volume 17 Number 8 1989
Nucleic Acids Research
Sequences that promote formation of catenated intertwines during termination of DNA
replication
Shawn C.Fields-Berry+ and Melvin L.DePamphilis*§
Department of Biological Chemistry, Harvard Medical School, Boston, MA 02115, USA
Received September 19, 1988; Revised and Accepted March 8, 1989
ABSTRACT
The normal sequence at which SV40 DNA replication terminates (TER) is
unusual in that it promotes formation of catenated intertwines when two
converging replication forks enter to complete replication (Weaver et al., 1985).
Here we show that yeast centromeric sequences also exhibit this phenomenon.
CEN3 caused accumulation of late replicating intermediates and catenated dimers in
plasmids replicating in mammalian cells, but only when it was located in the
termination region (180° from on'), and only when cells were subjected to
hypertonic shock to reduce topoisomerase II activity. Therefore, formation of
catenated intertwines during termination of DNA replication was sequence
dependent, suggesting that topoisomerase II acts behind replication forks in the
termination region to remove intertwines generated by unwinding DNA rather
than acting after replication is completed and catenates are formed. Under normal
physiological conditions, CEN3 did not promote formation of catenated dimers in
either mammalian or yeast cells. Therefore, CEN does not maintain association of
sister chromatids during mitosis in yeast by introducing stable catenated intertwines
during replication.
INTRODUCTION
Circular DNA molecules such as SV40 and polyoma virus chromosomes
replicate bidirectionally with two replication forks traveling in opposite directions
from a unique origin sequence (ori). Separation of sibling chromosomes does not
require a unique sequence, because termination still occurs approximately 180° from
ori even when the normal termination site is absent or moved to another location
relative to ori (reviewed in 1-3). Catenated intertwines are generated during
termination of DNA replication when replication proceeds by separating the DNA
templates (helicase activity) without concomitant unwinding of the two template
DNA strands (topoisomerase activity). If replication is completed before unwinding
is completed, then the two sibling molecules will be linked by one catenated
intertwine for each DNA helix turn that was not removed. If unwinding and
replication are completed simultaneously, then sibling molecules are released as
© I R L Press
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monomers with a small gap in the nascent DNA strand within the termination
region. Therefore, catenated intertwines may exist transiently in late replicating
intermediates ("8-structures"), but whether or not they are passed on in the form of
catenated dimers depends on how efficiently these intertwines are removed from
replicating intermediates. Evidence that specific DNA sequences promote
formation of catenated intertwines by retarding DNA unwinding during
termination of replication comes from studies on SV40.
Sundin and Varshavsky (4) observed that exposing SV40-infected cells to
hypertonic medium resulted in the accumulation of newly replicated SV40 DNA as
catenated dimers that were subsequently separated into monomers when the cells
were returned to isotonic medium. Since replicating intermediates, the direct
precursor to formation of catenated dimers, did not accumulate in these studies, it
appeared that production of catenated dimers was a normal consequence of DNA
replication and that hypertonic shock inhibited only the decatenation process that
occurred once replication was completed. However, Weaver et al. (3) observed that
hypertonic shock caused a reversible accumulation of both late replicating
intermediates that were about 90% completed (RI*) and catenated dimers, suggesting
that hypertonic shock inhibits DNA unwinding specifically in the normal
termination region of the SV40 genome (referred to as "TER" for convenience).
Specificity for TER was evident from the facts that replicating intermediates at
earlier stages of replication were not arrested, and that neither RI* nor catenated
dimers accumulated in the presence of hypertonic medium when replication
terminated at sequences other than TER (3). If the same enzyme that is required to
separate catenated dimers when they form is also required to unwind DNA in the
termination region, then inhibition of that enyzme would cause both the formation
and subsequent accumulation of catenated dimers. Apparently, this enzyme
interacts poorly with sequences normally found in the termination region of SV40.
This enzyme appears to be topoisomerase II.
Topoisomerase II is required specifically for termination of DNA replication,
and its inhibition produces catenated intertwines. Temperature-sensitive yeast
mutants in topoisomerase n accumulate catenated plasmid DNA (5) and interlocked
nuclei (6) at the restrictive temperature. In contrast, neither drugs nor mutations
that inhibit topoisomerase I cause formation of catenated dimers. Drugs that inhibit
topoisomerase II activity cause the accumulation of both RI* and catenated dimers
during SV40 DNA replication in a manner that mimics the effects of hypertonic
shock (7). In fact, VM26, a potent inhibitor of topoisomerase II, can inhibit
completion of SV40 RI* without causing accumulation of catenated dimers (8),
indicating that catenated dimers are formed only when unwinding activity in the
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termination region is reduced. Similar results are obtained during DNA replication
in vitro using cell extracts depleted of topoisomerase n (9). Since topoisomerase n is
also required to resolve catenated dimers into monomers, conditions that inhibit
the activity of this enzyme will not only generate catenated intertwines in the
termination region, but prevent their subsequent removal as well (3).
Since both topoisomerase II activity and a specific DNA sequence, the
centromere, are required for separation of chromosomes during mitosis in yeast (6,
10), it has been postulated that yeast centromeres may maintain association of sister
chromatids until anaphase by preventing resolution of catenated intertwines that
form during termination of DNA replication (4, 11). To test this hypothesis, we
examined the ability of yeast CEN3 to promote formation of catenated dimers in
mammalian cells using the same techniques that proved successful with SV40, since
identification of replicating intermediates is very difficult in yeast. We then
determined whether or not catenated dimers normally accumulate during plasmid
replication in yeast cells. The yeast CEN3 region behaved like the SV40 TER region
in promoting catenated intertwines during replication in mammalian cells, but
plasmids containing CEN3 did not form stable catenated dimers when propagated in
yeast. Therefore, CEN and TER represent two examples of specific sequences that
promote formation of catenated dimers when present at a termination site for DNA
replication, but not when located at regions outside the termination site, regardless
of the presence of absence of cell specific proteins. The results with CEN confirm
and extend our previous observations on sequence dependent formation of
catenated intertwines during termination of replication, and reveal that
topoisomerase II is required at the termination region to remove topological
intertwines behind replication forks rather than waiting until after catenated dimers
are formed to carry out this function.
RESULTS
Separation of Sibling Chromosomes in Mammalian Cells
To determine whether or not a yeast centromeric sequence promoted
formation of catenated intertwines during DNA replication, two plasmids were
constructed in which the yeast CEN3 sequence was positioned either 84 (pSVcen84)
or 180° (pSVcenl80) from the SV40 origin of bidirectional DNA replication that is
located at a specific site within ori (Fig. 1). These two plasmids and the parent
plasmid containing SV40 ori but lacking CEN3 (pSVori) were used to transfect
monkey cells that express SV40 large tumor antigen (T-Ag) and therefore allow
SV40 on-dependent DNA replication. To insure that the mechanism by which
sibling chromosomes are separated was not affected by the efficiency of DNA
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SV40 on
. Nrul
p S V o r i \\<pSVcen84)
Amp1
Dral (pSVcen180)
SV40 TER region
3'terminus of E-mRNA
180° from OBR
VP-1 gene
T-Ag gene
•4-
TER
3Merminus of L-mRNA
t
yeast CEN3 region
180°
Alul
(41)
In pSVcen180
Figure 1. Structure of plasmids containing yeast CEN3 and a comparison of the yeast
CEN3 region with the SV40 termination region (TER). A 301 bp Alul fragment
containing CEN3 was isolated from plasmid A195p6 (26) and joined to Sacl linkers
(27). Sacl linkers were also attached to pSVori (28) at either the Nrul site
(nucleotide 643) or the Dral site (nucleotide 1512). Orientation of the inserted
fragment was determined by restriction site analysis and contructs were chosen in
which region 2 of CEN3 was either 84° or 180° from the SV40 origin of bidirectional
replication (OBR; 29). The SV40 TER and yeast CEN3 regions are diagramed with
the fraction of AT bp, nucleotide map positions, and key landmarks indicated. A
sequence comparison made between the two regions with a stringency of 70% or
greater revealed a single region of homology.
replication, studies were carried out in both COS-1 cells containing an integrated oridefective SV40 genome and CMT-3 cells containing an integrated copy of SV40 T-ag
gene driven by the metallothionin promoter (11). CMT-3 cells induced with heavy
metals produce five-fold more T-Ag than COS-1 cells.
Under normal physiological conditions [IX Dulbecco's modified Eagle's
medium (DMEM)], the rate of DNA replication for all three plasmids was 2 to 3 fold
greater in CMT-3 cells than in COS-1 cells (Table I). This difference was due to the
increased level of T-Ag because the rate of wild-type SV40 DNA replication was
essentially the same in both cell lines (Table I). When either cell line was
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Table I. Effect of T-Antigen Levels on DNA Replication
Cell Line
COS-1
CMT-3
Relative Rates of DNA Synthesis in IX DMEM
SV40
pSVori
pSVcen84 pSVcen!80
(1.0)
0.25
0.17
0.17
1.2
0.52
0.48
0.40
Cell Line
COS-1
CMT-3
Ratio of DNA Synthesis in IX DMEM to 2X DMEM
SV40
pSVori
pSVcen84 pSVcen!80
2.1
1.9
1.6
1.7
2.2
2.2
2.6
2.3
DNA in transfected cells was radiolabeled at 44 hr post-transfection in IX or 2X
DMEM for 30 minutes as described in figure 2, and the small molecular weight
DNA extracted and then precipitated in acid to measure total plasmid or viral
PH]DNA.
transfected with DNA, incubated for 44 hr to allow DNA replication to reach its
maximum rate and then subjected to hypertonic shock by changing the medium to
2X DMEM, the rates of DNA synthesis were transiently reduced about 50% relative
to the rates observed in IX medium (Table I). Thiu inhibition of DNA synthesis by
hypertonic medium, previously observed with SV40 infected CV-1 cells (3, 4), was
independent of the DNA sequence in the termination region.
Each form of newly replicated DNA was identified by several criteria as we (3)
and others (4, 12) have previously done for SV40 infected cells and cells transfected
with plasmids utilizing SV40 ori to initiate replication. First, each DNA form had a
recognizable mobility during gel electrophoresis relative to DNA standards. Second,
addition of ethidium bromide to the electrophoresis buffer or introduction of
phosphodiester bond interruptions in the DNA sample dramatically altered the
mobility of covalently-closed DNA monomers and dimers. Finally, the mobility of
covalently-dosed, catenated dimers was confirmed by electron microscopy of DNA
isolated after gel electrophoresis. As previously observed with circular DNA
molecules in which replication terminated at sequences other than the SV40 TER
region (3), catenated dimers were not detected in pSVori even when the transfected
cells were placed in hypertonic medium (Fig. 2). DNA replication products
consisted of almost exclusively Forms I and II DNA, regardless of whether cells
were incubated in IX or 2X DMEM (Table n). The same result was observed with
newly synthesized pSVcen84 and pSVcenl80 when transfected cells were
radiolabeled in isotonic medium (Fig. 2; Table I). However, significant differences
were observed when cells transfected with these plasmids were transferred to
hypertonic medium.
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COS-1
CMT-3
pSVcen84
pSVcen180
pSVcen84
COS-1
pSVcemBO
Relative Concentration of DMEM
1
1.6 1.8
2
1
2
2
2
2.2
1 2
(nicked)
1 2
2
Hi.-*.
mm
.*-•. "
1
20'
MM
M
" ~~ ~ Jt
' 15' 20'
251 '
20'
'
Incubation Time
Figure 2. Sequence-dependent formation of catenated dimers in mammalian cells.
COS-1 or CMT-3 cells were transfected with viral or plasmid DNA which was later
radiolabeled as previously described (3). Data shown are from DEAE-dextran
mediated transfection but the same results were also obtained with the calcium
phosphate procedure. Medium for CMT-3 cells was supplemented with 100 uM
ZnCl2 and 1 uM CdSO4 to induce the metallothionine promoter driving T-ag
synthesis (12). Transfected monolayers were incubated for the times indicated in IX,
1.2X, 1.4X, 1.6X, 1.8X, 2X or 2.2X concentrations of Dulbecco's Modified Eagle's
Medium (DMEM) supplemented with [ 3 H]thymidine. Only the extreme
concentrations are shown for simplicity. Plasmid DNA was fractionated by agarose
gel electrophoresis, and the different forms of newly synthesized [3H]DNA detected
by fluorgraphy (3).
Radiolabeling of pSVcen84 in hypertonic medium (1.6X to 2.0X DMEM) did
not result in accumulation of catenated dimers, although 2X DMEM inhibited
formation of Form I DNA and increased the fraction of RI* in CMT-3 cells (Fig. 2,
Table II). In contrast, when cells transfected with pSVcenl80 were placed in
hypertonic medium, the products of DNA replication changed markedly (Fig. 2,
Table II). First, a large fraction of nascent pSVcenl80 accumulated as late replicating
intermediates (RI*) and catenated dimers (cat-D). The fraction of radiolabel in RI*
was greatest with shorter labeling periods (15 min) than at longer labeling periods
(20 and 25 min). In the presence of 2.2X DMEM, most of the pSVcenl80 DNA
accumulated as newly synthesized RI*. When samples of pSVcenl80 [ 3 H]DNA
labeled in hypertonic medium were stored at 4°C for several weeks, one or more
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Table II. Effect of Termination Region on the Products of DNA Replication
DNA
[Mediuml
RI*
Fraction of Nascent DNA in:
Form I
cat-D
Form II
pSVori
pSVori
IX
2X
6
4
<1
<1
28
35
41
45
pSVcen84
pSVcen84
IX
2X
9
21
<1
<1
23
63
61
5
pSVcenl80
pSVcenl80
pSVcenl80
IX
2X
2.2X
8
10
67
<1
19
6
27
14
7
41
44
2
SV40
SV40
SV40
IX
1.8X
2X
5
25
60
1
5
9
5
9
4
79
39
3
Data are taken from densitometer tracings of several autoradiograms exposed for
different periods of time so that the measured peak was proportional to the amount
of [3H]DNA (3).
phosphodiester bond interruptions were introduced ("nicked DNA") that resulted
in conversion of Form I to Form II DNA. In addition, a ladder of bands appeared
above Form II DNA that corresponded to relaxed catenated dimers containing one
to eight intertwines as described by Sundin and Varshavsky (13). Results obtained
in COS-1 and CMT-3 cell lines were essentially the same.
The accumulation of RI* and catenated dimers in the presence of hypertonic
medium was completely reversible; returning the transfected cells to isotonic
medium allowed conversion of RI* and catenated dimers into Form I and Form II
DNA, as previously observed with SV40 DNA (3). Therefore, termination of DNA
replication within a yeast centromere sequence promoted formation of catenated
dimers in the presence of hypertonic medium but not in the presence of isotonic
medium. Since the data with pSVcenl80 were essentially the same as previously
observed with SV40 DNA (Table U), CEN3, like SV40 TER, promoted formation of
catenated dimers only when present at the termination site for replication.
Furthermore, CEN3, like SV40 TER, responded to increased hypertonic medium by
increasing the fraction of newly formed late replicating intermediates (RI* in Fig. 2),
revealing that the primary effect of these sequences was to retard the progress of
replication forks through the termination region under conditions that inhibit
topoisomerase II activity.
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Separation of Sibling Chromosomes in Yeast Cells
The failure of yeast CEN3 to promote formation of catenated dimers in
mammalian cells under normal physiological conditions may reflect the absence of
CEN3-specific proteins in mammalian cells. Unfortunately, the experimental
protocol used with mammalian cells was not adaptable to yeast cells, and we were
unable to detect plasmid replicating intermediates in yeast. Therefore, we looked for
CEN3-dependent production of catenated dimers. Synchronized cultures of yeast
strain A281.13 harboring a plasmid containing both ARS1 and CEN3 sequences
(A105pl; Fig. 1) were allowed to proceed through 1.5 cell cycles with portions of the
culture withdrawn periodically for analysis. Catenated dimers were not detected,
and only Form I and Form II plasmid DNA was observed throughout the time
course as cells progressed through one cell division (Fig. 3, "Time Course"). To
insure that the absence of catenated dimers was not an artifact of the experimental
protocol, several control experiments were carried out.
Isolation of DNA from yeast begins with preparation of spheroplasts. To
determine whether or not catenated dimers were produced during replication but
subsequently separated into monomers during spheroplast formation, this
experiment was repeated using a temperature-sensitive topoisomerase II mutant
(yeast strain A305) that also harbors 1-2 copies per cell of plasmid A105pl.
Synchronized strain A305 was grown at the permissive temperature, but spheroplast
formation and plasmid DNA preparation were carried out at the restrictive
temperature to prevent topoisomerase II from resolving any catenated dimers that
may have formed. The results were identical to those with strain A281.13.
To insure that catenated dimers were not resolved during mitosis by
attachment of spindle fibers to CEN3, synchronized yeast cultures were incubated in
the presence of nocodazole, a potent inhibitor of tubulin polymerization (10). The
presence of nocodazole in these cultures did not significantly alter the distribution
of DNA forms (Fig. 3, "Noc"). Nocodazole concentrations of 5 to 15 ug/ml resulted
in cells with the morphology expected for premitotically arrested cells as compared
to control cultures in which a majority of cells were observed undergoing mitosis.
At the lowest nocodazole concentration, the rate of [3H]uracil incorporation into
acid precipitable DNA was the same as in control cultures, while the highest
concentration used reduced the rate of DNA synthesis by 30%.
To insure that catenated dimers would have been observed had they been
formed, a synchronized culture of A305 was incubated for 1.5 cell cycles at the
restrictive temperature for topoisomerase II inorder to cause the formation of
catenated dimers. A broad band of plasmid DNA was observed that migrated more
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Std
1
Time
ts
Course
Noc. top2
''1 2 3 4 5"a b c i r n
I-
Figuie 3. Structure of CEN3-containing plasmid DNA during yeast cell cycle.
Saccharomyces cerevisiae strains A305 (MATa adel+/ade2 ura3-52, Ieu2-3.112, his3,
top2) and A281.13 (MATa, Ieu2, his3, trpl, ura3, canl, cirO), each containing plasmid
A105pl (Fig. 1), were grown at 26°C in SD medium supplemented with adenine
sulfate, uracil, L-histidine and leucine (30). Cells were synchronized with a-factor
pheromone (31) and progress through the yeast cell cycle was monitored by phase
contrast microscopy and by radiolabeling of nascent DNA in vivo with [5,6-3H]uracil
(10). Aliquots of A281.13 were withdrawn at 30, 60, 90, 120 and 150 min (lanes 1-5).
Nocodazole was added to other samples immediately after removal of a-factor
pheromone to prevent completion of mitosis (Nco., 5, 10 or 15 u.g/ml, lanes a-c).
Synchronized A305 was grown at 37°C for 2.5 hr to inactivate topoisomerase II (ts
top2). Total DNA was prepared (32) and fractionated by electophoresis in an 0.6%
agarose gel. Plasmid DNA was detected by blotting-hybridization using nicktranslated plasmid [32P]DNA as a probe (33). A sample of purified A105pl DNA was
fractionated in lane 1 to identify Form I and Form II plasmid DNA.
slowly than Form II DNA (Fig. 3, "ts top2"), corresponding to the catenated dimers
of plasmids previously observed under these conditions (5, 14). The larger size of
A105pl relative to pSVcenl80 contributed to a decrease in resolution of catentated
dimers from Form II DNA.
Taken together, these results demonstrate that CEN3 does not stablize
catenated dimers that may form in yeast cells. Therefore, yeast centromeric
sequences do not maintain association of sister chromatids during mitosis by
introducing catenated intertwines during replication. The same conclusion was
reached by Koshland and Harrwell (15) in their study of plasmid replication in
various yeast cell division cycle mutants.
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Termination Region (-250 bp)
1. Strand Separation
2. DNA Replication (10 bp/fork)
Figure 4. Process of formation and resolution of transient catenated intertwines at
DNA replication forks.
DISCUSSION
DNA sequences that Alter the Pathway for Separating Sibling chromosomes
Of seven sequences examined so far, only TER and CEN promoted
accumulation of RI* and catenated dimers when placed at the termination region
and the host cells subjected to either hypertonic shock (Fig. 2; ref. 3) or
topoisomerase II inhibitors (7, 8). CEN3 is typical of other yeast centromeric
sequences (16) and was equivalent to TER in its effects on DNA replication. Neither
sequence affected the pathway for separation when placed at other regions of the
genome or when DNA replication occurred under normal physiological conditions.
Molecules that terminated at sequences other than TER or CEN replicated with the
same efficiency as molecules that terminated at either TER or CEN. Replication was
not affected by the level of T-ag, the size of the plasmid, or the presence of TER or
CEN at locations outside the termination region. Therefore, some DNA sequences
promote formation of catenated dimers in the termination region, and other
sequences do not. If the effects of hypertonic shock only affected separation of
catenated dimers, as originally reported (4), then the catenated intertwines in these
dimers would have to be localized at the termination region in order to account for
the effect of specific sequences such as TER and CEN. In fact, the intertwines in
catenated dimers of SV40 chromosomes appear to be distributed throughout the
genome (17).
When catenated dimers are formed, hypertonic shock effectively blocks their
resolution into monomers. Thus, the fact that hypertonic shock does not result in
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accumulation of catenated dimers when termination occurs at sequences other than
TER or CEN demonstrates that catenated dimers are not formed at all termination
sites. Therefore,, catenated dimers are an alternative, not an obligatory, intermediate
in separation of sibling chomosomes. Similarly, the fact that termination at TER or
CEN during hypertonic shock resulted in a 10 - 15 fold accumulation of RI* while
termination at five other sequences did not demonstrates that inhibition is directed
specifically at the process of completing DNA replication that occurs in the
termination region of late replicating intermediates.
The Role of Topoisomerase H
in Separation of Sibling
Chromosomes
As replication forks advance, the two template strands are separated by
helicase activity, resulting in positive superhelical turns that must be removed by
topoisomerase activity either in front or behind the replication forks (18).
Topoisomerase I can relieve this topological strain in front of replication forks by
breaking and closing one of the template strands or it can relieve this strain at the
replication fork by passing the single-stranded template on one side of a fork
through the double-stranded arm on the other side (19). In fact, high concentrations
of topoisomerase I prevents formation of catenated dimers from replicating plasmid
DNA and instead leads to the appearance of form II and form I DNA (20).
However, since replication in eukaryotic cells requires topoisomerase II activity for
separation of sibling molecules (5, 7-9, 14), the target for topoisomerase I in
eukaryotic cells appears to be restricted to the double stranded DNA in front of forks.
Since this target diminishes as two replication forks approach one another in the
termination region, topoisomerase I becomes ineffective.
At this point,
topoisomerase II is required to relieve topological strain because topoisomerase II is
the only enyzme that can pass two double-stranded DNA molecules through one
another (the enzyme's decatenation activity, Fig. 4). If topoisomerase II can act
behind replication forks to relieve torsional strain as rapidly as it is generated, then
unwinding of the parental unreplicated duplex can continue to completion without
forming any catenated dimers (18), whereas if topoisomerase II can act only in front
of replication forks, then formation of catenated dimers becomes an obligatory step
in termination. Conditions that reduce topoisomerase II activity will prevent
release of topological strain that results from unwinding DNA and thus allow the
formation of one catenated intertwine behind the replication forks for each DNA
helical turn removed in front of the forks (Fig. 4). This increased topological strain
will reduce the rate of DNA replication in the termination region, causing
accumulation of late replicating intermediates. Replicating intermediates that do
complete replication under these conditions will produce catenated dimers that
accumulate because the same enzyme required for relieving topological strain in the
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termination region is also required to resolve catenated intertwines in
nonreplicating molecules.
Topoisomerase n interacts efficiently at most, but not all, DNA sequences (21).
Since the presence of either SV40 TER or yeast CEN3 sequences in the termination
region markedly stimulate both the accumulation of late replicating intermediates
and catenated dimers under conditions that reduce topoisomerase II activity, TER
and CEN must represent sequences with which topoisomerase II interacts poorly,
thus making removal of intertwines by topoisomerase II hypersensitive to
inhibitors (hypertonic shock, VM26) as replication forks pass over these sequences.
In order to observe formation of catenated dimers during termination at other
sequences, topoisomerase II must be eliminated completely from the reaction (9).
TER and CEN share in common large A/T rich regions (Fig. 1) that can give
rise to unusual secondary structure which may explain their effect on termination
of replication. The SV40 TER region represents the strongest example of DNA
bending in the SV40 genome (22, 23), a phenomenon associated with A/T rich
sequences (24). Perhaps bent DNA is a poor substrate for topoisomerase II. SV40
TER also forms nucleosomes of unusual stability (23, 25) that may interfere with
topoisomerase II action. We note that the A/T rich region of CEN3 is 86 bp long and
produced catenated dimers with 7 to 8 intertwines (Fig. 2), while the SV40
termination region contains 213 bp that are 71% A/T , interrupted by a 25 bp G/Crich sequence and produces 20 to 24 intertwines under the same conditions (4).
Thus, the number of catenated intertwines is proportional to the size of the A-T
rich, suggesting that the inability of topoisomerase II to interact with the
termination region begins only after the replication forks have entered the A/T rich
domains.
ACKNOWI F.nr.F.MF.NTS
We thank J. Szostak for yeast strains A305 and A281.13, and plasmid A105pl.
We also thank C. Holm for helpful discussions and suggesting the use of
nocodazole. This work was supported by grants from the National Institutes of
Health and the American Cancer Society.
*To whom correspondence should be addressed
Present addresses: + Department of Genetics, Harvard Medical School, 25 Shattuck Street, Boston,
MA 02115 and ^Department of Cell and Developmental Biology, Roche Institute of Molecular
Biology, Roche Research Center, Nutley, NJ 07110, USA
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