 1453-bp EcoRI yeast chromosomal fragment containing TRP1 and
ARS1. Ligated products were treated with topoisomerase II to
eliminate catenanes, and the resulting circular species containing two
and three copies of the TRP1 ARS1 fragment were purified
from agarose gels. Plasmids pRK-G1T1 (19) and YCp-G1hT1 (25) were
kindly provided by R. Kim and J. C. Wang (Harvard University).
DNA Isolation and Hybridization--
DNA from transformed yeast
cells was prepared from yeast spheroplast as described by Sherman
et al. (26). Blot hybridization was as described by Southern
(27), using 32P-labeled DNA probes obtained by random
priming of gel-purified DNA sequences.
Assay of Topoisomerase II Unlinking Activity--
Yeast DNA
topoisomerase II was purified from a S. cerevisiae strain
BCY123 harboring a yeast DNA topoisomerase II expression clone
YEpTOP2GAL1 as described by Worland and Wang (28). Knotted DNA rings
from P4-phage capsids were purified and used to assay the DNA unlinking
activity of topoisomerase II as described by Liu et al.
(29).
DNA Minicircles Strongly Multimerize in Topoisomerase-deficient
Yeast Cells--
DNA minicircles of different size containing an
autonomous replicating sequence (ARS1 or ARS-HO)
and a selective gene marker (TRP1 or URA3) were
constructed to transform the S. cerevisiae strain FY251 and
its derivative double mutant JCW28 ( Multimers Are DNA Rings Containing Direct Tandem Repeats of the
Monomeric Ring--
The ladder-type distribution of multimers
generated in the TOP1 Is Essential to Avoid Multimerization--
To test whether
the formation of multimers in the double mutant strain
To corroborate that multimerization was strictly dependent of
topoisomerase I deficiency, we examined minichromosome stability in the
double mutant strain Both Formation and Resolution of Multimers Take Place in
Topoisomerase-deficient Yeast Cells--
For any given DNA construct,
the relative amounts of monomers and multimers observed in
topoisomerase-deficient yeast cells was very similar in all the
transformants examined, and did not change in ongoing generations of
the cells. This suggested that the distributions of multimers were the
result of a steady-state equilibrium between generation and resolution
pathways. To examine this hypothesis, we transformed the JCW28
( Multimerization Can Be Reverted by the Extrachromosomal Expression
of Topoisomerase I--
Experiments in the above section indicated
that interconversion among different multimeric forms of
minichromosomes takes place rapidly in topoisomerase-deficient cells.
We hypothesized that the restoration of topoisomerase I expression, in
cells already containing multimeric minichromosomes, would shift the
distribution of multimers toward the monomeric form. In the experiment
depicted in Fig. 5, the double mutant
JCW28 ( Multimerization of plasmids is mediated by factors involved in
homologous recombination, and has frequently been observed in bacteria
(30-33) and yeast (34). We report here that in topoisomerase-deficient yeast cells, circular minichromosomes generate broad distributions of
multimeric forms, which consist of tandem copies of their monomeric sequences. By examining the presence of such minichromosome multimers in a set of isogenic yeast strains, with mutations in one or more of
the three known topoisomerases, we find that the main determinant for
multimerization is the absence of topoisomerase I. In TOP1 cells, the monomeric forms of minichromosomes remain stable regardless of additional mutations in the TOP2 or TOP3
genes. Conversely, incipient multimerization appears in
The enhancing effect of the top2-4 mutation on the
formation of multimers, in the The appearance of minichromosome multimers in the
topoisomerase-deficient yeast cells could be due either to the high
instability of monomeric forms, or to the increased stability of the
oligomers. We show that DNA circles, consisting of two or three tandem
copies of a given construct, are not stable when introduced in
topoisomerase-deficient cells. Therefore, the distributions of
multimers observed probably reflects a dynamic equilibrium of a
bidirectional pathway, in which oligomeric constructs are continuously
generated and then transformed back to simpler forms. In
topoisomerase-deficient cells, both directions of the process could be
enhanced, but the tendency to generate multimers surpasses the tendency
to reduce them. In support of that, we show that pre-existing
distributions of multimers can be efficiently reverted to the monomeric
form just by turning on the expression of the TOP1 gene.
The multimerization of minichromosomes has several similarities with
the instability of the rDNA gene cluster also observed in
topoisomerase-deficient yeast cells. In a double mutant
The instability of circular minichromosomes and that of rDNA genes may
be variations of the same process. If that is the case, a common threat
is making circular minichromosomes and the rDNA genes particularly
sensitive to topoisomerase deficiency. This thread does not alter,
however, the stability of tandem copies of other DNA sequences located
at the chromosomal level (18). The instability of rDNA genes has been
attributed to their exceptionally high transcriptional rate (19). DNA
supercoiling generated during their transcription would persist longer
when the combined relaxation activity of the cellular topoisomerases is
low. Supercoiling in general and negative supercoiling in particular
may stimulate recombination by unwinding the DNA duplex or disrupting
the chromatin structure. However, the rate of transcription in the
circular constructs examined here (which harbor the yeast
TRP1 or URA3 genes) is not high. Nevertheless, we
envisage that in circular minichromosomes any sporadic burst of DNA
supercoiling might be more intense or persist longer than in
chromosomal sequences, and that such an effect would be magnified in
topoisomerase-deficient cells. One reason is that a burst of DNA
supercoiling in a small domain cannot be diluted to the same extend as
in a large one. Another reason is that the amount of topoisomerase
activity required to modulate DNA supercoiling must correlate with the
dimensions of the topological domains in which cellular DNA is
organized, rather than with the total amount of cellular DNA.
Therefore, the smaller the size of a topological domain, the more
limited the diffusible topoisomerase activity that will reach it.
Circular minichromosomes, hence, would become quickly affected when
cellular levels of topoisomerase are low. This scheme would explain why the instability of the monomers is inversely proportional to their size; and also, why all the multimeric distributions are centered around circles of similar dimensions (about 5-10 kb, as seen in Fig.
1), regardless of the size of the monomeric construct that generated
them. Taken together, these results suggest that topoisomerase dosage
is finely adjusted by the cell for the removal of DNA supercoils. On
one hand, cells might not be allowed to surpass a threshold amount of
topoisomerase activity. Overexpression of topoisomerase I from
multicopy plasmids carrying the TOP1 gene rapidly leads to
cell death (35). On the other, subthreshold levels of topoisomerases compromise genome stability, which is promptly manifested as increased recombination in certain multicopy sequences (circular minichromosomes and the rDNA cluster). The reported findings will provide a suitable model to further study the interplay between DNA topology and genome stability, as well as mechanistic aspects of DNA recombination.
top1 cells, and is highly magnified in double mutant
top1 top2-4 cells. No multimers are observed in single
mutant top2-4 or
top3 cells, or in
top1 cells that express a plasmid-borne TOP1
gene. Interconversion among multimeric forms takes place rapidly in
double mutant
top1 top2-4 cells, and the multimeric distributions are readily reverted to the monomeric form when a
plasmid-borne TOP1 gene is expressed from an inducible
promoter. These observations are a new example of the interplay between DNA topology and genome stability, and suggest that the cell capacity to modulate DNA supercoiling is limited when DNA is organized in small
topological domains. Yeast minichromosome multimerization provides an
appropriate system in which to study mechanistic aspects of DNA recombination.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
top1 top2-ts, grown at permissive temperature, destabilization of the rDNA gene cluster was revealed by
the presence of over half of the rDNA genes as extrachromosomal rings
containing one or more copies of the 9-kb rDNA unit (19). These excised
rings were found to integrate back into the rDNA locus, presumably
through homologous recombination, when plasmid-borne TOP1 or
TOP2 genes were expressed. Recombination in tandem arrays of
repetitive nucleotide sequences, other than the rDNA cluster, appears
to be unaffected by reducing the cellular level of DNA topoisomerase I
or II (18). On the other hand, null top3 mutants show an
increased recombination between a variety of sequence repeats (5),
including subtelomeric sequences (20).
top1 top2-ts circular
minichromosomes strongly multimerize. These multimeric forms consist of
tandem copies of the monomeric rings, and their formation or resolution is readily affected by DNA topoisomerase I.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
200 leu2-
1 trp1-
63
ura3-52). JCW 26 (top2-4), harboring a
thermo-sensitive mutation in the TOP2 gene, was derived by
one-step gene replacement (21). JCW27 (
top1) and JCW28
(
top1 top2-4) were derived from FY251 and JCW26,
respectively, by the hit-and-run method of gene replacement to create a
null mutation in the TOP1 gene (22). JCW253
(
top3::TRP1) and JCW273 (
top3::TRP1
top1) were derived by
one-step gene replacement from JCW25 and JCW27, respectively, to create
a null mutation in the TOP3 gene. Yeast cells were grown in
synthetic selective media as described by Sherman (23). Cell
transformation was carried out using the lithium acetate method
described by Ito et al. (24).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
top1
top2-4), which carries a deletion in the TOP1 gene and
a ts mutation in the TOP2 gene. Transformants of the FY251
and JCW28 strains were obtained in selective media plates and then
grown in liquid cultures at 26 °C, a permissive temperature for the
top2-4 mutant. DNA samples from individual transformants
were analyzed by agarose gel electrophoresis, and the DNA minicircle
sequences were identified by blot-hybridization with
32P-labeled probes. Fig.
1a depicts the structure of
the DNA minicircles constructed, and Fig. 1b shows the
typical electrophoretic patterns obtained with these constructs
following yeast transformation. As DNA samples were analyzed by
electrophoresis in TBE buffer containing calculated amounts of
chloroquine, covalently closed DNA molecules produced characteristic
ladders of topoisomers. In the FY251 strain, most of the constructs
migrated as monomeric DNA circles (Fig. 1b, lane 1 in each
panel). In contrast, in the JCW28 (
top1
top2-4) strain most of these DNA migrated as a variety of
multimeric forms (Fig. 1b, lane 2 in each panel).
The degree of multimerization was inversely proportional to the size of
the rings. The smaller the monomeric rings, the stronger the
multimerization. Constructs Yp 1.4, a 1.4-kb ring with the
TRP1-ARS1 sequence, and Yp 1.5, a 1.5-kb ring with the
URA3 ARS-HO sequence, similarly generate multimeric forms.
We ruled out the possibility that the presence of multimeric forms
could be due to their preferred uptake by the
top1
top2-4 cells at the time of transformation, thus identical
results were obtained by transforming these cells with gel-purified
monomeric forms of each construct. The multimers were invariably found
in any single transformant of the double mutant
top1 top2-4 strain, and the distribution of
multimers did not change significantly in exponentially growing cells
or in cell-saturated cultures.
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Fig. 1.
Transformation of the FY251 and JCW28 strains
with DNA minicircles. A, structure of the DNA
minicircles Yp 0.8, Yp 1.4, Yp 1.5, Yp 2, Yp 3.4, and Yp 4.6. These
circles were constructed as described under "Materials and
Methods," and the number given to name them relates to their sizes in
kb. Double line arches correspond to yeast sequences, as
indicated: TRP1 or URA3 are filled in
black, and ARS1 or ARS-HO are filled
in white. Thin line arches represent DNA sequences derived
from pBR322. Restriction sites relevant during their construction are
indicated (R, EcoRI; S,
SacI; and H, HindIII). b,
electrophoretic patterns of DNA minicircles recovered from the yeast
strains FY251 and its derivative JCW28 ( top1
top2-4). DNA samples were obtained from individual transformants
of FY251 (lane 1 in each panel) and JCW28 (lane 2 in
each panel) harboring the construct indicated on top of each
panel. Electrophoresis were done in agarose gels containing Tris
borate buffer plus 0.6 µg/ml chloroquine. Blot hybridization of the
gels was done with 32P-labeled probes obtained by random
priming of purified TRP1 or URA3 DNA
fragments.
top1 top2-4 strain suggested
that these DNA forms consisted of several complete copies of the
monomeric rings. This was confirmed by cutting them at restriction
sites that were unique to the monomeric sequences. Fig.
2a shows that the digestion of
the multimeric forms of the Yp 1.4 construct with EcoRI
endonuclease generated a unique DNA fragment of 1.4 kb, which
corresponds to the linearized monomeric sequence. In the experiment
depicted in Fig. 2b, DNA extracted from the
top1 top2-4 strain, which contained
multimeric forms of the Yp 1.4 construct, was mixed with knotted DNA
rings derived from P4-phage capsids. These mixtures were then treated with purified yeast topoisomerase II, and analyzed by agarose gel
electrophoresis in the presence of ethidium (Fig. 2b,
left panel). The knotted DNA rings derived from P4-phage
capsids, which migrated as an smeary distribution (Fig. 2b,
K), were converted to the unknotted form (Fig.
2b, U) in the reaction containing ATP and
topoisomerase II. The same gel was then blotted and probed to visualize
the multimeric forms of the Yp 1.4 construct (Fig. 2b,
right panel). No alteration in the distribution of multimers was observed following topoisomerase II treatment in any of the reacted
samples. This indicated, therefore, that the multimeric forms, rather
than catenanes, were circular DNA molecules consisting of tandem
repeats of the monomeric ring.
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Fig. 2.
Treatment of multimeric forms with
restriction nucleases and topoisomerase II. a,
multimeric forms of the Yp 1.4 construct, obtained from the double
mutant ( top1 top2-4), were digested with the
restriction nuclease EcoRI. Digested DNA was examined by
agarose gel electrophoresis, which was run in Tris borate buffer
containing saturating amounts of ethidium. Blot hybridization of the
gel was done with a 32P-labeled TRP1 probe.
Lane 1, monomeric construct Yp 1.4; lanes 2 and
3, DNA samples from JCW28 harboring Yp 1.4 before and after
treatment with EcoRI, respectively. L indicates
the position of linear 1.4-kb DNA. b, purified DNA,
extracted from the mutant strain JCW28 (
top1
top2-4) harboring multimers of the Yp 1.4 construct, was mixed
with an excess amount of knotted DNA circles (10 kb), purified from
P4-phage capsids. These DNA mixtures were incubated in the absence or
presence of yeast topoisomerase II and ATP, as indicated on the
top of the panel. Reacted samples were examined
by electrophoresis in a 1% agarose gel, for 16 h at 50 V in TBE
buffer containing saturating amounts of ethidium. After verifying by UV
light illumination (left panel) the complete unknotting of
phage DNA in the reaction that contained ATP and topoisomerase (Topo
II), the gel was blotted and hybridized with a 32P-labeled
TRP1 probe (right panel). K and
U, indicate the position of knotted and unknotted forms of
P4-phage DNA, respectively.
top1 top2-4 was strictly related to the
alteration of the TOP1 or TOP2 gene, we examined
the stability of monomeric rings in the single mutant strains JCW26
(top2-4) and JCW27 (
top1). Fig.
3a shows the electrophoretic
patterns of DNA rings extracted from strains FY251, JCW26, JCW27, and
JCW28, transformed with either the Yp 1.4 construct (lanes
1-4) or Yp 1.5 construct (lanes 5-8). We observed
that in the JCW26 (top2-4) strain the monomeric forms of
these constructs were stable (lanes 2 and 6). No
multimers were observed, even when this strain was grown at
semipermissive temperature (30 °C) (data not shown). Multimeric
forms of the constructs, however, were apparent in the single mutant
strain JCW27 lacking topoisomerase I (lanes 3 and
7). The degree of multimerization in this strain was,
however, much lower than in the double mutant
top1
top2-4 (lanes 4 and 8). We also examined
the occurrence of multimerization in cells defective in topoisomerase
III. The single mutant strain JCW253 (
top3)
and the double mutant strain JCW273 (
top1
top3) were transformed with the Yp 1.5 construct and
the monomer stability of this ring was examined. As shown in Fig.
3a, lanes 9 and 10, the absence of
topoisomerase III, either by itself or in conjunction with the absence
of topoisomerase I, did not contribute to the generation of multimeric
forms. The degree of multimerization in the double mutant
top1
top3 (lane 10)
was comparable to that observed in the single mutant
top1 (lane 7).
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Fig. 3.
Minichromosome multimerization in yeast
strains defective in one or several topoisomerases. a,
DNA was extracted from the isogenic strains FY251, JCW26
(top2-4), JCW27 ( top1), and JCW28
(
top1 top2-4) harboring either the Yp 1.4 construct (lanes 1-4, respectively) or the Yp 1.5 construct
(lanes 5-8, respectively). DNA was also extracted from the
strains JCW253 (
top3) and JCW273
(
top1
top3) harboring the Yp 1.5 construct (lanes 9 and 10, respectively). These DNA samples
were examined by agarose gel electrophoresis as described in the legend
to Fig. 1b. b, the double mutant strain JCW28
(
top1 top2-4) was transformed with plasmid
Ycp50, or one of its two derivatives pRK-G1T1 and Ycp-G1hT1, which
carry the yeast TOP1 and the human TOP1 genes
under the yeast pGAL1 promoter, respectively. These cells
were grown in selective media containing 2% galactose to activate the
expression of the TOP1 gene, and then transformed with the
Yp 1.4 construct. DNA samples of these double transformants, which grew
in selective media containing 2% galactose, were examined by gel
electrophoresis as described in the legend to Fig. 1b.
Lane 1, JCW28 containing Ycp50 and Yp 1.4. Lane
2, JCW28 containing pRK-G1T1 and Yp 1.4. Lane 3, JCW28
containing Ycp50 and Yp 1.4. Lane 4, JCW28 containing
Ycp-hG1T1 and Yp 1.4.
top1 top2-4 containing
the plasmid pRK-G1T1 (19). This plasmid derives from the single-copy
yeast vector YCp50 and expresses the yeast TOP1 gene from an
inducible yeast promoter pGAL1. As shown in the experiment
depicted in Fig. 3b, multimerization did not occur in
top1 top2-4 cells expressing the
plasmid-borne yeast TOP1 gene (lanes 1 and
2). An identical result was also obtained when expressing a
plasmid-borne human TOP1 gene (lanes 3 and
4). We conclude, therefore, that topoisomerase I was
necessary and sufficient to confer monomer stability of the circular
minichromosomes. On the other hand, the alteration of topoisomerase II
function as a result of the top2-4 mutation had no effect
by itself, but it dramatically augmented multimerization when
topoisomerase I was absent.
top1 top2-4) strain with gel-purified
constructs that contained either 2 or 3 tandem repeats of the monomeric
sequence of Yp 1.4. As shown in Fig. 4,
in all the transformants examined, a broad distribution of multimers of
the Yp 1.4 construct was observed (lanes 2 and
5). Such distributions contained more or fewer repeats than
the initial dimeric or trimeric construct used to transform the cells
(lanes 1 and 4), and were indistinguishable from
the distributions previously obtained with the monomeric rings.
Digestion of these multimers with the restriction enzyme
EcoRI yielded a unique product of 1.4 kb (lanes 3 and 6), which confirmed that such multimers consisted of
direct repeats of the monomeric sequence.
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Fig. 4.
Stability of multimeric constructs in
topoisomerase-deficient yeast cells. Dimeric and trimeric
constructs of the Yp 1.4 sequence were constructed as described under
"Materials and Methods," and used to transform the double mutant
JCW28 ( top1 top2-4) strain. Lane
1, purified dimeric Yp 1.4. Lane 2, DNA from JCW28
transformed with dimeric Yp 1.4. Lane 3, sample of
lane 2 digested with EcoRI. Lane 4,
purified trimeric Yp 1.4. Lane 5, DNA from JCW28 transformed
with trimeric Yp 1.4. Lane 6, sample of lane 5 digested with EcoRI. DNA samples were examined by agarose
gel electrophoresis as described in the legend to Fig. 1b.
M1 to M6 indicate multimeric forms containing 1 to 6 copies
of the Yp 1.4 construct, respectively. L indicates linear Yp
1.4 construct.
top1 top2-4), containing multimeric
forms of the Yp 1.4 construct, was transformed with the plasmid
pRK-G1T1. The distribution of multimers was then examined following the
repression or the induction of the plasmid-borne TOP1 gene.
As shown in Fig. 5a, multimers were present as long as the
cells grew in media containing 2% dextrose, which represses the
expression of TOP1 (lane 1). When cells were
transferred for 50 generations to a medium containing 2% galactose,
which induces the expression of TOP1, the distribution of
multimers was shifted toward the monomeric form (lane 2).
When the cells were returned to a medium containing 2% dextrose, the
multimers reappeared (lane 3). These were again reverted to
the monomer when the cells were grown in 2% galactose (lane
4). This effect of topoisomerase I expression on the
directionality of multimer formation or resolution is similar to that
described for the excision and integration of extrachromosomal rDNA
rings, which also happens in
top1 top2-4 strains (19). Hence, in the same DNA samples analyzed above, we
examined whether the formation and resolution of minichromosome multimers occurred in parallel to the excision and integration of rDNA
rings. As shown in Fig. 5b, extrachromosomal rDNA rings were
present in the
top1 top2-4 strain when the
TOP1 gene was repressed (lanes 1 and
3). Upon induction of the TOP1 gene (lanes 2 and 4), the excised rDNA rings integrated back into
the chromosome. The excision of rDNA rings and the multimerization of
minichromosomes, therefore, take place concomitantly in the
topoisomerase-deficient cells, and both processes are reversed upon
expressing TOP1.
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Fig. 5.
TOP 1 expression reverts the multimerization
of minichromosomes, and concomitantly leads to the integration of
excised rDNA rings. a, the double mutant strain JCW28
( top1 top2-4), containing multimeric forms of
the Yp 1.4 construct, was transformed with pRK-G1T1. DNA samples of
these double transformants were analyzed after growing them,
sequentially, in selective media containing 2% dextrose or 2%
galactose. Initially, the cells grew 50 generations in dextrose medium
(lane 1), then transferred to galactose medium for 50 generations (lane 2), again transferred to dextrose medium
for 50 generations (lane 3), and transferred again to
galactose medium for a further 50 generations (lane 4). DNA
samples were examined by two-dimensional electrophoresis, carried out
in a 1% agarose gel, that was run in TBE buffer plus 0.6 and 3 µg/ml
chloroquine, respectively, in the first (top to bottom,
12 h at 60 V) and second (left to right, 6 h at 60 V) dimensions. The gel was blot hybridized with a
32P-labeled TRP1 probe to reveal the positions
of the Yp 1.4 construct. b, the same DNA samples obtained in
the above experiment (lanes 1-4) were also examined by
electrophoresis (24 h at 30 V) in a 0.6% agarose gel containing Tris
borate buffer with saturating amounts of ethidium. This gel was
blot-hybridized with a 32P-labeled probe that revealed the
positions the yeast rDNA genes. R, extrachromosomal rDNA
rings. C, chromosomal rDNA.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
top1 or
top1
top3 strains, and multimerization increases
dramatically in the
top1 top2-4 strain. The
dependence of multimerization on topoisomerase I activity is ratified
by showing that it does not occur in
top1
top2-4 cells expressing a plasmid-borne TOP1 gene.
top1
background, suggests that this phenomenon is related to the overall
decreased capacity of these cells to remove DNA supercoils.
Topoisomerase I and II provide the DNA swivel activity in yeast cells.
In the single mutant
top1, topoisomerase II
might closely compensate the absence of topoisomerase I. In the double
mutant
top1 top2-4, however, DNA swivel
activity must be severely reduced. The amount of topoisomerase II
activity detected in lysates from top2-4 cells, grown at
permissive temperature (26 °C), is at least 10-fold less than that
detected in lysates from TOP2
cells.2 In that regard, as
the main role of yeast topoisomerase II is to unlink the newly
replicated DNA molecules, we suspected that in the
top1 top2-4 strain the increased
multimerization was due to the incomplete unlinking of monomeric rings.
We found, however, no trace of catenanes among the multimeric forms.
This observation highlights a topoisomerase II function apart from DNA
unlinking. The fact that
top3 strains does not
contribute to the formation of multimers, further supports the
hypothesis that multimerization is related to the reduced efficiency
with which DNA supercoils are removed. Although
top3 strains are hyper-recombinogenic (5), topoisomerase III is the least abundant cellular topoisomerase and its
ability to remove DNA supercoils is weaker than that of topoisomerase I
or II (14).
top1 top2-4 strain, over half the rDNA genes
are present as extrachromosomal rings containing one or more copies of
the 9-kb rDNA unit. These excised rings integrate back into the rDNA
locus when a plasmid borne TOP1 or TOP2 gene is
expressed (19). Like minichromosome multimerization, the instability of
the rDNA gene cluster is a bidirectional process, in which both the
forward and reverse pathways are probably mediated by homologous
recombination. We have verified that in
top1
top2-4 strains both cases of instability coexist and are
analogously tuned. In topoisomerase-deficient cells, excision dominates
over integration in the case of rDNA genes, and multimerization dominates over resolution in the case of minichromosomes. The expression of a plasmid-borne TOP1 gene readily eliminates
the multimers as well as the excised rDNA rings.
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FOOTNOTES |
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* This work was supported in part by Grants PB95-0131 and PB98-0487 from the Ministry of Science and Education of Spain (to J. R.).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 predoctoral training fellowship from the Ministry
of Science and Education of Spain.
§ To whom correspondence should be addressed: IBMB, Consejo Superior de Investigaciones Científicas, Jordi Girona 18-26, 08034 Barcelona, Spain. Tel.: 34-93-4006178; Fax: 34-93-2045904; E-mail: jrbbmc@cid.csic.es.
Published, JBC Papers in Press, October 27, 2000, DOI 10.1074/jbc.M008930200
2 S. Trigueros and J. Roca, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: ts, temperature sensitive; bp, base pair(s); kb, kilobase(s).
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