Role of Topoisomerase I in the Stability of the Ribosomal DNA
of Saccharomyces cerevisiae
by
Fred S. Dietrich
B.S. Mathematics/Zoology, University of California - Davis
(1985)
Submitted to the Department of Biology
in partial fulfillment of the requirements for the
Degree of
Doctor of Philosophy
in Biology
at the
Massachusetts Institute of Technology
February, 1996
© Massachusetts Institute of Technology
All Rights Reserved
Signature of Author:
Fred S. Dietrich
Thesis Supervisor:
Dr. Gerald R. Fink
- sor of Biology
Certified by:
Frank Solomon
Professor of Biology
Chairman, Committee on Graduate Students
JUL 0 8 1996
Role of Topoisomerase I in the Stability of the Ribosomal DNA
of Saccharomyces cerevisiae
by
Fred S. Dietrich
Submitted to the Department of Biology
on Feb 1, 1996 in partial fulfillment of the requirements for the Degree of
Doctor of Philosophy in Biology
ABSTRACT
Mitotic recombination in the rDNA cluster is suppressed by DNA
topoisomerase I and II. Strains containing either a null allele of the TOP1
gene, or a temperature sensitive allele (top2-1) of the TOP2 gene have a rate
of mitotic recombination in the rDNA approximately 100 fold higher than
do wildtype strains. These mutations have little, if any, effect on
recombination at other loci. Using pulse field gel electrophoresis I have
further investigated the role of topoisomerases in rDNA. Under a variety of
running conditions chromosome XII DNA (containing the rDNA repeat) in
a topI strain fails to enter the gel, where as this chromosome enters the gel
normally in DNA made from an isogenic TOPi strain. This aberrant
migration of chromosome XII is observed only in DNA preparations made
from logarithmically growing cells.
Thesis Supervisor: Dr. Gerald R. Fink
Title: Professor of Biology
Acknowledgments
I am grateful to all the wonderful members of the Fink lab for their
assistance and tolerance. I particularly want to acknowledge the assistance of
Cora Styles and of Mike Christman in matters technical and Chris Dreft for
many constructive discussions of the materials in this thesis. I would also like
to thank Gerry Fink for his guidance and support.
I also wish to thank my parents, Sam and Mary Dietrich for their
unfaltering support.
Table of Contents
Title Page ............................................................................
1
Abstract.......................................................................................
.....
2
Acknow ledgm ents..........................................................................
3
Table of Contents.............................................................................
4
List of Figures........................................................
5
List of Tables ................................................
6
.........................
Chapter I - Introduction ............................
7-43
The DNA Rotation Problem in Replication........
8
The DNA Rotation Problem in Transcription....
10
Topoisomerases ..........................................................
11
Biological Functions of Topoisomerases......... .
15
Topoisomerse Mutants...................................
17
Ribosomal DNA and Topoisomerases............
18
Examination of Yeast Chromosomes.............
23
References
25
...............................
Chapter II - Mitotic Recombination in the rDNA of
S. cerevisiae is suppressed by the combined
action of DNA topoisomerase I and II................
44-57
Chapter III - The ribosomal DNA array has an altered
structure in topoisomerase I mutants of
S. cerevisiae..........
.....................
58-77
List of Figures
Chapter I
Figure 1 - Supercoiling by the DNA replication
m achinery .............................................................
Figure 2 - Overall arrangement of the ribosomal DNA
of S. cerevisiae ........................................................
Chapter II
Figure 1 - Loss of a URA3 Insertion in the rDNA via
Mitotic Recombination...............................
Figure 2 - Segregation of Form I/Form II rDNA
Polymorphisms after Recombination.............
Figure 3 - DNA Probe Used to Distinguish between the
Form I and Form II rDNA Polymorphisms.......
Figure 4 - Southern Analysis of Tetrads to Follow the
Segregation of Form I/FormII rDNA
Polymorphisms in Mitosis and Meiosis in
topl/topl and TOP1/TOP1 Backgrounds.........
Figure 5 - Segregation of rDNA Markers at Meiosis
following Unequl Sister Chromatid Exchange
in the rDNA During Meiosis.............................
Chapter III
Figure 1 - Pulsed field gel electrophoresis of yeast
chromosomal DNA................................
Figure 2 - Pulsed field gel electrophoresis of yeast
restriction digested DNA.....................................
Figure 3 - Construction of a strain containing rDNA on
chrom osom e III................................. . ............
Figure 4 - Chromosome preparations from top1 and TOP1
strains grown to stationary phase.......................
Figure 5 - Chromosomes from top2 strains.............................
10
19
46
48
48
49
52
63
65
66-67
69
71
List of Tables
Chapter II
Table 1 - Yeast Strains..................................................................
46
Table 2 - Frequency and Rate of Mitotic Recombination in
rDNA in TOP1 and top1 Strains......................
47
Table 3 - Frequency of Mitotic Recombination in rDNA in
rDNA in top2-1(ts) and TOP2 Strains................
49
Table 4 - Mitotic Recombination at Non-rDNA Loci in top1
and TOP1 Strains........................
..............
50
Table 5 - Mitotic Recombination at Non-rDNA Loci in
top2-1(ts) and TOP2 Strains.............................
50
Table 6 - Frequency of Mitotic Recombination in rDNA in
rad52 strains.............................................
..........
51
Table 7 - Meiotic Recombination in rDNA in topl/topl
and TOP1/TOP1 Backgrounds...................
52
Chapter I
Introduction
Intoduction
The subject of my thesis is how a specific class of yeast enzymes, the
topoisomerases, interact with a specific portion of the yeast genome, the
ribosomal DNA. Both of these subjects, topoisomerases and ribosomal DNA,
have been extensively studied; the following introduction covers the more
relevant aspects of these fields.
The double helix structure of DNA imposes certain restrictions on the
mechanisms of DNA replication, transcription, and recombination. This
restriction, arising out of the necessity to unwind the strands, was first
realized to be a problem soon after the elucidation of the structure of DNA
(Watson and Crick 1953; Delburck 1954). Though early on it was realized that
a DNA swivel was needed for unwinding the DNA, it was not until 1971 that
such a swivel, in the form of an enzyme now referred to as a topoisomerase,
was first reported (Wang 1971).
The DNA Rotation Problem in Replication
To replicate DNA it is first necessary to separate the two DNA strands in order
for the replication complex to form. This process has been observed through
sensitivity of the origin region, e.g. oriA (Schnos et al. 1988), to single strand
nucleases such as S1 and P1. For DNA replication to proceed either the
replication complex must rotate, following the rotation of the parental DNA,
or a swivel must be introduced into the DNA to allow rotation around the
DNA axis (See figure 1). Rotation of the DNA complex results in two highly
intertwined daughter molecules which in the case of a circular DNA
molecule is resolvable only by breaking one daughter molecule and passing
the other daughter molecule through the break. A swivel necessitates at least
one additional swivel after the replication fork to reintroduce the twists that
were removed ahead of the replication fork.
DNA molecules can be underwound (i.e. less tightly twisted than a relaxed
molecule) or overwound (more tightly twisted) relative to the relaxed
molecule. Underwinding or overwinding is generally referred to as
supercoiling; an underwound molecule is said to be negatively supercoiled
and an overwound molecule is said to be. positively supercoiled.
Topoisomerases are important in the regulation of supercoiling. Supercoils
can be introduced into DNA in a number of ways. In Escherichia coli DNA
gyrase acts specifically to introduce negative supercoils (Gellert et al. 1976); in
eukaryotes there is no known comparable enzyme that specifically introduces
supercoils. A number of other processes, discussed below, can potentially
affect the degree of supercoiling either by introducing or removing supercoils.
I
I
OC13
B.
C.
D.
Figure 1. Supercoiling by the DNA replication machinery(represented by
the oval). As replication proceeds from (A) to (B) the DNA becomes
underwound behind the point of replication and overwound ahead of the
point of replication (assuming the polymerase does not rotate). Similarly, as
the transcription machinery moves from (C) to (D) overwinding occurs ahead
of the transcription and underwinding occurs after.
The DNA Rotation Problem in Transcription
The structure of DNA necessitates that transcription, like replication, can
proceed only if there is rotation of the DNA or rotation of the transcription
machinery around the DNA. Because of the size of the transcription complex
its rotation has generally been considered implausible. The more widely
favored model has been that transcription induces positive supercoiling in
front of the transcription fork, and negative supercoiling after the
transcription fork (Liu and Wang 1987). If the number of positive supercoils
removed ahead of the transcription machinery does not exactly match the
number of negative supercoils removed after the transcription machinery,
then a net change in the degree of supercoiling will result from transcription.
Evidence that transcription affects the supercoiling of circular plasmid DNA
in Saccharomyces cerevisiae comes from experiments in top1 top2 double
mutant strains where inducible transcription of a plasmid results in
alterations in supercoiling (Brill and Sternglanz 1988; Giaever and Wang
1988). When multiple transcription complexes move in tandem through a
region, the positive supercoil induced in front of the trailing transcription
complex can cancel out the negative supercoils produced by the leading
transcription complex.
Topoisomerases
Topoisomerases are classified into 2 types. Type I topoisomerases are those
able to transiently break a DNA strand, pass another strand through the break,
and reseal the break. Type II topoisomerases are able to break both strands of a
DNA molecule, pass another double stranded DNA molecule through the
break, and reseal the break. The type 1 topoisomerase of E. coli was the first
topoisomerase characterized (Wang 1971). The type I topoisomerases can
further be classified as "topA like" and "TOP1 like". While both of these
groups relax DNA by producing and resealing a single strand nick and do not
require ATP, there are several significant differences between them. The
"topA like" enzymes , such as E. coli topA, only relax negatively supercoiled
DNA (Wang 1971) or DNA molecules with single-stranded regions
(Kirkegaard and Wang 1985) where as the "TOP1 like" enzymes, such as S.
cerevisiae top1, can relax both positive and negative supercoils (Champoux
and Dulbecco 1972). At the reaction level, E. coli topA attaches to the 5' end of
the nicked DNA (Depew et al. 1978; Kirkegaard et al. 1984) where as the
eukaryotic TOP1 attaches to the 3' end (Champoux 1977; Champoux 1978). An
additional specialized type I topoisomerase differing in properties from both
the "topA like" or the "TOP1 like" enzymes has been characterized from the
thermophilic archaebacterium Sulfolobus acidocaldariuswhich is ATP
dependent and can introduce positive supercoils (Kikuchi and Asai 1984;
Mirambeau et al. 1984).
The original assay for determining degree of supercoiling of closed circular
DNA molecules was measurement of sedimentation rate of relaxed versus
supercoiled DNA by ultracentrifugation(Wang 1971). Several more
convenient assays involving filter binding (relying on the affinity of single
stranded regions of negatively supercoiled DNA) and nuclease sensitivity
have been used as topoisomerase assays (Wang and Kirkegaard 1981) though
the standard assay now is gel electrophoresis (Keller 1975). This standard assay
for topoisomerase I measures relaxation of supercoiled plasmid DNA by
taking advantage of the fact that plasmids of different degrees of supercoiling
can easily be resolved on an agarose gel. This assay is specific for type I
topoisomerases because it is done without ATP which is required by type II
topoisomerases.
A number of type 1 topoisomerases have been cloned and sequencedl.In S.
cerevisiae and S. pombe topoisomerase I has been shown to be non-essential
though strains carrying deletions of these genes have slightly longer
generation times (Thrash et al. 1985; Uemura et al. 1987). Deletions of the E.
coli topA gene are lethal, and overexpression causes slow growth (Wang and
Becherer 1983) but can be compensated for by second site mutations in the
gyrase genes (DiNardo et al. 1982; Pruss et al. 1982), at the uncharacterized toc
(topoisomerase one compensatory) locus (Raji et al. 1985), by enhanced
expression of the gyrA and gyrB homologues parC and parE (Kato et al. 1990),
or by an inhibitor of gyrase (Hammond et al. 1991). In addition the E. coli
topA mutation can be complemented by the yeast topoisomerase I gene
(Bjornsti and Wang 1987). Sequence comparisons of the type I topoisomerases
have revealed that the "topA like" and the "TOP1 like" enzymes constitute
two distinct classes on the basis of sequence similarity. In the "topA like" class
in addition to E. coli topA and Salmonella typhimurium topA are thetopB
gene of E. coli (DiGate and Marians 1989) and the S. cerevisiae TOP3 gene
(Wallis et al. 1989). The topB gene product is able to decatenate concatenated
double strand DNA circles which contain a single stranded region or a nick.
The topB gene is also able to relax negatively supercoiled DNA circles as does
topA. This relaxation activity can be detected in strains lacking topA
(Srivenugopal et al. 1984; DiGate and Marians 1988). No biochemical activity has yet
been ascribed to the TOP3 gene product from S. cerevisiae.
1 E. coli topA (Wang and Becherer 1983), Salmonella typhimurium (partial sequence only)
(Ostrowski et al. 1987), yeast S. cerevisiae (Goto and Wang 1985; Thrash et al.
1985),Schizosaccharomyces pombe (Uemura et al. 1987), rat (Durban et al. 1988), vaccinia
(Shuman and Moss 1987), Shope fibroma virus (Upton et al. 1990), and human (D'Arpa et al.
1988).
Unlike the type I topoisomerase, all type II topoisomerases that have been
sequenced so far 2 share extensive sequence homology. The main distinction
among the various type II topoisomerases is between over- and
underwinding enzymes. Bacterial gyrase is able to introduce negative
supercoils (Gellert et al. 1976; Gellert et al. 1976; Liu and Wang 1978) whereas
the eukaryotic type II topoisomerases found thus far cannot introduce
negative supercoils but can relax either positively or negatively supercoiled
DNA. The Eukaryotic type II topoisomerases are encoded by a single gene,
where as the T4 equivalent is specified by three genes and the E. coli and B.
subtilis equivalents by two. It has been shown that the Drosophila
topoisomerase II can complement S. cerevisiae top2 mutations (Wyckoff and
Hsieh 1988), that antibodies against purified Drosophila topoisomerase II can
cross react with the S. cerevisiae Topoisomerase II (Heller et al. 1986), and that
the TOP2 genes from S. cerevisiae and S. pombe can compliment in the
heterologous yeast. In E. coli the product of the parC and parE loci are gyrA
and gyrB homologues and can compensate for mutations in topA. The
standard assay for topoisomerase II is the ATP dependent decatenation of
kinetoplast DNA from trypanosomes (Marini et al. 1980). This assay is specific
for type II topoisomerases because type I topoisomerases are unable to
decatenate concatenated doubled stranded DNA circles.
In addition to the general topoisomerases, a number of enzymes can act as site
specific topoisomerases. Lambda int (Nash et al. 1981), and yeast S. cerevisiae
2 E. coli gyrase (Adachi et al. 1987; Swanberg and Wang 1987), phage T4 (Huang 1986; Huang
1986), Mycoplasma pneumoniae (Colman et al. 1990), Staphylococcus aureus (Hopewell et al.
1990), Haloferax sp.(Holmes and Dyall-Smith 1991), Neisseria gonorrheae (Stein et al. 1991),
Bacillus subtilis (Moriya et al. 1985), Klebsiella pneumoniae (Dimri and Das 1990),
Pseudomonas putida (Parales and Harwood 1990), Crithidiafasciculata (Pasion et al. 1992), S.
cerevisiae (Giaever et al. 1986), Trypanosoma brucei (Strauss and Wang 1990), S. pombe
(Uemura et al. 1986), Drosophila (Wyckoff et al. 1989), human (Tsai et al. 1988)
flp protein (Beatty et al. 1986; Volkert and Broach 1986). both can alter the
supercoiling of their respective substrates but differ from TOPI or TOP2
because they recognize specific sequences in the DNA. Similarly transposable
element resolvases such as that of Tn3 (Krasnow and Cozzarelli 1983;
Krasnow et al. 1983) can affect DNA supercoiling. Broken DNA strands either
from DNA damage or excision repair mechanisms can also relax supercoiled
DNA.
Biological Functions of Topoisomerases
Although a fair amount is known about the structure and biochemical
activities of various topoisomerases, the question of their specific biological
role is still open. One clue to the biological role of topoisomerases comes from
sub-cellular localization studies. A number of studies have localized
topoisomerase I to transcriptionally active regions. In Drosophila, antibodies
against topoisomerase I stain the induced but not the uninduced heat shock
loci (Fleischmann et al. 1984). One of the most heavily transcribed regions of a
eukaryotic genome is the cluster of genes encoding the ribosomal RNA
(rRNA), known as the ribosomal DNA (rDNA), found in the nucleolus.
Recently in situ hybridization has shown that the S. cerevisiae nucleolus is as
in other organisms composed of ribosomal DNA (Dvorkin et al. 1991).
Topoisomerase I has been localized to the nucleolus in Drosophila
(Fleischmann et al. 1984) a chicken lymphoblastoid cell line (Muller et al.
1985), rat (Thiry et al. 1991), and S. cerevisiae (Giroux et al. 1989). That DNA
supercoiling can affect transcription as has been shown by studies in which
the degree of supercoiling of the substrate is altered. In vitro studies of closed
circular lambda DNA showed that more negatively supercoiled molecules
have a greater rate of initiation of transcription (Botchan et al. 1973). In vivo
synthesis of DNA gyrase has been shown to be higher when the template is
relaxed than when it is negatively supercoiled(Menzel and Gellert 1983).
Studies mapping DNA nicks resulting from interaction of the topoisomerase
I specific inhibitor camptothecin have also shown a high level of
topoisomerase I in transcriptionally active regions (Gilmour and Elgin 1987;
Gilmour and Elgin 1987; Stewart et al. 1990).
The transcription rate in the nucleolus is very high and in a number of
systems topoisomerase I has been shown to localize to this region. The first
work in this area was an examination of DNA-relaxing activity in isolated
nucleoli compared with total DNA (Higashinakagawa et al. 1977) DNArelaxing activity co-segregated with RNA polymerase activity and appeared to
be largely associated with the nucleoli. Because of the assay conditions this
polymerase associated DNA-relaxing activity is most likely topoisomerase I.
Transcription in vitro of rat rDNA appears to be reduced upon addition of the
specific topoisomerase I inhibitor camptothecin. (Garg et al. 1987). This
inhibition could be due to two different mechanisms. One possibility is that
reduced transcription results from the decrease in topoisomerase activity. The
other possibility, frequently not addressed, is that the camptothecin-DNAtopoisomerase complex blocks transcription directly. Blockage of transcription
by this complex has been shown to occur under some conditions (Bendixen et
al. 1990). This second possibility was ruled out in Garg et. al. by addition of
purified topoisomerase I which restored the pre-camptothecin level of
expression. A similar experiment in which anti-topoisomerase I antibodies
were used instead of camptothecin also showed that inhibition of
topoisomerase I inhibits ribosomal transcription in a way that can be bypassed
by adding exogenous topoisomerase I (Egyhazi and Durban 1987).
Topoisomerse Mutants
In both S. cerevisiae (Brill et al. 1987) and S. pombe (Yamagishi and Nomura
1988) top1 top2-ts double mutant strains display greatly reduced ribosomal
RNA synthesis at restrictive temperature for top2-ts. This phenotype is not
seen in strains carrying either a top1 or top2-ts single mutation, suggesting
that these topoisomerases have at least partially overlapping function.
Additionally, topl top2-ts double mutant S. cerevisiae strains contain a large
number of free rDNA circles (Kim and Wang 1989). These free rDNA circles
are seen in large numbers only in the double mutant strains, not in strains
carrying either a topl or a top2 mutation alone. In top1 top2-4 double mutant
strains more than half of the rDNA was shown to be in the form of
extrachromosomal rings, where as in top1 top2-1 double mutant strains few
free rDNA circles were seen.
In both S. cerevisiae and S. pombe top1 deletions are viable, although they
grow more slowly than wildtype (Thrash et al. 1985; Uemura et al. 1987). One
explanation for this phenotype is that topoisomerase I plays an important
role, but one which can be substituted for by other topoisomerases.
Topoisomerase II function appears to be essential for mitotic chromosome
segregation, but not for DNA synthesis. Evidence for this comes from cell
synchronization experiments in S. cerevisiae where temperature sensitive
mutations lead to inviability at the time when the cells are undergoing
mitosis, which is clearly after DNA synthesis (DiNardo et al. 1984; Holm et al.
1985). Additional evidence is that inhibition of topoisomerase II with VM-26,
a specific inhibitor of topoisomerase II (Chen et al. 1984), does not affect DNA
replication but delays formation of mitotic chromosomes (Charron and
Hancock 1990). Topoisomerase I is also not required for DNA replication. If
topoisomerase I plays an important role in DNA replication then it's function
must be interchangeable with that of another gene product because yeast
strains carrying topi deletions are viable. Although single mutations in either
topoisomerase I or topoisomerase II do not block DNA replication, double
mutant strains lacking both topoisomerases are blocked in mitotic DNA
replication (Brill et al. 1987; Kim and Wang 1989). Similarly, in S. cerevisiae
topoisomerase II appears to be required for meiotic chromosome segregation,
but not pre-meiotic DNA replication (Rose et al. 1990). Likewise in surf clam
oocytes the topoisomerase II inhibitor VM-26 can block both meiotic and
subsequent mitotic chromosome condensation (Wright and Schatten 1990).
The various models for the role of topoisomerases are somewhat clouded by
the recent discoveries of additional topoisomerase genes, including topB
(DiGate and Marians 1989) and parC-parE(Kato et al. 1990) in E. coli, and top3
(Wallis et al. 1989) and hprl (Aguilera and Klein 1990) in S. cerevisiae. Little is
known about the roles of these enzymes, but their existence suggests that
there may be additional mechanisms for regulating supercoiling.
Ribosomal DNA and Topoisomerases
The ribosomal DNA of S cerevisiae is composed of 50-200 (Retal and Planta
1968; Schweizer et al. 1969; Zamb and Petes 1982) tandemly repeated units of
approximately 9Kb each (Cramer et al. 1976) located on chromosome XII (Petes
1979; Petes 1979; Petes and Smolik 1979; Szostak and Wu 1979) which behaves
as a single Mendelian trait (Petes and Botstein 1977) and whose replication in
meiosis and mitosis is similar to that of the rest of the genome (Brewer et al.
1980). Most or all of this region has been sequenced 3 though not all from the
same strain and there remain a few ambiguities. Some of the main features of
S. cerevisiae rDNA are shown in figure 2.
A.
Unit Repeat
5S
35S
5S
35S
35S
B.
NTS1
35S
T
E
NTS2
5S
A
P
35S
Figure 2. Overall arrangement of the ribosomal DNA of S. cerevisiae is 50-200
copies of the repeat shown in (A). 5S and 35S are the small and large ribosomal transcription
units respectively. Details of the spacer regions are shown in in (B). Features are as follows:
NTS1, nontranscribed spacer region 1; NTS2, nontranscribed spacer region 2; T, Putative
Topoisomerase I binding site; E, 35S enhancer; A, ARS; P 35S promoter.
Ribosomal DNA transcription in S. cerevisiae has been extensively studied.
Each repeat contains two transcripts, the small 5S and the large 35S transcript
which are divergently transcribed (Phillippsen et al. 1978). Promoter,
enhancer, and terminator sequences necessary for transcription of the 35S
(Bayev et al. 1980; Elion and Warner 1984; Elion and Warner 1986; Mestel et
3 (Bell
et al. 1977; Olson et al. 1977; Valenzuela et al. 1977; Kramer et al. 1978; Skryabin et al.
1979; Skryabin et al. 1979; Bayev et al. 1980; Rubtsov et al. 1980; Veldman et al. 1980; Veldman
et al. 1980; Bayev et al. 1981; Georgiev et al. 1981; Swanson and Holland 1983; McMahon et al.
1984; Piper et al. 1984; Skryabin et al. 1984; Nazar and Wong 1985; Swanson et al. 1985;
Jemtland et al. 1986; Mankin et al. 1986; Vincent and Petes 1986; Riggs and Nomura 1990)
al. 1989) ribosomal RNA have been characterized. Expression of the 5S RNA,
which is transcribed by RNA polymerase III, is less well characterized, though
appears to be independent of the RNA polymerase I enhancer (Neigeborn and
Warner 1990).
Each of the rDNA repeats contains an ARS element located between the 5'
ends of the 5S and 35S transcription units (Skryabin et al. 1984). This ARS
unit was first characterized by its ability to stabilize a circular plasmid (Szostak
and Wu 1979; Kouprina and Larionov 1983), and has subsequently been
verified to be a chromosomal origin of replication by electron microscopy
(Saffer and Miller 1986) and by two dimensional agarose gel electrophoresis.
Two separate two-dimensional gel electrophoresis techniques have been
developed. One approach allows the characterization of the forked and bubble
structures which identify a replication origin by separating the DNA in the
native form in the first dimension, denaturing the DNA, and separating the
single stranded DNA in the second dimension (Linskens and Huberman
1988). The second approach involves separating the DNA in the native form
in both dimensions. The second dimension is run under conditions in which
forked and bubble structures migrate slower relative to lineat molecules than
they do in the first dimension (Brewer et al. 1988). The electron microscopic
analysis of rDNA replication by Saffer and Miller reveals that replication
occurs in transcriptionally active rDNA. It has been noted that in E. coli the
replication and transcription of the rDNA proceed in the same direction
(Nomura et al. 1977) and it has been hypothesized that this is necessary to
avoid collisions between the DNA and RNA polymerases. This may also be
the case in S. cerevisiae where it has been that DNA replication is blocked at
the 3' end of the 35S transcription unit, as seen by the accumulation of
replication forks at this point (Brewer and Fangman 1988) suggesting that the
majority of the rDNA is replicated by movement of the replication fork in the
same direction as transcription of the 35S rRNA. It is interesting to note that
figures 4 and 5 in the paper by Saffer and Miller (Saffer and Miller 1986) show
three fairly clear examples of actively transcribed rDNA units with replication
forks located at their 3' ends.
The presence of an ARS element within each rDNA repeat explains the
existence of extrachromosomal rDNA circles which have been reported and
are generally referred to as 3ýp circles (Clark-Walker and Azad 1980; Larionov
et al. 1980). In wildtype cells these free circles appear to compose at most a
small fraction of the total rDNA (Szostak and Wu 1979), though in the
absence of topoisomerase they constitute a large fraction of the ribosomal
DNA (Kim and Wang 1989).
Tetrad analysis of a yeast strain carrying a restriction site polymorphism in
the ribosomal DNA revealed of 14 tetrads no meiotic recombinants but two
examples of mitotic recombination events that occurred before meiosis (Petes
and Botstein 1977). The suggestion in these data that mitotic recombination
occurs at a higher frequency than meiotic recombination is contrary to what is
usually found for non-rDNA. A subsequent random screen for
recombination stimulating sequences has shown that the rDNA fragments
containing the 35S promoter and enhancer together can act as a mitotic (but
not meiotic) recombination hot spot and are referred to as HOT1 (Keil and
Roeder 1984; Voelkel-Meiman et al. 1987). It appears that HOT1 is cis-acting
and that the stimulation of recombination is due to RNA polymerase I
(Stewart and Roeder 1989). Analysis of HOT1 has been done at non-rDNA
loci, so it is still not clear if RNA polymerase stimulates recombination
within the rDNA. The recent development of a S. cerevisiae strain containing
RNA polymerase II transcribed 35S RNA in which RNA polymerase I is
dispensable (Nogi et al. 1991) should make it possible to determine if HOT1
stimulates mitotic recombination in the rDNA and if so if it is RNA
polymerase I dependent.
A feature of the ribosomal DNA of Tetrahymena and Dictyostelium is the
presence of topoisomerase I binding sites (Bonven et al. 1985; Ness et al. 1988).
Although topoisomerase I will bind and relax any DNA sequence, the
enzyme has certain preferred cutting sites (Been et al. 1984; Champoux et al.
1984; Kirkegaard et al. 1984). These rDNA high affinity binding sites however
have an even greater affinity for topoisomerase I, roughly 3 orders of
magnitude greater than random DNA sequences (Busk et al. 1987; Thomsen
et al. 1987). In S. cerevisiae there is a homologous site between the 3' end of
the 35S transcript and the RNA polymerase I enhancer sequence. However,
this sequence has not yet been shown to be a binding site. No role has been
shown for any of these binding sites, nor is it known if localization of
topoisomerase I to the nucleolus is due to the presence of these binding sites.
One report suggests that S. cerevisiae topoisomerase I is capable of binding to
the Tetrahymena high affinity binding sites (Bonven et al. 1985).
Recombination of repeated units in S. cerevisiae has been studied in both
mitosis and meiosis both between naturally occurring repeats and synthetic
repeats (Petes 1980; Szostak and Wu 1980; Jackson and Fink 1981; Jackson and
Fink 1985; Jinks-Robertson and Petes 1985; Jinks-Robertson and Petes 1986;
Kupiec and Petes 1988). Recombination in repeated tandem arrays such as the
rDNA can apparently occur by multiple mechanisms, including a RAD52independent pathway (Ozenberger and Roeder 1991).
There appears to be an interesting relationship between topoisomerases and
repeated DNA in S. cerevisiae. Mutations in several topoisomerases and
topoisomerase homologues preferentially elevate the level of recombination
between specific repeated elements. A mutation known as edrl (now called
top3) was identified which enhanced recombination between repeated 8
elements flanking the SUP4-o marker (Rothstein 1984; Wallis et al. 1989).
Another topoisomerase homologue was identified by a screen for mutations
which increase recombination between synthetic direct repeats (Aguilera and
Klein 1988; Aguilera and Klein 1989). This mutation, called hprl is in a gene
homologous to TOPI (Aguilera and Klein 1990). Strains carrying either topl
or top2 mutations show an elevated level of recombination in the rDNA of S.
cerevisiae but not at a number of other loci (Christman et al. 1988). There also
appears to be a relationship between recombination and transcription. In nonrDNA recombination has been shown to be enhanced by transcription from
an RNA polymerase I promoter (Voelkel-Meiman et al. 1987) and also to be
stimulated by RNA polymerase II transcription (Thomas and Rothstein 1989).
It is possible that the affect of transcription on recombination may be due to
changes in supercoiling arising from the transcription process.
Examination of Yeast Chromosomes
Yeast chromosomes are much smaller than those of higher eukaryotes and it
is only within the last few years that techniques have been developed that
allow entire chromosomes to be examined. One technique is the meiotic
spread preparation (Dresser and Giroux 1988) in which the structure of
condensed meiotic chromosomes can be examined. The other technique is
that of pulse field gels (Schwartz and Cantor 1984; Carle and Olson 1985).
These techniques provide a possible assay for mutations which affect long
range structures in DNA. Supercoiling of DNA and the effects of
topoisomerase on supercoiling are one of these long range affects.
In my research I have tryed to address a number of questions about
topoisomerases in yeast. What is the relationship between topoisomerases
and recombination? Is the elevated level of recombination found in rDNA
specific to rDNA or is it found elsewhere at other loci? In particular, do yeast
strains carrying topoisomerases have an elevated level of recombination in
other tandemly repeated genes such as CUP1. Another issue I have addressed
is whether large scale changes in the rDNA are detectable by pulse field gel
electrophoresis.
References
Adachi, T., M. Mizuuchi, E.Robinson, E.Appella, M. O'dea, M. Gellert and K.
Mizuuchi (1987). "DNA sequence of the E. coli gyrB gene: Application
of a new sequencing strategy." Nuc. Acids Res. 15: 771-783.
Aguilera, A. and H. L. Klein (1988). "Genetic control of intrachromosomal
recombination in Saccharomyces cerevisiae. I. Isolation and genetic
characterization of hyper-recombination mutations." Genetics 119: 779790.
Aguilera, A. and H. L. Klein (1989). "Genetic and molecular analysis of
recombination events in Saccharomyces cerevisiae occurring in the
presence of the hyper-recombination mutation hprl." Genetics 122:
503-517.
Aguilera, A. and H. L. Klein (1990). "HPR1, a novel yeast gene that prevents
intrachromosomal excision recombination, shows carboxy-terminal
homology to the Saccharomyces cerevisiae TOP1 gene." Mol. Cell. Biol.
10: 1439-1451.
Bayev, A., O. I. Georgiev, A. A. Hadjiolov, N. Nikolaev, K. G. Skryabin and V.
M. Zakharyev (1981). "The structure of the yeast ribosomal RNA genes.
3. Precise mapping of the 18 S and 25 S rRNA genes and structure of the
adjacent regions." Nuc. Acids Res. 9: 789-799.
Bayev, A. A., O. I. Georgiev, A. A. Hadjiolov, M. B. Kermekchiev, N.
Nikolaev, K. G. Skryabin and V. M. Zakharyev (1980). "The structure of
the yeast ribosomal RNA genes. 2. The nucleotide sequence of the
initiation site for ribosomal RNA transcription." Nuc. Acids Res. 8:
4919-4926.
Beatty, L. G., C. D. Babineau, C. Hogrefe and P. D. Sadowski (1986). "FLP sitespecific recombinase of yeast 2-micron plasmid. Topological features of
the reaction." J. Mol. Biol. 188: 529-544.
Been, M. D., R. R. Burgess and J. J.Champoux (1984). "Nucleotide sequence
preference at rat liver and wheat germ type 1 DNA topoisomerase
breakage sites in duplex SV40 DNA." Nuc. Acids Res. 12: 3097-3114.
Bell, G. I., L. J. Degennaro, D. H. Gelfand, R. J. Bishop, P. Valenzuela and W. J.
Rutter (1977). "Ribosomal RNA genes of Saccharomyces cerevisiae: I.
Physical map of the repeating unit and location of the regions coding
for 5S, 5.8S, 18S, and 25S ribosomal RNAs." J. Biol. Chem. 252: 81188125.
Bendixen, C., B. Thomsen, J. Alsner and 0. Westergaard (1990).
"Camptothecin-stabilized topoisomerase I-DNA adducts cause
premature termination of transcription." Biochemistry 29: 5613-5619.
Bjornsti, M. A. and J. C. Wang (1987). "Expression of yeast DNA
topoisomerase I can complement a conditional-lethal DNA
topoisomerase I mutation in Escherichia coli." Proc. Natl. Acad. Sci. 84:
8971-8975.
Bonven, B. J., E. Gocke and 0. Westergaard (1985). "A high affinity
topoisomerase I binding sequence is clustered at DNAase I
hypersensitive sites in Tetrahymena R-chromatin." Cell 41: 541-551.
Botchan, P., J. C. Wang and H. Echols (1973). "Effect of circularity and
superhelicity on transcription from bacteriophage lambda DNA." Proc.
Natl. Acad. Sci. 70: 3077-3081.
Brewer, B. J.and W. L. Fangman (1988). "A replication fork barrier at the 3'
end of yeast ribosomal RNA genes." Cell 55: 637-643.
Brewer, B. J., E. P. Sena and W. L. Fangman (1988). "Analysis of replication
intermediates by two-dimensional agarose gel electrophoresis." Cancer
Cells 6: 229-234.
Brewer, B. J., V. A. Zakian and W. L. Fangman (1980). "Replication and
meiotic transmission of yeast ribosomal RNA genes." Proc. Natl. Acad.
Sci. 77: 6739-6743.
Brill, S. J., S. DiNardo, M. K. Voelkel and R. Sternglanz (1987). "DNA
topoisomerase activity is required as a swivel for DNA replication and
for ribosomal RNA transcription." NCI Monogr. 1987: 11-15.
Brill, S. J. and R. Sternglanz (1988). "Transcription-dependent DNA
supercoiling in yeast DNA topoisomerase mutants." Cell 54: 403-411.
Busk, H., B. Thomsen, B. J.Bonven, E. Kjeldsen, O. F. Nielsen and 0.
Westergaard (1987). "Preferential relaxation of supercoiled DNA
containing a hexadecameric recognition sequence for topoisomerase I."
Nature 327: 638-640.
Carle, G. F. and M. V. Olson (1985). "An electrophoretic karyotype for yeast."
Proc. Natl. Acad. Sci. 82: 3756-3760.
Champoux, J. J. (1977). "Strand breakage by the DNA untwisting enzyme
results in covalent attachment of the enzyme to DNA." Proc. Natl.
Acad. Sci. 74: 3800-3804.
Champoux, J. J.(1978). "Mechanism of the reaction catalyzed by the DNA
untwisting enzyme: Attachment of the enzyme to 3' -terminus of the
nicked DNA." J. Mol. Biol. 118: 441-446.
Champoux, J. J. and R. Dulbecco (1972). "An activity from mammalian cells
that untwists superhelical DNA-A possible swivel for DNA
replication." Proc. Natl. Acad. Sci. 69: 143-146.
Champoux, J. J., W. J. McCoubrey and M. D. Been (1984). "DNA structural
features that lead to strand breakage by eukaryotic type-I
topoisomerase." CSH Symp. Quant. Biol. 49: 435-42.
Charron, M. and R. Hancock (1990). "DNA topoisomerase II is required for
formation of mitotic chromosomes in Chinese hamster ovary cells:
studies using the inhibitor 4'-demethylepipodophyllotoxin 9-(4,6-0thenylidene-beta-D-glucopyranoside)." Biochemistry 29: 9531-9537.
Chen, G. L., L. Yang, T. C. Rowe, B. D. Halligan, K. M. Tewey and L. F. Liu
(1984). "Nonintercalative antitumor drugs interfere with the breakagereunion reaction of mammalian DNA topoisomerase II." J. Biol.
Chem. 259: 13560-13566.
Christman, M. F., F. S. Dietrich and G. R. Fink (1988). "Mitotic recombination
in the rDNA of S. cerevisiae is suppressed by the combined action of
DNA topoisomerases I and II." Cell 55: 413-425.
Clark-Walker, G. D. and A. A. Azad (1980). "Hybridizable sequences between
cytoplasmic ribosomal RNAs and 3 micron circular DNAs of
Saccharomyces cerevisiae and Torulopsis glabrata." Nuc. Acids Res. 8:
1009-1022.
Colman, S. D., P. C. Hu and K. F. Bott (1990). "Mycoplasma pneumoniae DNA
gyrase genes." Mol. Microbiol. 4: 1129-1134.
Cramer, J. H., F. W. Farrelly and R. H. Rownd (1976). "Restriction
endonuclease analysis of ribosomal DNA from Saccharomyces
cerevisiae." Mol. Gen. Genet. 148: 233-241.
D'Arpa, P., P. S. Machlin, H. Ratrie 3d, N. F. Rothfield, D. W. Cleveland and
W. C. Earnshaw (1988). "cDNA cloning of human DNA topoisomerase
I: catalytic activity of a 67.7-kDa carboxyl-terminal fragment." Proc. Natl.
Acad. Sci. 85: 2543-2547.
Delburck, M. (1954). "On the the replication of deoxyribonucleic acid (DNA)."
Proc. Natl. Acad. Sci. 40: 783-788.
Depew, R. E., L. F. Liu and J. C. Wang (1978). "Interaction between DNA and
Escherichia coli protein omega. Formation of a complex between
single-stranded DNA and omega protein." T.Biol. Chem. 253: 511-518.
DiGate, R. J. and K. J. Marians (1988). "Identification of a potent decatenating
enzyme from Escherichia coli." I Biol Chem 263: 13366-73.
DiGate, R. J. and K. J. Marians (1989). "Molecular cloning and DNA sequence
analysis of Escherichia coli topB, the gene encoding topoisomerase III."
J. Biol. Chem. 264: 17924-17930.
Dimri, G. P. and H. K. Das (1990). "Cloning and sequence analysis of gyrA gene
of Klebsiella pneumoniae." Nuc. Acids Res. 18: 151-156.
DiNardo, S., K. Voelkel and R. Sternglanz (1984). "DNA topoisomerase II
mutant of Saccharomyces cerevisiae: topoisomerase II is required for
segregation of daughter molecules at the termination of DNA
replication." Proc. Natl. Acad. Sci. 81: 2616-2620.
DiNardo, S., K. A. Voelkel, R. Sternglanz, A. E.Reynolds and A. Wright
(1982). "Escherichia coli DNA topoisomerase I mutants have
compensatory mutations in DNA gyrase genes." Cell 31: 43-51.
Dresser, M. E. and C. N. Giroux (1988). "Meiotic chromosome behavior in
spread preparations of Yeast." J. Cell Bio. 106: 567-573.
Durban, E., M. Bramucci and R. Cook (1988). "Partial amino acid sequence of
rat topoisomerase I: comparison with the predicted sequences for the
human and yeast enzymes." Biochem. Biophys. Res. Commun. 154:
358-364.
Dvorkin, N., M. W. Clark and B. A. Hamkalo (1991). "Ultrastructural
localization of nucleic acid sequences in Saccharomyces cerevisiae
nucleoli." Chromosoma 100: 519-523.
Egyhazi, E.and E. Durban (1987). "Microinjection of anti-topoisomerase I
immunoglobulin G into nuclei of Chironomus tentans salivary gland
cells leads to blockage of transcription elongation." Mol. Cell. Biol. 7:
4308-4316.
Elion, E. A. and J. R. Warner (1984). "The major promoter element of rRNA
transcription in yeast lies 2 kb upstream." Cell 39: 663-673.
Elion, E.A. and J. R. Warner (1986). "An RNA polymerase I enhancer in
Saccharomyces cerevisiae." Mol. Cell. Biol. 6: 2089-2097.
Fleischmann, G., G. Pflugfelder, E.K. Steiner, K. Javaherian, G. C. Howard, J.
C. Wang and S. C. Elgin (1984). "DrosophilaDNA topoisomerase I is
associated with transcriptionally active regions of the genome." Proc.
Natl. Acad. Sci. 81: 6958-6962.
Garg, L. C., S. DiAngelo and S. T. Jacob (1987). "Role of DNA topoisomerase I
in the transcription of supercoiled rRNA gene." Proc. Natl. Acad. Sci.
84: 3185-3188.
Gellert, M., K. Mizuuchi, M. H. O'Dea and H. A. Nash (1976). "DNA gyrase:
An enzyme that introduces superhelical turns into DNA." Proc. Natl.
Acad. Sci. 73: 3872-3876.
Gellert, M., M. H. O'Dea, T. Itoh and J. I. Tomizawa (1976). "Novobiocin and
coumermycin inhibit DNA supercoiling catalyzed by DNA gyrase."
Proc. Natl. Acad. Sci. 73: 4474-4478.
Georgiev, O. I., N. Nikolaev, A. A. Hadjiolov, K. G. Skryabin, V. M.
Zakharyev and A. A. Bayev (1981). "The structure of the yeast
ribosomal RNA genes. 4. Complete sequence of the 25 S rRNA gene
from Saccharomyces cerevisae." Nuc. Acids Res. 9: 6953-6958.
Giaever, G., R. Lynn, T. Goto and J.C. Wang (1986). "The complete nucleotide
sequence of the structural gene TOP2 of yeast DNA topoisomerase II." L
Biol. Chem. 261: 12448-12454.
Giaever, G. N. and J. C. Wang (1988). "Supercoiling of intracellular DNA can
occur in eukaryotic cells." Cell 55: 849-856.
Gilmour, D. S. and S. C. Elgin (1987). "Association of topoisomerase I with
transcriptionally active loci in Drosophila." NCI Monogr. 1987: 17-21.
Gilmour, D. S. and S. C. Elgin (1987). "Localization of specific topoisomerase I
interactions within the transcribed region of active heat shock genes by
using the inhibitor camptothecin." Mol. Cell. Biol. 7: 141-148.
Giroux, C. N., M. E.Dresser and H. F. Tiano (1989). "Genetic control of
chromosome synapsis in yeast meiosis." Genome 31: 88-93.
Goto, T. and J.C. Wang (1985). "Cloning of yeast TOP1, the gene encoding
DNA topoisomerase I, and construction of mutants defective in both
DNA topoisomerase I and DNA topoisomerase II." Proc. Natl. Acad.
Sci. 82: 7178-7182.
Hammond, G. G., P. J. Cassidy and K. M. Overbye (1991). "Novobiocindependent topA deletion mutants of Escherichia coli." J. Bacteriol. 173:
5564-5567.
Heller, R. A., E. R. Shelton, V. Dietrich, S. C. Elgin and D. L. Brutlag (1986).
"Multiple forms and cellular localization of Drosophila DNA
topoisomerase II." J. Biol. Chem. 261: 8063-8069.
Higashinakagawa, T., H. Wahn and R. H. Reeder (1977). "Isolation of
Ribosomal Gene Chromatin." Dev. Biol. 55: 375-386.
Holm, C., T. Goto, J. C. Wang and D. Botstein (1985). "DNA topoisomerase II
is required at the time of mitosis in yeast." Cell 41: 553-563.
Holmes, M. L. and M. Dyall-Smith (1991). "Mutations in DNA gyrase result in
novobiocin resistance in halophilic archaebacteria." J. Bacteriol. 173:
642-648.
Hopewell, R., M. Oram, R. Briesewitz and L. M. Fisher (1990). "DNA cloning
and organization of the Staphylococcus aureus gyrA and gyrB genes:
Close homology among gyrase proteins and implications for 4quinolone action and resistance." J. Bacteriol. 172: 3481-3484.
Huang, W. M. (1986). "The 52-protein subunit of T4 DNA topoisomerase is
homologous to the gyrA-protein of gyrase." Nuc. Acids Res. 14: 73797390.
Huang, W. M. (1986). "Nucleotide sequence of a type II DNA topoisomerase
gene. Bacteriophage T4 gene 39." Nuc. Acids Res. 14: 7751-7765.
Jackson, J. A. and G. R. Fink (1981). "Gene conversion between duplicated
genetic elements in yeast." Nature 292: 306-311.
Jackson, J. A. and G. R. Fink (1985). "Meiotic recombination between
duplicated genetic elements in Saccharomyces cerevisiae." Genetics 109:
303-332.
Jemtland, R., E. Maehlum, O. S. Gabrielsen and T. B. Oyen (1986). "Regular
distribution of length heterogeneities within non-transcribed spacer
regions of cloned and genomic rDNA of Saccharomyces cerevisiae."
Nuc. Acids Res. 14: 5145-5158.
Jinks-Robertson, S. and T. D. Petes (1985). "High-frequency meiotic gene
conversion between repeated genes on nonhomologous chromosomes
in yeast." Proc. Natl. Acad. Sci. 82: 3350-3354.
Jinks-Robertson, S. and T. D. Petes (1986). "Chromosomal translocations
generated by high-frequency meiotic recombination between repeated
yeast genes." Genetics 114: 731-752.
Kato, J., Y. Nishimura, R. Imamura, H. Niki, S. Hiraga and H. Suzuki (1990).
"New topoisomerase essential for chromosome segregation in E. coli."
Cell 63: 393-404.
Keil, R. L. and G. S. Roeder (1984). "Cis-acting, recombination-stimulating
activity in a fragment of the ribosomal DNA of S. cerevisiae." Cell 39:
377-386.
Keller, W. (1975). "Characterization of purified DNA-relaxing enzyme from
human tissue culture cells." Proc. Natl. Acad. Sci. 72: 2250-2554.
Kikuchi, A. and K. Asai (1984). "Reverse gyrase--a topoisomerase which
introduces positive superhelical turns into DNA." Nature 309: 677-681.
Kim, R. A. and J. C. Wang (1989). "Function of DNA topoisomerases as
replication swivels in Saccharomyces cerevisiae." I. Mol. Biol. 208: 257267.
Kim, R. A. and J. C. Wang (1989). "A subthreshold level of DNA
topoisomerases leads to the excision of yeast rDNA as
extrachromosomal rings." Cell 57: 975-985.
Kirkegaard, K., G. Pflugfelder and J. C. Wang (1984). "The cleavage of DNA by
type-I DNA topoisomerases." CSH Symp. Quant. Biol. 49: 411-419.
Kirkegaard, K. and J. C. Wang (1985). "Bacterial DNA topoisomerase I can
relax positively supercoiled DNA containing a single-stranded loop." L.
Mol. Biol. 185: 625-637.
Kouprina, N. Y. and V. L. Larionov (1983). "The study of a rDNA replicator in
Saccharomyces." Curr. Genet. 7: 433-438.
Kramer, R. A., P. Philippsen and R. W. Davis (1978). "Divergent transcription
in the yeast ribosomal RNA coding region as shown by hybridization to
separated strands and sequence analysis of cloned DNA." J.Mol. Biol.
123: 405-416.
Krasnow, M. A. and N. R. Cozzarelli (1983). "Site-specific relaxation and
recombination by the Tn3 resolvase: recognition of the DNA path
between oriented res sites." Cell 32: 1313-1324.
Krasnow, M. A., A. Stasiak, S. J. Spengler, F. Dean, T. Koller and N. R.
Cozzarelli (1983). "Determination of the absolute handedness of knots
and catenanes of DNA." Nature 304: 559-560.
Kupiec, M. and T. D. Petes (1988). "Meiotic recombination between repeated
transposable elements in Saccharomyces cerevisiae." Mol. Cell. Biol. 8:
2942-2954.
Larionov, V. L., A. V. Grishin and M. N. Smirnov (1980). "3 micron DNA - an
extrachromosomal ribosomal DNA in the yeast Saccharomyces
cerevisiae." Gene 12: 41-49.
Linskens, M. H. K. and J.A. Huberman (1988). "Organization of replication of
ribosomal DNA in Saccharomyces cerevisiae." Mol. Cell. Biol. 8: 49274935.
Liu, L. F. and J. C. Wang (1978). "Micrococcus luteus DNA gyrase: active
components and a model for its supercoiling of DNA." Proc. Natl.
Acad. Sci. 75: 2098-2102.
Liu, L. F. and J.C. Wang (1987). "Supercoiling of the DNA template during
transcription." Proc. Natl. Acad. Sci. 84: 7024-7027.
Mankin, A. S., K. G. Skryabin and P. M. Rubtsov (1986). "Identification of ten
additional nucleotides in the primary structure of yeast 18S rRNA."
Gene 44: 143-143.
Marini, J. C., K. G. Miller and P. T. Englund (1980). "Decatenation of
kinetoplast DNA by topoisomerases." J.Biol. Chem. 255: 4976-4979.
McMahon, M. E., D. Stamenkovich and T. D. Petes (1984). "Tandemly
arranged variant 5S ribosomal RNA genes in the yeast Saccharomyces
cerevisiae." Nuc. Acids Res. 12: 8001-8016.
Menzel, R. and M. Gellert (1983). "Regulation of the genes for E. coli DNA
gyrase: homeostatic control of DNA supercoiling." Cell 34: 105-113.
Mestel, R., M. Yip, J. P. Holland, E. Wang, J. Kang and M. J. Holland (1989).
"Sequences within the spacer region of yeast rRNA cistrons that
stimulate 35S rRNA synthesis in vivo mediate RNA polymerase Idependent promoter and terminator activities." Mol. Cell. Biol. 9: 12431254.
Mirambeau, G., M. Duguet and P. Forterre (1984). "ATP-dependent DNA
topoisomerase from the archaebacterium Sulfolobus acidocaldarius.
Relaxation of supercoiled DNA at high temperature." 1. Mol. Biol. 179:
559-563.
Moriya, S., N. Ogasawara and H. Yoshikawa (1985). "Structure and function of
the replication origin of the Bacillus subtilis chromosome. III.
Nucleotide sequence of some 10,000 base pairs in the origin region."
Nuc. Acids Res. 13: 2251-2265.
Muller, M. T., W. P. Pfund, V. B. Mehta and D. K. Trask (1985). "Eukaryotic
type I topoisomerase is enriched in the nucleolus and catalytically
active on ribosomal DNA." EMBO J. 4: 1237-1243.
Nash, H. A., K. Mizuuchi, L. W. Enquist and R. A. Weisberg (1981). "Strand
exchange in lambda integrative recombination: genetics, biochemistry,
and models." CSH Symp. Quant. Biol. 1: 417-428.
Nazar, R. N. and W. M. Wong (1985). "Is the 5S RNA a primitive ribosomal
RNA sequence?" Proc. Natl. Acad. Sci. 82: 5608-5611.
Neigeborn, L. and J.R. Warner (1990). "Expression of yeast 5S RNA is
independent of the rDNA enhancer region." Nuc. Acids Res. 18: 41794184.
Ness, P. J., T. Koller and F. Thoma (1988). "Topoisomerase I cleavage sites
identified and mapped in the chromatin of Dictyostelium ribosomal
RNA genes." J. Mol. Biol. 200: 127-139.
Nogi, Y., R. Yano and M. Nomura (1991). "Synthesis of large rRNAs by RNA
polymerase II in mutants of Saccharomyces cerevisiae defective in
RNA polymerase I." Proc. Natl. Acad. Sci. 88: 3962-3966.
Nomura, M., E.A. Morgan and S. R. Jaskunas (1977). "Genetics of bacterial
ribosomes." Annu. Rev. Genet. 11: 297-347.
Olson, M. V., D. L. Montgomery, A. K. Hopper, G. S. Page, F. Horodyski and B.
D. Hall (1977). "Nucleotide sequence of the yeast 5S ribosomal RNA
gene and adjacent putative control regions." Nature 267: 641-643.
Ostrowski, J., G. Burdzy-Jagura and N. M. Kredich (1987). "DNA sequences of
the cysB regions of Salmonella typhimurium and Escherichia coli." L
Biol. Chem. 262: 5999-6005.
Ozenberger, B. A. and G. S. Roeder (1991). "A unique pathway of doublestrand break repair operates in tandemly repeated genes." Mol. Cell.
Biol. 11: 1222-1231.
Parales, R. E.and C. S. Harwood (1990). "Nucleotide sequence of the gyrB gene
of Pseudomonas putida." Nuc. Acids Res. 18: 5880-5880.
Pasion, S. G., J. C. Hines, R. Aebersold and D. S. Ray (1992). "Molecular
cloning and expression of the gene encoding the kinetoplast-associated
type II DNA topoisomerase of Crithidiafasciculata." Mol. Biochem.
Parasitol. 50: 57-67.
Petes, T. D. (1979). "Meiotic mapping of yeast ribosomal deoxyribonucleic acid
on chromosome XII." J. Bact. 138: 185-192.
Petes, T. D. (1979). "Yeast ribosomal DNA genes are located on chromosome
XII." Proc. Natl. Acad. Sci. 76: 410-414.
Petes, T. D. (1980). "Unequal meiotic recombination within tandem arrays of
yeast ribosomal DNA genes." Cell 19: 765-774.
Petes, T. D. and D. Botstein (1977). "Simple Mendelian inheritance of the
reiterated ribosomal DNA of yeast." Proc. Natl. Acad. Sci. 74: 5091-5095.
Petes, T. D. and U. S. Smolik (1979). "Evidence that the ribosomal DNA genes
of yeast are not on chromosome I." Mol. Gen. Genet. 175: 187-193.
Phillippsen, P., M. J. Thomas, R. A. Kramer and R. W. Davis (1978). "Unique
arrangement of coding sequences for 5S, 5.8S, 18S and 25S ribosomal
RNA in Saccharomyces cerevisiae as determined by R-loop and
hybridization analysis." J. Mol. Biol. 123: 387-404.
Piper, P. W., A. Lockheart and N. Patel (1984). "A minor class of 5S rRNA
genes in Saccharomyces cerevisiae X2180-1B, one member of which lies
adjacent to a Ty transposable element." Nuc. Acids Res. 12: 4083-4096.
Pruss, G. J., S. H. Manes and K. Drlica (1982). "Escherichia coli DNA
topoisomerase I mutants: increased supercoiling is corrected by
mutations near gyrase genes." Cell 31: 35-42.
Raji, A., D. J. Zabel, C. S. Laufer and R. E. Depew (1985). "Genetic analysis of
mutations that compensate for loss of Escherichia coli DNA
topoisomerase I." J. Bact. 162: 1173-1179.
Retal, J. and R. J. Planta (1968). "The investigation of the ribosomal RNA sites
in yeast DNA by hybridization technique." Biochim. Biophys. Acta. 169:
416-429.
Riggs, D. L. and M. Nomura (1990). "Specific transcription of Saccharomyces
cerevisiae 35 S rDNA by RNA polymerase I in vitro." J. Biol. Chem.
265: 7596-7603.
Rose, D., W. Thomas and C. Holm (1990). "Segregation of recombined
chromosomes in meiosis I requires DNA topoisomerase II." Cell 60:
1009-1017.
Rothstein, R. (1984). "Double-strand-break repair, gene conversion, and
postdivision segregation." CSH Symp. Quant. Biol. 49: 629-637.
Rubtsov, P. M., M. M. Musakhanov, V. M. Zakharyev, A. S. Krayev, K. G.
Skryabin and A. A. Bayev (1980). "The structure of the yeast ribosomal
RNA genes. I. The complete nucleotide sequence of the 18S ribosomal
RNA gene from Saccharomyces cerevisiae." Nuc. Acids Res. 8: 57795794.
Saffer, L. D. and 0. L. J. Miller (1986). "Electron microscopic study of
Saccharomyces cerevisiae rDNA chromatin replication." Mol. Cell.
Biol. 6: 1148-1157.
Schnos, M., K. Zahn, R. B. Inman and F. R. Blattner (1988). "Initiation protein
induced helix destabilization at the X origin: A prepriming step in
DNA replication." Cell 52: 385-395.
Schwartz, D. C. and C. R. Cantor (1984). "Separation of yeast chromosomesized DNAs by pulsed field gradient gel electrophoresis." Cell 37: 67-75.
Schweizer, E., C. MacKechnie and H. O. Halvorson (1969). "The redundency of
ribosomal and transfer RNA genes in Saccharomyces cerevisiae." Mol.
Biol. 40: 261-277.
Shuman, S. and B. Moss (1987). "Identification of a vaccinia virus gene
encoding a type I DNA topoisomerase." Proc. Natl. Acad. Sci. 84: 74787482.
Skryabin, K. G., M. A. Eldarov, V. L. Larionov, A. A. Bayev, J. Klootwijk, V. C.
de Regt, G.M. Veldman, R. J. Planta, O. I. Georgiev and A. A.
Hadjiolov (1984). "Structure and function of the nontranscribed spacer
regions of yeast rDNA." Nuc. Acids Res. 12: 2955-2968.
Skryabin, K. G., A. S. Kraev, P. M. Rubtsov and A. A. Baev (1979). "Complete
nucleotide sequence of the spacer region situated between the 18S and
5.8S RNAs of yeasts." Dokl. Biol. Sci. 247: 278-281.
Skryabin, K. G., V. M. Zakhararev, P. M. Rubtsov and A. A. Baev (1979).
"Nucleotide sequence of the presumed region of initiation of
transcription of the yeast ribosomal operon." Dokl. Biochem. 247: 288290.
Srivenugopal, K. S., D. Lockshon and D. R. Morris (1984). "Escherichia coli
DNA topoisomerase III: purification and characterization of a new type
I enzyme." Biochemistry 23: 1899-906.
Stein, D. C., R. Danaher and T. M. Cook (1991). "Characterization of a gyrB
mutation responsible for low level nalidixic acid resistance in Neisseria
gonorrheae." Antimicrob. Agents Chemother. 35: 622-626.
Stewart, A. F., R. E. Herrera and A. Nordheim (1990). "Rapid induction of cfos transcription reveals quantitative linkage of RNA polymerase II
and DNA topoisomerase I enzyme activities." Cell 60: 141-149.
Stewart, S. E. and G. S. Roeder (1989). "Transcription by RNA polymerase I
stimulates mitotic recombination in Saccharomyces cerevisiae." Mol.
Cell. Biol. 9: 3464-3472.
Strauss, P. R. and J. C. Wang (1990). "The TOP2 gene of Trypanosoma brucei: a
single-copy gene that shares extensive homology with other TOP2
genes encoding eukaryotic DNA topoisomerase II." Mol. Biochem.
Parasitol. 38: 141-150.
Swanberg, S. L. and J. C. Wang (1987). "Cloning and sequencing of the
Escherichia coli gyrA gene coding for the A subunit of DNA gyrase." L
Mol. Biol. 197: 729-736.
Swanson, M. E. and M. J. Holland (1983). "RNA polymerase I-dependent
selective transcription of yeast ribosomal DNA. Identification of a new
cellular ribosomal RNA precursor." J. Biol. Chem. 258: 3242-3250.
Swanson, M. E., M. Yip and M. J. Holland (1985). "Characterization of an RNA
polymerase I-dependent promoter within the spacer region of yeast
ribosomal cistrons." J. Biol. Chem. 260: 9905-9915.
Szostak, J. W. and R. Wu (1979). "Insertion of a genetic marker into the
ribosomal DNA of yeast." Plasmid 2: 536-554.
Szostak, J. W. and R. Wu (1980). "Unequal crossing over in the ribosmal DNA
of Saccharomyces cerevisiae." Nature 284: 426-430.
Thiry, M., U. Scheer and G. Goessens (1991). "Localization of nucleolar
chromatin by immunocytochemistry and in situ hybridization at the
electron microscopic level." Electron Microsc. Rev. 4: 85-110.
Thomas, B. J. and R. Rothstein (1989). "Elevated recombination rates in
transcriptionally active DNA." Cell 56: 619-630.
Thomsen, B., S. Mollerup, B. J. Bonven, R. Frank, H. Blocker, O. F. Nielsen
and 0. Westergaard (1987). "Sequence specificity of DNA topoisomerase
I in the presence and absence of camptothecin." EMBO J. 6: 1817-1823.
Thrash, C., A. T. Bankier, B. G. Barrell and R. Sternglanz (1985). "Cloning,
characterization, and sequence of the yeast DNA topoisomerase I gene."
Proc. Natl. Acad. Sci. 82: 4374-4378.
Tsai, P. M., L. F. Liu, A. A. Liu, K. M. Tewey, P. J. Whang, T. Knutsen, K.
Huebner, C. M. Croce and J. C. Wang (1988). "Cloning and sequencing
of cDNA encoding human DNA topoisomerase II and localization of
the gene to chromosome region 17q21-22." Proc. Natl. Acad. Sci. 85:
7177-7181.
Uemura, T., K. Morikawa and M. Yanagida (1986). "The nucleotide sequence
of the fission yeast DNA topoisomerase II gene: structural and
functional relationships to other DNA topoisomerases." EMBO T.5:
2355-2361.
Uemura, T., K. Morino, S. Uzawa, K. Shiozaki and M. Yanagida (1987).
"Cloning and sequencing of Schizosaccharomyces pombe DNA
topoisomerase I gene, and effect of gene disruption." Nuc. Acids Res.
15: 9727-9739.
Upton, C., A. Opgenorth, P. Traktman and G. McFadden (1990). "Identification
and DNA sequence of the Shope fibroma virus DNA topoisomerase
gene." Virology 176: 439-447.
Valenzuela, P., G. I. Bell, A. Venegas, E. T. Sewell, F. R. Masiarz, L. J.
Degennaro, F. Weinberg and W. J. Rutter (1977). "Ribosomal RNA
genes of Saccharomyces cerevisiae: II. Physical map and nucleotide
sequence of the 5S ribosomal RNA gene and adjacent intergenic
regions." J. Biol. Chem. 252: 8126-8135.
Veldman, G. M., R. C. Brand, J. Klootwijk and R. J. Planta (1980). "Some
characteristics of processing sites in ribosomal RNA of yeast." Nuc.
Acids Res. 8: 2907-2920.
Veldman, G. M., J. Klootwijk, P. de Jonge, R. J. Leer and R. J. Planta (1980).
"The transcription termination site of the ribosomal RNA operon in
yeast." Nuc. Acids Res. 8: 5179-5192.
Vincent, A. and T. D. Petes (1986). "Isolation and characterization of a Ty
element inserted into ribosomal DNA of the yeast Saccharomyces
cerevisiae." Nuc. Acids Res. 14: 2939-2949.
Voelkel-Meiman, K., R. L. Keil and G. S. Roeder (1987). "Recombinationstimulating sequences in yeast ribosomal DNA correspond to
sequences regulating transcription by RNA polymerase I." Cell 48: 10711079.
Volkert, F. C. and J. R. Broach (1986). "Site-specific recombination promotes
plasmid amplification in yeast." Cell 46: 541-550.
Wallis, J. W., G. Chrebet, G. Brodsky, M. Rolfe and R. Rothstein (1989). "A
hyper-recombination mutation in S. cerevisiae identifies a novel
eukaryotic topoisomerase." Cell 58: 409-419.
Wang, J. C. (1971). "Interaction between DNA and an Escherichia coli protein
omega." J. Mol. Biol. 55: 523-533.
Wang, J. C. and K. Becherer (1983). "Cloning of the gene topA encoding for
DNA topoisomerase I and the physical mapping of the cysB-topA-trp
region of Escherichia coli." Nuc. Acids Res. 11: 1773-1790.
Wang, J. C. and K. Kirkegaard (1981). "DNA topoisomerases." Gene
Amplification & Analysis 2: 455-473.
Watson, J. D. and F. H. C. Crick (1953). "Genetic implications of the structure
of deoxyribonucleic acid." Nature 171: 964-967.
Wright, S. J. and G. Schatten (1990). "Teniposide, a topoisomerase II inhibitor,
prevents chromosome condensation and separation but not
decondensation in fertilized surf clam (Spisula solidissima) oocytes."
Dev. Biol. 142: 224-232.
Wyckoff, E. and T. S. Hsieh (1988). "Functional expression of a Drosophila
gene in yeast: genetic complementation of DNA topoisomerase II."
Proc. Natl. Acad. Sci. 85: 6272-6276.
Wyckoff, E., D. Natalie, J. M. Nolan, M. Lee and T. Hsieh (1989). "Structure of
the Drosophila DNA topoisomerase IIgene. Nucleotide sequence and
homology among topoisomerases II." J. Mol. Biol. 205: 1-13.
Yamagishi, M. and M. Nomura (1988). "Deficiency in both type I and type II
DNA topoisomerase activities differentially affect rRNA and ribosomal
protein synthesis in Schizosaccharomyces pombe." Curr. Genet. 13: 305314.
Zamb, T. J. and T. D. Petes (1982). "Analysis of the junction between ribosomal
RNA genes and single-copy chromosomal sequences in the yeast
Saccharomyces cerevisiae." Cell 28: 355-364.
Chapter II
Mitotic Recombination in the rDNA of
S. cerevisiae is suppressed by the combined
action of DNA topoisomerase I and II
Cell, Vol. 55, 413-425, November 4, 1988, Copyright @ 1988 by Cell Press
Mitotic Recombination in the rDNA of S. cerevisiae
Is Suppressed by the Combined Action of
DNA Topoisomerases I and II
Michael F. Christman, Fred S. Dietrich,
and Gerald R. Fink
Whitehead Institute for Biomedical Research
Nine Cambridge Center
Cambridge, Massachusetts 02142
and Department of Biology
Massachusetts Institute of Technology
Cambridge, Massachusetts 02139
Summary
We have found that mitotic recombination within the
S. cerevisiae rDNA cluster (200 tandemly repeated 9.1
kb units) is strongly suppressed and that this suppression requires the combined action of DNA topoisomerases I and II. Strains with a null mutation in the
TOP1 gene (encoding topoisomerase I) or a ts mutation in the TOP2 gene (encoding topoisomerase II)
grown at a semipermissive temperature show 50- to
200-fold higher frequencies of mitotic recombination
in rDNA relative to TOP + controls. Suppression of
recombination is specific to the rDNA because the
recombination frequency at another tandem array, the
CUP1 locus, at a simple HIS4 duplication, or among
dispersed repeats (MAT and HML or HMR) is not
elevated in top1 or top2 mutants. The high frequency
of mitotic recombination within the rDNA cluster in
topoisomerase mutants shows that both TOP1 and
TOP2 are required for suppression of recombination in
this region of the genome.
et al., 1969). If the meiotic recombination frequency in the
rDNA cistrons were similar to that in other chromosomal
regions, the distance from one end of the cluster to the
other would be on the order of 800 map units. However,
it has been estimated to be about 3 cM (Zamb and Petes,
1982). Two types of experiments have been used to measure the recombination frequency in the rDNA at meiosis.
In one set of experiments two strains whose rDNA cistrons
differ by a restriction site polymorphism were crossed and
the progeny were examined for recombinants (Petes and
Botstein, 1977). The frequency of recombination was less
than 1% of that expected based on the number of recombination events per genome and the fraction of the genome that is rDNA. An independent measure of the frequency of meiotic recombination using markers at either
end of the rDNA array (Zamb and Petes, 1982) indicated
that the recombination frequency was at least 15-fold
lower than expected. Both of these experiments suggested that meiotic recombination is suppressed in rDNA
cistrons.
Previous studies estimated the frequency of mitotic
recombination inthe rDNA by following the loss of an indicator marker that has been introduced into the rDNA array
on chromosome XII. The loss is presumed to result from
mitotic recombination between the homologous rDNA
regions flanking the marker. A marker in the rDNA is typically lost at a rate of roughly 5 x 10- 4 per cell division
(Szostak and Wu, 1980). In this report we show that this
level of mitotic recombination in rDNA is a result of a specific recombination suppression and that both topoisomerase I and topoisomerase II are required for the suppression.
Introduction
Results
The homologous recombination system of the yeast Saccharomyces cerevisiae is extremely active in both meiosis
and mitosis. The frequency of recombination at meiosis
established for many chromosomal regions is about 1
map unit per 2 kb, or about 100 times greater than that
found for human meiosis. The role of the homologous
recombination system in controlling genome size is evident from studies on artificial duplications constructed by
transformation. Transformation of yeast cells with a circular plasmid containing a yeast gene for which there is homology on the chromosome results in a duplication of that
cloned segment (Hinnen et al., 1978). These duplications
are unstable in both meiosis and mitosis. Loss of one of
the copies occurs by a homologous recombination event
that leaves a single copy of the gene on the chromosome
(Hicks et al., 1979). The absence of substantial repeated
DNA in yeast has been ascribed to the activity of this homologous recombination system.
Despite the presence of this active recombination system, yeast cells are able to maintain the highly reiterated
cluster of ribosomal DNA genes (rDNA) on chromosome
XII. The 9.1 kb rDNA cistron encoding the ribosomal RNAs
is tandemly repeated approximately 200 times (Schweizer
Topoisomerase I Is Required for Suppression
of Mitotic Recombination in rDNA
The frequency of mitotic recombination in rDNA can be
monitored by following the frequency of loss of a marker
gene that has been inserted into the rDNA cluster by
transformation. A marker gene in the rDNA array can be
lost by any one of a variety of recombination events between rDNA repeats: unequal sister chromatid exchange,
intrachromosomal excision, or gene conversion (either
inter- or intrachromatid). Because loss of the marker gene
will result from only a fraction of these recombination
events (many events may not involve the particular rDNA
repeat in which the marker resides), the frequency of loss
of Ura + is a minimum estimate of the overall frequency of
mitotic recombination in rDNA.
A MATa strain containing a URA3 insertion in the rDNA
(CY163) was crossed by a MATa strain containing the
same URA3 insertion and a topl-8::LEU2 mutation
(CY205, cross 50). The frequency of loss of Ura + (mitotic
recombination in rDNA) was measured for the TOP1 and
topl::LEU2 spores by scoring the frequency of 5-fluoroorotic acid (5FOA) resistance. In S. cerevisiae, URA3 cells
Topoisomerases Suppress Mitotic rDNA Recombination
415
Table 2. Frequency and Rate of Mitotic Recombination in rDNA in TOP1 and topi Strains
Strain
Genotypea
Frequency of Ura-3
Rate of Ura-
CY117
CY119
TOPI rDNA::URA3-1
topl-7::LEU2 rDNA::URA3-1
1.5 x 10 (1)
2.1 x 10- 1 (140)
3.5 x 10- 4 (1)
1.9 x 10-2 (54)
CY118
CY120
TOP1 rDNA::URA3-2
topl-7::LEU2 rDNA::URA3-2
1.6 x 10- 3 (1)
3.2 x 10-1 (200)
2.7 x 10- 4 (1)
1.4 x 10-2 (53)
The frequency and rate of loss of Ura+ were determined as described in the text. Numbers in parentheses represent the fold increase in a top1
background as compared with TOP1. Each frequency represents the average of ten independent measurements. Rates were determined from
the Ura+lUra - sectoring of at least 5000 colonies in each case.
aOnly the relevant genotype for this experiment is shown for each strain. See Table 1 for complete genotypes.
To be sure that the enhancement of URA3 loss in topi
mutants was not a consequence of the presence of 5FOA,
we also measured the frequency of segregation of Ura +
to Ura- in these strains in the absence of 5FOA. Strains
were first streaked selectively on complete medium lacking uracil. From this medium, independent Ura + colonies
were then streaked nonselectively on complete medium
(containing uracil) to permit loss of URA3 from the rDNA
during mitotic growth of the colony. Colonies arising on
the nonselective plates were then examined for the fraction of Ura + and Ura- cells in that colony. This type of
quantitative analysis for both CY117/CY119 and CY118/
CY120 pairs is presented inTable 2. Ten independent colonies were examined from each isogenic pair for the fraction of Ura- cells, and the average frequency for each set
of ten was used to compare the frequencies of mitotic
rDNA recombination. The topi mutant strains show 140and 200-fold higher frequencies of mitotic recombination
in rDNA compared with the isogenic TOP1 strains. Southern analysis of four independent Ura- clones derived
from CY117 and CY119 and four from CY118 and CY120 indicate that in each case the lacZ DNA sequences present
in CB32 were also lost from the rDNA array (data not
shown). The co-loss of URA3 and lacZ excludes the possibility that the recombinant strains became Ura- by virtue
of a gene conversion event between the URA3 gene in the
rDNA and the ura3-52 allele at the resident URA3 locus,
and shows that loss of Ura+ results from recombination
between flanking rDNA sequences.
The recombination rate in rDNA in topi mutants is so
high that conventional methods of fluctuation analysis are
difficult. We took advantage of this high rate to devise a
simple fluctuation analysis of individual colonies that allows us to determine the fraction of cells that have not undergone a recombination event in the rDNA (i.e., the P(0)
term of the Poisson; Luria and Delbrtck, 1943). Strains
were grown overnight in complete medium lacking uracil
to select for cells that retain the URA3 gene. Cells were
diluted and plated for single colonies on rich medium
(YPD). The colonies that grew on YPD were then printed
to complete medium lacking uracil. Each colony was then
examined for the presence of a Ura- sector. A colony that
was one-half Ura ÷ and one-half Ura- was assumed to
have incurred a recombination event in the rDNA at the
first mitotic division in the growth of that colony. Unsectored colonies or colonies that were only partially sectored
(less than one-half Ura-) were assumed to have had no
recombination event at the first mitotic division in the
growth of that colony. By determining which colonies had
an event at the first division and which did not, we could
determine the rate of Ura- segregants from the P(0) term
of the Poisson. The data, presented in Table 2, show that
top1 mutants have 53- to 54-fold higher rates of mitotic
recombination in rDNA compared with their otherwise isogenic parents.
Mitotic recombination in rDNA was measured by a third
method that did not require an insertion in rDNA to signal
the recombination event. Diploid strains were constructed
that are heterozygous for an EcoRI restriction site polymorphism in rDNA. Most yeast strains contain seven
EcoRI sites per rDNA cistron (form I strains), but some
contain only six EcoRI sites per rDNA cistron (form II
strains; Petes et al., 1978). If such a diploid were to undergo a recombination event at the four-strand stage in mitosis (and most mitotic recombination events in rDNA occur at the four-strand stage, since the primary mitotic
event is sister chromatid exchange [Szostak and Wu,
1980]), then subsequent cell division would frequently
produce diploid progeny that contained an unequal mixture of the two rDNA forms (Figure 2A). When such a
recombinant was induced to undergo meiosis, four spores
would be produced, two spores each with a mixture of
forms I and II and two spores each with only form I(or with
only form II; see Figure 2A). This analysis assumes that
no recombination in rDNA occurs during meiosis (see below). Because recombination can take place anywhere
within the rDNA array, an enormous variety of different
form i/form II mixtures is possible for the four meiotic
spores derived from a diploid that undergoes recombination at the four-strand stage of mitosis. However diverse
these events may be, all mitotic events have one characteristic pattern: they generate only two patterns of form 11I
mixtures among the four meiotic spores. By contrast,
meiotic recombination events in rDNA could yield tetrads
in which either three of the four spores or all four of the
spores have different form I/I1patterns (Figure 2B).
We have constructed form 1/11
diploids where the MATa
parents are the same and the MATa parents differ by only
the disruption of the TOPi gene (CY159, CY160, CY161,
CY162; Table 1). The segregation of form I and form II
rDNAs was analyzed by Southern analysis for 32 fourspored tetrads from CY159 and CY161 (TOP1/topl) and 26
Topoisomerases Suppress Mitotic rDNA Recombination
417
TOP 1/top1
a b c d
topl/topi
a
b
c
Table 3. Frequency of Mitotic Recombination in rDNA in top2-1 (ts)
and TOP2 Strains
d
ale
M
---..
40
-
-`)-
me 0 ý04M
404
-- q
a
9
10
Frequency of UraSpores
0
0
24 C
30 C
top2-1 (ts)
39-60
39-3D
39-18B
10101-14B
10102-78
10101-20
TOP2
1.7
5.1
1.3
9.7
3.8
1.7
x
x
x
x
x
x
10- 3
10- 4
10- 3
10- 4
10- 3
10- 3
(1)
(1)
(1)
(1)
(1)
(1)
4.0
2.3
3.2
6.7
1.7
1.0
x
x
x
x
x
x
10-1
10-'
10-2
10-2
10-1
10-1
39-8D
39-4A
10101-1A
10102-8C
10101-4C
6.7
7.2
4.0
2.9
2.5
x
x
x
x
x
10- 4
10- 4
10- 3
3
1010- 3
(1)
(1)
(1)
(1)
(1)
6.8
2.9
1.2
1.6
3.8
x
x
x
x
x
10- 4 (1)
10- 3 (4)
10- 3 (0.3)
10- 3 (0.6)
10-4(0.2)
(235)
(451)
(25)
(69)
(45)
(59)
Each frequency is the average of four independent measurements of
the frequency of Ura- segregants. Numbers in parentheses represent
the fold increase at 300C relative to 24 0C.
05
Figure 4. Southern Analysis of Tetrads to Follow the Segregation of
Form IIForm IIrDNA Polymorphisms in Mitosis and Meiosis in toplltopl
Backgrounds
and TOP1/70TOP1
The bands marked X', B,and C in panel I correspond to the EcoRI fragments shown schematically in Figure 3. Panels 1-5 are tetrads from
TOPIlTOP1 diploids, and panels 6-10 are tetrads from toplltopl
diploids. Panels 1 and 2 are tetrads from CY161; panels 3, 4, and 5 are
tetrads from CY159; panels 6, 7, and 8 are tetrads from CY160; and
panels 8, 9, and 10 are tetrads from CY162. In each panel the probe
was the 2.7 kb Hindlll fragment of yeast rDNA (see Figure 3).
the same frequencies of mitotic recombination inrDNA at
240C as the five wild-type spores, as shown in Table 3.
However, at 300C the top2-1 (ts)-containing spores have
greatly ir,creased frequencies of mitotic recombination in
rDNA relative to the TOP2 spores. The recombination frequency ,n rDNA in a top1 top2-1 (ts) double mutant is
about the same as that in either topl or top2-1 (ts) single
mutants (data not shown), although inall cases the recombination frequency is near the theoretical limit of 1.The interpretation of the results is not as clear as with the topi
disruption because the top2-1 (ts) mutation affects viability
(50%-80% of the cells in colonies grown at 30PC fail to
form colonies on rich medium at 240C). However, the top21 (ts) mutation does not cause a general increase in all
types of recombination events at 300C (see subsequent
section).
Mitotic Recombination at Other Loci Is Unaffected
in top1 or top2 Mutants
CUP1 Repeat
The CUP1 locus in S. cerevisae encodes a metallothionein-like protein that mediates copper resistance (Fogel et
al., 1983). Strains containing a single copy of CUP1 are
sensitive to killing by copper, whereas strains containing
ten tandemly repeated copies of CUPI are copper resistant. Most laboratory yeast strains contain about ten
directly repeated copies of CUP1 on the left arm of chromosome VIII (Fogel and Welch, 1982).
We inserted a URA3 marker into the CUP1 region of a
strain (CY72) that has 10-12 tandemly repeated CUP1
genes (Southern analysis not shown) to monitor the frequency of mitotic recombination. Four independent URA3
insertions were generated at CUP1 by transformation of
CY72 using plasmid FDp110. Southern analysis showed
that each of the insertions was at a different site within the
CUP1 repeat (data not shown). The strains carrying
CUP1::URA3 were subsequently transformed with Hindllldigested CB25 to make disruptions of the TOP1 gene
marked with LEU2. Each of the four isogenic pairs of TOP1
and topl insertions was tested for the loss of URA3 from
the CUP1 repeat by assaying the frequency of 5FOAresistant colonies. As shown inTable 4, there is no difference inthe frequency of 5FOA-resistant colonies inTOP1
versus topl strains for any of the four different CUP1::URA3
insertions.
One of the CUP1::URA3 insertion strains (FDY64) was
crossed to a top2-1 strain (CY206, cross F88-24), and the
appropriate spores were tested for the frequency of 5FOA
resistance at both 240C and 300C. As shown inTable 5, the
three different top2-1 (ts) spores tested at both 240C or
300C show no difference from TOP2 spores in the frequency of 5FOA-resistant segregants.
Simple Duplications
A simple duplication of the HIS4 region was constructed
by transforming strain CY72 to Ura ÷ with plasmid CB30,
which carries both HIS4 and URA3. The plasmid was first
digested with Bglll inorder to direct integration to the HIS4
locus. Southern analysis of several transformants showed
that the chromosomal arrangement in the transformants
was a HIS4 duplication flanking a single copy of the URA3
gene (data not shown). One of these transformants, desig-
Topoisomerases Suppress Mitotic rDNA Recombination
419
nated CY125 (Table 1), was then transformed to Leu + with
the 5.0 kb Hindlll fragment of CB25 to disrupt the TOP1
gene with LEU2. Southern blots confirmed the structure
of several transformants (data not shown). One of these,
CY126 (HIS4::URA3::HIS4 topl), was compared with the
isogenic CY125 (HIS4::URA3::HIS4 TOPI) for the frequency of loss of URA3 (5FOA resistance). As shown in
Table 4, the frequency of 5FOA resistance is approximately the same for both CY125 (TOPi) and CY126
(topl::LEU2).
A strain similar to CY125 was constructed (CY127, see
Experimental Procedures) except that each of the duplicated HIS4 copies contained a 20 nucleotide oligomer
representing the yeast version of the highly conserved
topoisomerase I binding site from yeast rDNA. This experiment was designed to test whether the synthetic
topoisomerase I binding site from rDNA was sufficient to
cause topoisomerase I-mediated suppression of recombination. An isogenic topi derivative of CY127 (designated
CY128) was constructed by transformation (see Experimental Procedures). As shown in Table 4, there is no
difference in the frequency of 5FOA resistance between
CY125 and CY127, and, therefore, no effect of top1 deficiency on recombination between these repeats. Southern analysis of four independent 5FOA-resistant derivatives of CY127 and four from CY128 demonstrate that the
URA3 gene is missing from HIS4 in the recombinants and
not simply gene-converted to Ura- from the resident
ura3-52 allele (data not shown).
Strains containing the simple HIS4 duplication and
top2-1 (ts) (generated by crossing CY125 and CY206, cross
F88-21) were assayed for their frequency of 5FOA resistance at both 240C and 300C. As shown in Table 5, there
is no difference between top2-1 and wild-type strains in
the frequency to 5FOA resistance at either 240C or 300C.
The frequency of 5FOA resistance in three different top2-1
(ts) spores is essentially the same at both 240C and 300C,
as shown in Table 5.
MAT Switching
Mitotic recombination between the silent mating type cassettes (HMR and HML) and the active cassette (MAT) is
suppressed (Klar et al., 1981). Although the suppression
is known to involve the SIR genes, the mechanism is unclear. We tested the ability of otherwise isogenic TOP1
(CY72 and CY66) and topi mutant strains (CY112 and
CY64) to switch mating types spontaneously (Table 4). The
frequency of switching is the same for both pairs of strains,
indicating no role for topoisomerase I in suppression of
mitotic recombination events between the repeated mating type cassettes. We have also tested several top2-1
strains for the ability to switch mating types spontaneously
at both 240C and 300C. As shown in Table 5, there is no
difference in either MATa-to-MATd or MATa-to-MATa
switching frequency at either 240C or 300C in top2-1
versus wild-type strains.
Mitotic Recombination in rDNA Is Not
Dependent on RAD52
The RAD52 gene product is required for mitotic recombination events involving gene conversion (Jackson and
Table 6. Frequency of Mitotic Recombination in rDNA in rad52 strains
Strain
Genotype
Frequency of Ura-
CY210
CY211
CY212
CY213
RAD52 TOP1
topl-7::LEU2
rad52::TRP1
rad52::TRP1 topl-7::LEU2
3 x 10 (1)
2 x 10-' (67)
8.5 x 10- 3 (2.8)
4.2 x 10-2 (14)
3
Numbers in parentheses represent the fold increase relative to the frequency in a RAD52 TOP1 background. Each frequency is the average
of ten independent measurements.
Fink, 1981). Aset of strains have been constructed (CY210,
CY211, CY212, and CY213; see Experimental Procedures)
consisting of otherwise isogenic pairs of RAD52 and
rad52::TRP1 in both TOP1 and topl::LEU2 genetic backgrounds. Mitotic recombination in rDNA was measured in
these strains as described above, and the results are
presented in Table 6. A disruption of RAD52 does not decrease rDNA recombination in TOP1 strains. topi rad52
strains have an apparent 4.7-fold decrease in the frequency of rDNA recombination as compared with top1
RAD52. The interpretation of this result should be tempered by the observation that top1l rad52 strains grow
much more slowly than either topi or rad52 single-mutant
strains.
Meiotic Recombination in rDNA in toplltopl Mutants
Using strains polymorphic for an EcoRI restriction site in
the rDNA cistron, Petes and Botstein (1977) showed that
meiotic recombination in the rDNA cistrons is suppressed
300-fold relative to the rest of the nuclear genome. They
observed no meiotic recombination in 12 tetrads despite
the fact that the rDNA array is the size of a typical yeast
chromosome and, therefore, should show an average of
five meiotic recombination events per meiosis. We have
constructed diploids that are heterozygous for an EcoRI
site polymorphism in rDNA and are either TOPlltopl
(CY159, CY161) or toplltopl (CY160, CY162; see Experimental Procedures and Table 1). We were unable to determine whether topoisomerase II is required for suppression of rDNA recombination during meiosis because
recent data indicate that topoisomerase II is required for
the first meiotic division (Connie Holm, personal communication). The toplltopl diploids were induced to sporulate, and their meiotic progeny were examined by Southern analysis to monitor the segregation of the rDNA
polymorphism.
A meiotic event is characterized by either three or four
different form 1/11
patterns among the four meiotic progeny
(see Figure 28). In the homozygous toplitopl mutants
(CY160 and CY162), seven of the 32 tetrads examined
gave meiotic patterns of this type (Figure 4, panels 6 and
7). In each of the seven tetrads that display meiotic recombination events, the event appears to have been nonreciprocal. For example, in Figure 4, panel 7, spore d appears to be a form I spore that has acquired-some form
II rDNA. However, neither of the form IIspores (a or b)has
acquired any additional form I DNA or appears to have lost
Topoisomerases Suppress Mitotic rDNA Recombination
421
(Petes, 1980). The frequencies of these events in TOP11
TOP1 (15%, 10/65) and toplltopl (6%, 6/98) strains issimilar to that reported previously (10%; Petes, 1980).
Reciprocal meiotic crossovers also occur at equal frequencies in TOP1lTOP1 and toplltopl diploids. Reciprocal
recombination is highly repressed in rDNA relative to the
rest of the nuclear genome. Petes and Botstein (1977) did
not observe a reciprocal event in 12 tetrads using the
EcoRI restriction polymorphism as a marker. Our data
show that reciprocal recombination occurs in both TOP11
TOP1 and toplltopl backgrounds at a frequency of 8%
(5/65) and 5% (5/98), respectively. This level of recombination is still low because a segment of DNA the size of
the rDNA cistrons should have an average of five crossover events per meiosis.
We observed four gene conversion events in the topll
topi diploid and none in the TOPI/TOP1 diploid. We also
observed an apparent increase in gene conversions in
toplitopl strains using the EcoRI restriction polymorphism (see above). Clearly, more tetrad data are required
to determine whether a deficiency of topoisomerase I
causes any significant difference in meiotic gene conversion frequency.
In agreement with the results from analysis of haploid
cells and the form I/form II polymorphism, the toplltopl
strain (relative to the TOPIlTOP1 control) shows a great increase in mitotic recombination events in rDNA preceeding meiosis. Most of the patterns that are presumed to be
mitotic could also arise via double meiotic events. However, the absence of a large number of single meiotic
events argues that these patterns represent mitotic
events.
We have noticed a slight growth defect that segregates
2:2 in 16 tetrads from CY182 (toplltopl). In each tetrad, the
two slow-growing colonies have the same rDNA genotype
as determined by the indicator genes (that is, the two slow
growers are both HIS3 or both URA3). This result can be
explained if the growth defect occurs via an event in mitosis that is linked to the rDNA cistrons. Since no growth defect is generated in TOP1ITOP1 diploids (CY180), we ascribe the slow-growth phenotype to the enhanced rDNA
mitotic recombination in toplltopl cells. Perhaps the loss
of amplification resulting from rDNA recombination is the
cause of the slower growth rate.
Discussion
Yeast strains defective in either topoisomerase I or
topoisomerase II activity (topi and top2-1 (ts) mutants)
have much higher levels of mitotic recombination in the
rDNA than do TOP + controls. Mitotic recombination in
other duplicated regions of the genome is not affected by
top1 or top2-1 (ts). We interpret this enhancement of rDNA
recombination in top1 and top2 strains to mean that one
function of topoisomerases in yeast is to suppress recombination specifically inthe rDNA. A large number of genes
have been identified in yeast that are involved in the
recombination process (Orr-Weaver and Szostak, 1985);
however, most of these affect recombination throughout
the genome, whereas the effects of the topi and top2 mutations appear to be restricted to the rDNA.
The existence of a system to suppress mitotic recombination in the rDNA is somewhat unexpected because mitotic recombination is usually considered a rare event.
However, in the absence of topoisomerase I or II,the frequency of rDNA recombination jumps as high as one
recombinant in every three cells. At these high frequencies, successive amplification and deletion of the rDNA
array by recombination could be deleterious to growth. Although we have no direct evidence for deletion or amplification, we have observed a growth defect that could result
from an alteration in the number of rDNA cistrons. Tetrad
analysis of toplltopl diploids revealed a growth defect
linked to the rDNA cistrons. This slow growth could be a
consequence of the increased frequency of deletion or
amplification of the rDNA array in the toplltopl background. The slow-growing colonies gave rise spontaneously to faster-growing cells at high frequency, making
analysis difficult. Presumably, repeated cycles of unequal
sister chromatid exchange could give rise to fastergrowing segregants by restoring the array to its original
size (Szostak and Wu, 1980). Since diploids as well as
haploids require topoisomerase function for suppression
of rDNA recombination, any defect in growth because of
recombination has an added consequence for meiosis.
Since each mitotic diploid is developmentally capable of
undergoing meiosis, deleterious alterations of the rDNA
can be transmitted to meiotic progeny.
It is known that meiotic recombination in the rDNA is
strongly suppressed relative to the rest of the nuclear genome in both yeast and Neurospora (Petes and Botstein,
1977; Russell et al., 1988). Our results indicate that
tcpoisomerase I is not required for this suppression in
yeast. Although the rDNA array is the size of a typical
yeast chromosome (1000 kb) and should have about five
crossovers per meiosis, homozygous toplltopl mutants
gave only one crossover in 20 meioses as assayed with
rDNA marked with URA3 and HIS3. Furthermore, no
reciprocal crossovers were obtained in 32 tetrads from
topl/topl diploids heterozygous for an rDNA restriction
site polymorphism. We conclude that an alternative system is required for suppressing meiotic reciprocal recombination in the rDNA. Although the data are not conclusive, our results indicate a possible differential effect of
topoisomerase Ion meiotic gene conversion as compared
with reciprocal recombination. Using the rDNA polymorphism, we detected 7/32 tetrads with conversions in
topl/topl mutants as compared with 0/26 in TOP1/topl
strains. And using the URA31HIS3 markers, we detected
4/98 tetrads with conversions in toplltopl versus 0/65 in
TOPI/TOP1 diploids.
The overall interpretation of the top2 results is not as
straightforward as that for topi. For example, we could not
determine the meiotic effect of the top2 mutation, because
top2/top2 diploids fail to undergo normal meiosis. Moreover, it is not possible to study the effects of a null allele
of TOP2 (as was done for TOP1) because TOP2 is required
for life. Although the top2-1 (ts) allele that we used is deficient in topoisomerase II activity at the semipermissive
Topoisomerases Suppress Mitotic rDNA Recombination
423
suppression of recombination between the directly
repeated termini of the Ty retrotransposon (Rothstein,
1984). The mechanism by which the SIR genes and the
EDR1 gene suppress recombination is not known. However, our data show that neither topoisomerase I nor
topoisomerase IIis required for recombination suppression between the mating type cassettes, and recent experiments show that they are not required for delta-delta
recombination (unpublished observations). In many organisms meiotic recombination is suppressed incentromeric regions. InS.cerevisiae there is some evidence for
suppression of recombination in centromeric regions
(Lambie and Roeder, 1988). Perhaps this suppression is
mediated by a topoisomerase activity that is among the
ensemble of proteins that constitute the centromere.
Experimental Procedures
Strain Constructions
CY119 and CY120
These strains are single-step disruptions of the TOP1 gene marked by
LEU2. Plasmid CB25 was digested with Hindill and then used to transform CY117 and CY118 to Leu + . The large Hindill fragment of CB25
contains the entire TOP1 coding region from which 849 internal nucleotides have been deleted and into which the LEU2 gene was inserted
(Thrash et al., 1985).
CY159, CY160, CY161, and CY162
CY159 and CY160 are diploids that are heterozygous for an EcoRi restriction site polymorphism in rDNA. The MATa parents are the same
for the two strains (CY167), and the MATa parents (derived from CY72)
are isogenic except that the TOP1 gene has been disrupted using
CB25 in the case of CY160. CY159 and CY160 contain the same URA3
insertion in the rDNA. CY161 and CY162 have the same MATa form II
parent (CY168) and MATa parents that are isogenic except for a top1
disruption (CY64 for CY162 and CY66 for CY161). CY161 and CY162
contain no foreign insertions in the rDNA.
CY125, CY126, CY127, and CY128
+
CY125 was made by transformation of CY72 to Ura with CB30 (which
contains an internal 2.2 kb Xbal-Xhol fragment of the HIS4 gene
cloned into YIP5; see Donahue et al. 119821 for a restriction map). The
TOP1 gene was disrupted in CY125 using CB25 digested with Hindlli
to generate CY126. CY127 was made in two steps, as follows: First,
CY72 was cotransformed with YEp13 and CB26 (which had been
digested with Xbal and Xhol). CB26 contains an internal 2.2 kb
Xbal-Xhol fragment from the HIS4 gene into which a single 20 nucleotide oligomer containing the conserved topoisomerase I binding site
from yeast rDNA had been cloned. The sequence of the oligonucleotide was 5S'CGATTTCTTTCTAAGTGGAT-3'. The complementary oligonucleotide was made such that, upon annealing, the double-stranded
molecule contains a 2 nucleotide overhang at each 5' end, forming
Clal-compatible ends. Leu + transformants were screened for His-.
+
Two His- colonies were found among 2300 Leu transformants. These
were grown nonselectively in order to lose the cotransforming Leu+
plasmid. Both of the His- transformants had polar his4A mutations as
determined by complementation. This is expected, since the oligonucleotide was cloned into the Clal site in the HIS4A region. One of these
was designated CY113. In the second step, CY113 was transformed to
Ura+ with CB31. CB31 contains the Xbal-Xhol fragment from CB26
+
cloned into YIP5. Therefore, Ura transformants arise via duplication
of the his4 region flanking the URA3 gene. Each his4 copy contains a
topoisomerase I rDNA olionucleotide at the Clal site. CY128 was de+
rived from CY127 by tranformation to Leu using CB25 that had been
digested with Hindill such that the TOP1 gene was disrupted and
marked by LEU2.
CY210, CY211, CY212, CY213
CY210 is a TOP1 spore derived from cross 39. It contains a single URA3
insertion in the rDNA. CY211 is isogenic to CY210 except for the disruption of the RAD52 locus. The disruption was made using the 1.97 kb
BamHI fragment from CB15, which contains the TRP1 marker. CY212
is a topl-8::LEU2 spore from cross 39. CY213 is isogenic to CY212 except for the same rad52::TRP1 disruption.
FDY62-FDY69
CY72 was transformed to Ura+ with Kpnl-digested FDp110. Four independent transformants were designated FDY62, FDY64, FDY66, and
FDY68. The URA3 marker was shown to have integrated at the CUP1
locus by both tetrad analysis and Southern blot analysis (data not
shown). The Southern analysis showed that each of the four transformants contained a URA3 insertion at a different location in CUP1. Each
of these strains was then transformed to Leu+ with Hindlll-digested
CB25 in order to introduce the topl-7::LEU2 mutation. These transformants were designated FDY63, FDY65, FDY67, and FDY69.
CY180 and CY182
CY180 is a diploid made by mating CY143 and CY147. CY143 is a Ura+
transformant of CY141 using CB32 (which contains rDNA sequences
and the URA3 gene). CY147 isa His + transformant of CY142 (an otherwise isogenic MATd derivative of CY141) using CB39 (which contains
rDNA sequences and the HIS3 gene). Therefore, CY180 is a diploid
with a URA3 insert in the rDNA of one parent and a HIS3 insert in the
rDNA of the other. CY182 was made by mating CY151 and CY155.
CY151 is the top1 derivative of CY143, and CY155 is the top1 derivative
of CY147 (both made by single-step disruption using Hindlll-digested
CB25). Therefore, CY182 is isogenic to CY180 except that it is toplltopl
whereas CY180 is TOP1/TOP1.
Plasmid Constructions
CB32
This plasmid contains the 6.4 kb Hindlll fragment from yeast rDNA
cloned into pMC303 (Crabeel et al., 1985), which was partially digested
with Hindlll. pMC303 is 11kb in size and carries an ARG3::IacZ fusion,
the URA3 gene, and pBR322 backbone.
CB25
This plasmid carries the 5.1 kb HindIll fragment containing the TOP1
gene from which 849 internal nucleotides have been deleted and the
LEU2 gene has been inserted (Thrash et al., 1985). The disrupted topi7::LEU2 HindIll fragment is 5.1 kb long and is cloned into pUC18 to
make CB25 (also designated pCT80; Thrash et al., 1985).
CB21
This plasmid carries a 2.2 kb Xbal-Xhol fragment containing an internal portion of the HIS4 gene cloned into the Xbal-Xhol sites in the
pUC18 polylinker.
CB30
This plasmid carries a 2.2 kb Xbal-Xhol fragment containing an internal portion of the HIS4 gene cloned into the Nhel-Sall sites in YIP5
(which carries the URA3 gene).
CB26
This plasmid was constructed by digesting CB21 with Clal and cloning
into that site a 20 nucleotide oligomer containing the putative topoisomerase I binding site from rDNA (see DNA sequence above). The
oligomer was made with Clal ends.
CB31
This plasmid was constructed by cloning the 2.2 kb Xbal-Xhol fragment from CB26 (which contains an internal portion of the HIS4 gene
with the 20 nucleotide oligomer carrying the putative yeast rDNA
topoisomerase I site cloned into the Clal site) into the Nhel-Sall sites
in YIP5.
CB15
This plasmid carries a disrupted copy of the RAD52 gene in the BamHI
site of pBR322. The 2 kb BamHI fragment of RAD52 (Schild et al.,
1983) is disrupted by a BamHI-Bglll fragment containing TRP1 from
YRp7. Thus, digestion of CB15 with BamHI releases a fragment containing the rad52::TRP1 disruption and a pBR322 backbone.
CB39
This plasmid carries a pBR322 backbone into which the 6.4 kb Hindill
fragment from yeast rDNA has been cloned in the Hindill site. It also
carries a 1.8 kb BamHI fragment containing the HIS3 gene in the
BamHI site of pBR322.
FDpl04
The Kpni fragment of JW6 (see Fogel and Welch 11982] for a restriction
map) was cloned into the Kpnl site in pUC18.
Topoisomerases Suppress Mitotic rDNA Recombination
425
post division segregation. Cold Spring Harbor Symp. Quant. Biol. 49,
629-637.
Russell, P.J., Petersen, R.C., and Wagner, S. (1988). Ribosomal DNA
inheritance and recombination in Neurospora crassa. Mol. Gen. Genet. 211, 541-544.
Schild, D., Konforti, B., Perez, C., Gish, W., and Mortimer, R. (1983).
Isolation and characterization of yeast DNA repair genes. Curr. Genet.
7,85-92.
Schweizer, E., MacKechnie, C., and Halvorson, H. 0. (1969). The
redundancy of ribosomal and transfer RNA genes in Saccharomyces
cerevisiae. J. Mol. Biol. 40, 261-277.
Szostak, J. W., and Wu, R. (1980). Unequal crossing over in the
ribosomal DNA of Saccharomyces cerevisiae. Nature 284, 426-430.
Thrash, C., Voelkel, K., DiNardo, S., and Sternglanz, R.(1984). Identification of Saccharomyces cerevisiae mutants deficient in DNA topoisomerase I activity. J. Biol. Chem. 259, 1375-1377.
Thrash, C., Bankier, A. T., Barrell, B. G., and Sternglanz, R. (1985).
Cloning, characterization, and sequence of the yeast DNA topoisomerase I gene. Proc. Natl. Acad. Sci. USA 82, 4374-4378.
Voelkel-Meiman, K., Keil, R. L., and Roeder, G. S. (1987). Recombination-stimulating sequences in yeast ribosomal DNA correspond to sequences regulating transcription by RNA polymerase I. Cell 48,
1071-1079.
Wang, J. C. (1985). DNA topoisomerases. Annu. Rev. Biochem. 54,
665-697.
Zamb, T. J., and Petes, T. D. (1982). Analysis of the junction between
ribosomal RNA genes and single-copy chromosomal sequences in the
yeast Saccharomyces cerevisiae. Cell 28, 355-364.
Chapter III
The ribosomal DNA array has an altered
structure in topoisomerase I mutants of
S. cerevisiae
The ribosomal DNA array has an altered
structure in topoisomerase I mutants of S.
cerevisiae
(Pulse field gel/topl)
Fred S. Dietrich*t, Michael F. Christman*, and Gerald R. Fink*t
*Whitehead Institute for Biomedical Research, 9 Cambridge Center,
Cambridge MA 02142 and tDepartment of Biology, Massachusetts
Institute of Technology, Cambridge, MA 02139
Yeast mutants lacking the only type I topoisomerase activity
detectable in crude extracts (topl mutants) display only a minor slow
growth phenotype. We have shown previously that topl mutants
have an elevated rate of mitotic recombination in the ribosomal
DNA (rDNA) array but not at other loci. We now report that in
strains deleted for TOP1, chromosome XII (the rDNA containing
chromosome) specifically fails to migrate into a pulsed field gel. The
other 15 chromosomes do not migrate differently in top1 mutant
versus wild-type strains. The aberrant migration of chromosome XII
is caused by the presence of rDNA. When chromosome XII from top1
strains is cut into large fragments with a restriction enzyme, the
fragment containing rDNA fails to enter the gel, whereas those
without rDNA migrate normally. Furthermore, when rDNA is
transferred to chromosome III, chromosome III migrates aberrantly in
topl strains. These data demonstrate that the rDNA array has an
unusual structure in topl mutants and suggest that topoisomerase I
may have a specific role in rDNA metabolism.
The TOP1 gene of Saccharomyces cerevisiae encodes a type I
topoisomerase (1,2). Strains carrying a deletion of the TOP1 gene are
viable, exhibiting only a minor growth defect. (2). This is surprising
because the TOP1 gene encodes the only biochemically detectable type
I topoisomerase activity in yeast; an activity that has been proposed to
be required for several crucial processes in the cell including
transcription and DNA replication. Recently we have shown that
strains carrying a topi deletion have a 50-200 fold higher level of
mitotic recombination in the ribosomal DNA (rDNA) than do
wildtype strains (3). This elevated level of recombination occurs only
in the rDNA, which consists of about 200 copies of a 9.1 kb unit
encoding all four mature ribosomal RNAs (25, 18, 5.8, and 5S), and
not in other repeated or single copy genes (3). Furthermore,
topoisomerase I has been shown recently to be localized to the
nucleolus (the site of rDNA transcription and ribosome biogenesis) in
both yeast (4) and in animal cells (5).
MATERIALS AND METHODS
Preparation of Yeast Chromosomal DNA. Yeast chromosomes
were prepared from logarithmically growing cells using the method
of Carle and Olson (6). DNA to be cut with restriction enzymes was
washed extensively in 1X restriction enzyme buffer (approximately 10
washes in 5 ml buffer) while the DNA was still imbedded in low melt
agarose The DNA plug was then digested in a minimal amount of 1X
buffer overnight at 370 with 50 - 100 units of enzyme.
Running Pulse Field Gels. Pulse field gels were run using 0.5X
TBE, 1% agarose in a CHEF apparatus (7). Gel buffer was cooled by
circulation through a refrigerating water bath. Gels were stained with
ethidium bromide and blotted to nitrocellulose by standard
techniques (8). Southern Blots were probed using hexamer labeled
probes (9).
RESULTS
In addition to the high frequency of recombination that occurs in
the rDNA in topi mutants we report here that there is a physical
alteration in the rDNA array. When chromosomes are isolated from
isogenic TOP1 and top1 strains and separated by pulsed field
electrophoresis (10), chromosome XII from the topi strain fails to
enter the gel. The 15 other chromosomes that make up the
chromosome set migrate normally in topl mutants and are present at
levels equal to those found in the TOP1 strain (Fig. 1A). This
phenomenon is found in many different DNA preparations, in
strains from several different genetic backgrounds, and by two
different DNA separation methods (7,11). Even when the DNA was
subjected to conditions that separate very large chromosomes (12) or
lig. I
lulsed field gel ele~trophoresis of yea'st
chromosomal DNA made from isogenic T( )1'I and tl,1p
strains. (A) Pulsed field electrophoresis performed at 12 ( ,121)
volts, with a 5 minute switching time for 32 hours (standard
conditions).The gel was stained with ethidium bromide. (B)
Pulsed field electrophoresis performed at 1() C(, 5() volts, with
a 30) minute switching time for 122 hours. The gel was stained
with ethidium bromide.
A
a
WELL
XII
IV
B
XII-
was extensively treated with RNase , chromosome XII from topi
strains failed to enter the gel (Fig. 1B).
A number of different experiments indicate that the rDNA repeat
on chromosome XII is responsible for the aberrant migration of
chromosome XII. When the chromosome preparations are cut with
NotI , a restriction enzyme that fails to cut in the rDNA array but cuts
chromosome XII into several large fragments, the rDNA containing
NotI fragment fails to migrate into the gel but a non-rDNA fragment
of chromosome XII containing the ILV5 gene migrates into the gel
normally (Fig. 2A). The failure to observe chromosome XII on CHEF
gels is not a consequence of preferential degradation of rDNA because
cleavage of the same chromosome preparations with an enzyme that
cuts once in each of the 200 rDNA repeats (Pstl) reveals equal
amounts of rDNA in TOPI and topI strains (Fig. 2B). Furthermore,
the amount and migration of rDNA isolated by standard procedures
for Southern analysis on standard agarose gels was identical in TOP1
and topI strains (3).Thus, the aberrant migration of rDNA in topi
strains is not observed when the DNA is cut into single repeat units
(9kb). This result also demonstrates that the physical alteration in the
array requires the presence of more than a single repeat unit, as
individual units migrate similarly in topl mutant and TOP1 strains.
Fig 2 (A) Chromosome preparations were digested with
Notl, run under standard conditions., and transferred to
nitrocellulose filters. The filters were then hvbridized to a
mixture of 32P labelled DNA containing both rDNA and the
ILV5 gene.(B) Chromosome preparations were digested with
Pstl, run under standard conditions, and probed with the h.3
kb Hindll fragment of rDNA.
A
WELL- ·
..
B
'
ýI
-IIF
&
rDNA
NotI -W
fragment
rDlNA
repeat
ILV5
NotI
i
-
4iaiLnc III
65
Fig.3 Construction of a strain containing rDNA on
chromosome II and its behavior in pulsed field gels (A) The
initial strain contains two different leu2 alleles, one on
chromosome I and one inserted into rDNA on chromosome
XII.(13) A strain carrying a Ieu2 fragment integrated into the
rDNA can undergo a crossing over between leu2 in the rDNA
and the resident leu2 allele giving rise to a Leu + strain
carrying a reciprocal translocation (first arrow, 13). A second
recombination event (second arrow) between the rDNAs on
chromosome III::XII and chromosome XII::III reconstituted
chromosome XII and generated a chromosome III containing
a duplication of Ieu2 flanking the rDNA. (B)Chromosome
preparations from logarithmically growing cultures were
from strains CY215 which carries a III::rDNA
chromosome(TOPl)and CY216 (isogenic to CY215 except that
it contains a topl-7 mutation introduced by transformation).
Pulsed field gel electrophoresis, transfer to nitrocellulose, and
hybridization to 32 p labelled rDNA were performed under the
same conditions as described in Figure 1.
A
rDNA leu2 rDNA
XII
III-
leu2
-------- --- --leu2
XII::III
III::XII
XII'
LEU2
-----, ,
-rDNA
rDNA
66
w
c,.
ell
Wcl
-
-
!•
XII
Ilil::rDNA -
67
When rDNA is translocated to chromosome III, a chromosome
that does not normally contain any rDNA, chromosome III now fails
to enter a pulsed field gel in a topl strain (Fig. 3B). The structure of
the rDNA repeats permits the transfer of an rDNA segment to
chromosome III by genetic means (see Figure 3A for details). A
chromosome III reconstituted by this event will have an rDNA array
at the site of the original translocation (LEU2 locus). It should be
noted that no other segment of chromosome XII should be transposed
to chromosome III by this event (Fig 3A). Chromosome III containing
rDNA transposed to the LEU2 locus by this method fails to enter the
gel in a top1 mutant. The rDNA cistrons on chromosome III also
show increased recombination in top1 strains- an ADE2 marker
inserted into the rDNA on chromosome III shows and approximately
80-fold increased loss in a top1 strain as compared with a isogenic
TOP1 strain.
The aberrant migration of chromosome XII in pulsed field gels is
observed in top1 strains only when they are growing exponentially
(Fig. 4). Chromosome XII from top1 cells that have entered stationary
phase migrates into the gel normally compared to chromosome
preparations from a TOP1 strain. This result is important in view of
the high frequency of recombination in top1 mutants because an
explanation for the unusual behavior of chromosome XII might be
Fig 4 Chromosome preparations from topi and TOP1
strains grown to stationary phase. DNA was run under
standard conditions, blotted to nitrocellulose, and probed with
the 6.3 kb Hindl fragment of rDNA.
O
0
WELLXII-
69
that the excessive recombination produces a great degree of size
heterogeneity in chromosome XII, some copies containing more and
some containing less rDNA. These chromosomes might be spread out
over the gel so that no distinct band is observed. The restoration of a
single chromosome XII of normal size in stationary phase makes this
explanation unlikely.
Mutants defective in DNA topoisomerase II do not show aberrant
migration of chromosome XII in pulsed field gels although they
exhibit an elevated rate of recombination in the rDNA (Fig 5). This
result suggests that the elevated rate of recombination in rDNA in
topi and top2 mutants occurs through different mechanisms. Both
of these experiments were performed with a ts allele of TOP2 (top2-1)
at a semi-permissive temperature (300C), because the TOP2 gene is
essential for viability. At 300C top2-1 strains exhibit elevated
recombination in the rDNA. However, the interpretation of this
experiment is not simple, because the top2-1 allele may provide
sufficient activity at 300C to mask a migration defect.
DISCUSSION
Our data demonstrate that the rDNA array has an altered structure in
strains carrying the top1 mutation.This structure must be transient
because chromosome XII migrates normally when isolated from
stationary phase cells. The migration defect seems unlikely to be
Fig 5
Chromosomes were prepared from strain
MFC39-6D (top2-1) growing logarithmically at 24 C or
30°C.and electrophoresed under standard conditions
Southern blot was probed with the 6.3 kb Hindlll fragment of
rDNA.
0
WELL
XII
4
0
caused by unresolved recombination intermediates because both topi
and top2 mutants show elevated levels of recombination in the
rDNA (3), but only top1 mutants show altered mobility of
chromosome XII.
Another possible explanation for the observed phenotype is that
the absence of topoisomerase I specifically interferes with replication
of the rDNA array. Topoisomerases have been proposed to act as
swivels to relieve the torsional stress that accompanies the
movement of a replication fork. Perhaps a slower replication of the
rDNA in top1 mutants allows replication bubbles to persist through
much of the cell cycle. DNA containing these replication bubbles
might fail to enter the pulsed field gel. In stationary phase when the
cells are no longer replicating their DNA, chromosome XII would
enter the gel normally because it would no longer have these bubbles.
This explanation is attractive because it has been shown that during
any given round of rDNA replication only one in every 3-5
replication origins is used (14,15) and the altered structure that we
observe in topl mutants requires more than one rDNA unit. Perhaps
the migration defect is cause by an aberrant migration of the 3-5 unit
replication bubbles, whereas when the DNA is cut into single unit
pieces these altered structures are destroyed.
Alternatively, the altered mobility could result from a
transcriptional defect in the topl mutant. Ribosomal DNA is highly
transcribed as compared to most single copy genes. Transcription
occurs divergently with RNA polymerase I generating a 35S transcript
and RNA polymerase III producing a 5S transcript from the opposite
strand (16). Divergent transcription has been postulated to introduce
positively and negatively supercoiled domains (17), which can be
relaxed by the actions of DNA topoisomerases. In the absence of
topoisomerase I accumulation of supercoiled regions may lead to the
rDNA becoming physically altered in such a way that large rDNA
regions are unable to migrate into a pulsed field gel.
In addition to the specific elevation of rDNA recombination in
topl mutants (3) and the localization of the TOPI protein to the
nucleolus (4) the physical alteration of the rDNA in top1 mutants
further suggests that topoisomerase I has some specific role in rDNA
metabolism. Determination of precisely what that role is will require
further experiments.
We thank Tom Petes from the translocation strain, and Nikki Levin,
David Pellman, and F. Scott Kieff for critical reviews of the
manucript M.F.C. was supported by a grant from the Jane Coffin
Childs Memorial Fund for Medical Research. The work was
supported by grant GM35010. G.R.F. is an American Cancer Society
Professor of Genetics.
REFERENCES AND NOTES
1. Goto, T., Laipis, P. & Wang, J. C. (1984) J. Biol. Chem. 259, 1042210429.
2. Thrash, C., Voelkel, K., DiNardo, S. & Sternglanz, R. (1985) Proc.
Natl. Acad. Sci. U.S.A. 82, 4374- 4378.
3. Christman, M. F., Dietrich, F. S. & Fink, G. R. (1988) Cell 55, 413425.
4. Giroux, C. N., Dresser, M. E. & Tiano, H. F. (1989) Genome 31, 8894.
5. Muller, M. T., Pfund, W. P., Mehta, V. B. & Trask, D. K. (1985)
EMBO J. 4, 1237- 1243
6. Carle, G. F. & Olson, M. V. (1987) Methods in enzymology 155, 468482.
7. Chu, G., Vollrath, D. & Davis, R. W. (1986) Science 234, 1582- 1585.
8. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning
(CSHL, Cold Spring Harbor, NY)
9. Feinberg, A. & Vogelstein, B. (1983) Analytical Biochemistry 132, 613.
10.
Schwartz, D. C. & Cantor, C. R. (1984)Cell 37, 67- 75.
11.
Carle, G. F. & Olson, M. V. (1985) Proc. Natl. Acad. Sci. U.S.A. 82,
3756- 3760.
12.
Smith, C. L., Matsumoto, T., Niwa, O., Klco, S., Fan, J. B.,
Yanagida, M. & Cantor, C. R. (1987) Nucleic Acids Research 15,
4481- 4489.
13.
Mikus, M. D. & Petes, T. D. (1982)Gentetics 101, 369-404.
14.
Walmsley, R. M., Johnston, L. H., Williamson, D. H. & Oliver, S.
G. (1984) Mol. Gen. Genet. 195, 260- 266.
15.
Saffer, L. D. & Miller, O.L. Jr. (1986) Mol. Cell. Biol. 6, 1148 - 1157.
16.
Bell, G. I., DeGennaro, L. J., Gelfand, D. H., Bishop, R. J.,
Valenzuela, P. & Rutter, W. J. (1977)
J. Biol. Chem. 252, 8118-
8125.
17. Liu, L. F. & Wang, J. C. (1987) Proc. Natl. Acad. Sci. U.S.A. 84,
7024- 7027.