From the Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021
Received for publication, November 3, 2002, and in revised form, December 30, 2002
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ABSTRACT |
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topB, encoding topoisomerase III, was
identified as a high copy suppressor of the temperature-sensitive
parC1215 allele, encoding one of the subunits of
topoisomerase IV. Overexpression of topoisomerase III at the
nonpermissive temperature was shown subsequently to restore timely
chromosome decatenation and suppress lethality in strains carrying
either temperature-sensitive parE or parC alleles. By developing an assay in vitro for precatenane
unlinking, we demonstrated directly that both topoisomerase III and
topoisomerase IV were efficient at this task, whereas DNA gyrase was
very inefficient at precatenane removal. These observations suggest
that precatenane unlinking is sufficient to sustain decatenation of
replicating daughter chromosomes in the cell.
Escherichia coli has four DNA topoisomerases, two type
IIA DNA gyrase and topoisomerase
(Topo)1 IV, and two type IA
Topo I and Topo III. The roles in the cell played by three of these
proteins is reasonably well defined (1-4). DNA gyrase is the major
facilitator of DNA replication, acting to convert the positive
supercoils generated directly to negative ones. Mutations in either
gyrA or gyrB, encoding the subunits of gyrase,
are conditionally lethal, and incubation of such mutated strains at the
non-permissive temperature causes a rapid cessation of DNA replication.
Topo IV is the cellular decatenase, responsible for unlinking the
daughter chromosomes so that chromosome segregation is accomplished in
a timely manner. Mutations in either parE or parC, encoding the subunits of Topo IV, are also
conditionally lethal. Incubation of either parE or
parC temperature-sensitive strains at the non-permissive
temperature results in the elaboration of a par phenotype,
where a large DNA mass accumulates in the center of an elongated cell.
This DNA mass results from repeated initiations of replication on
chromosomes that have not segregated. Topo IV appears also to be able
to support replication fork progression at about one-half the rate
supported by gyrase (5). Topo I acts as a balancing force to gyrase,
such that the net negative superhelicity of the chromosome is
maintained within an optimal range. Inactivation of Topo I is tolerated
only in the presence of compensatory mutations in gyrase that act to
reduce its activity. In the case of Topo III, there is more known about
its biochemical activities than its role in the cell.
Topo III was detected originally as a superhelical DNA relaxing
activity present in extracts prepared from cells deficient in Topo I
(6) and as an activity that bound to novobiocin affinity columns (7).
We purified Topo III as an activity capable of decatenating replicating
daughter chromosomes (8) after we determined that gyrase was
inefficient at this task (9). Whereas Topo III was inefficient at
superhelical DNA relaxation, requiring high temperature for optimal
activity, it was very efficient at decatenating multiply linked
daughter chromosomes as long as one of the DNA circles contained a
small gap. These observations suggested that the productive binding
site for the enzyme was single-stranded DNA (10), which has been borne
out by all subsequent studies.
Cloning of the gene for Topo III (topB (10)) indicated that
the protein had considerable identity to Topo I, although it lacked the
zinc finger region of the latter enzyme. Rothstein and colleagues (11)
reported contemporaneously that EDR1, a gene in yeast that,
when mutated, caused enhanced deletion of SUP4-o when it was flanked by Disruption of topB was noted originally not to cause any
obvious phenotype (10). Subsequently, Schofield et al. (13)
showed that mutR, a gene that, when mutated, caused a 5-fold
increase in RecA-independent recombination between short directly
repeated sequences, was allelic with topB. To date, this
observation describes the only known phenotype of topB
deficiencies over an otherwise wild-type background. Interestingly, the
situation in eukaryotes is very different, where the phenotypes of
deficiencies in Top3 clearly indicate that the enzyme plays a
significant role in the maintenance of genomic integrity. Deletion of
TOP3 in Saccharomyces cerevisiae causes a slow
growth phenotype, the hyper-recombination phenotype mentioned above
(11), and sensitivity to several DNA-damaging agents as a result of
disruption of the intra-S phase checkpoint (14). In
Schizosaccharomyces pombe, TOP3 is an essential
gene. Cells deleted for TOP3 undergo only a few rounds of abnormal
nuclear division before death (15, 16). In this organism, dysregulation of Top3 function has been linked to low Cdc2-cyclin B activity, causing
a defect in the late stages of homologous recombination (17). In
vertebrates, there are two Top3 isozymes, Here we show that E. coli Topo III can act as the principal
cellular decatenase, capable of unlinking replicating daughter chromosomes in vivo, and provide evidence that this
decatenation proceeds via the removal of precatenanes.
Enzymes, Reagents, Proteins, and Antibodies--
Restriction
enzymes and bacteriophage T4 DNA ligase were from New England Biolabs.
Pfu polymerase was from Stratagene. Hybond ECL
nitrocellulose membrane and ECL-Western blotting detection reagents
were from Amersham Biosciences. DNA polymerase I, RNase H, and E. coli DNA ligase were from Roche Molecular Biochemicals. Topo III
was prepared from a topA Standard Microbiological Techniques--
W3110parE10
and C600parC1215, described previously by Kato and
colleagues (24, 25), were obtained from the laboratory of N. Cozzarelli
(University of California, Berkeley). W3110 was from the E. coli Genetic Stock Center. DH5- Fluorescence Microscopy--
Cells were grown in LB with
appropriate antibiotics to an A600 of
0.5. Equal volumes of cell culture and a solution of 4% paraformaldehyde and 1 µg/ml 4,6-diamidino-2-phenylindole in 1× phosphate-buffered saline were mixed, and the cells were fixed for 30 min at 4 °C on a rotator. Aliquots (40 µl) of the fixed cells were
spread on polylysine-coated glass slides and allowed to air-dry.
Samples were observed with an Olympus AX-70 microscope. Fluorescence
and differential interference contrast images were acquired with an
Hamamatsu Orca-ER CCD camera using MetaMorph software (Universal
Imaging). Figures were prepared for publication using Adobe Photoshop
7.0 software.
Plasmid Constructions--
The construction of the pBR-kan-inc3
suppressor library has been described (26). pBR-suppressor-6 is a
plasmid from the pBR-kan-inc3 suppressor library carrying the genomic
insert described under "Results."
pBR-suppressor-6( Construction of W3110 Purification of ParEC316Y Topo IV and Assays for
Activity--
The sequence of the parE gene in
W3110parE10 was determined by sequencing a PCR fragment made
from the genomic DNA. The only amino acid substitution present was
C316Y. The overexpression plasmid pET21a-parE10 was
constructed by introducing a PCR fragment made from
W3110parE10 genomic DNA carrying the parE ORF
flanked by NdeI and BamHI sites. ParEC316Y
expressed from this plasmid was insoluble. Therefore, we transferred
the expression cassette from pET3C-parC (23) to
pBR-kan-inc3, which is compatible with pET21a, to give
pBR-kan-inc3-(pET)parC, and we co-overexpressed the mutant
ParE with wild-type ParC. This proved effective and yielded soluble
Topo IV. ParEC316Y Topo IV was then purified from BL21(DE3)(pET21a-parE10, pBR-kan-inc3-(pET)parC)
after soluble lysis by sequential chromatography on Q-Sepharose,
phosphocellulose, and Superose 6. In the presence of excess ParC, the
specific activities of ParEC316Y and wild-type Topo IV were essentially
identical in the superhelical DNA relaxation assay. Titrations were
used to determine the amount of Topo IV required to effect either 80% superhelical DNA relaxation or decatenation. Mutant and wild-type Topo
IV was then heated (in 5-µl aliquots) at 20-fold this concentration at 50 °C for the times indicated in Fig. 3 and then chilled on ice.
Superhelical DNA relaxation assays (27) and kinetoplast DNA
decatenation (29) assays were then as described previously with 1 µl
of the heat-treated Topo IV being added to 20-µl reaction mixtures.
The extent of the reactions was quantitated using Bio-Rad Quantity One
software from digital images of the ethidium bromide-stained gels.
Precatenane Unlinking Assay--
Precatenated DNA was prepared
in oriC replication reactions increased in volume 40-fold
(30) using Identification of topB as a High Copy Suppressor of
parC1215--
We previously conducted a screen for high copy
suppressors of the conditional lethality of W3110parE10
(26), which revealed an interaction between Topo IV and the
The ends of the genomic DNA insert in pBR-suppressor-6 were determined
using primers flanking the BamHI insertion site in the
vector. These sequence data were then used to position the genomic
insert on the sequence of the E. coli chromosome (34). The
genomic insert was found to be 5,769 bp long, spanning nucleotides 1,842,971-1,848,740 of the E. coli chromosome. This
chromosomal region included the genes topB, selD,
ydjA, and sppA. Given that topB was,
of this group of genes, the most likely candidate for the suppressor,
we digested pBR-suppressor-6 with both ScaI and HpaI and religated to form pBR-suppressor-6
(
If rescue by Topo III was a result of substitution of its topoisomerase
activity for that of Topo IV, it seemed reasonable to expect that the
various suppressor plasmids would also rescue the temperature
sensitivity of W3110parE10. This was the case (Table I). For
reasons that are unclear, rescue was more complete for each plasmid in
the parE mutant strain compared with the parC mutant strain.
Characterization of the Rescue of W3110parE10 by Overexpression of
Topo III--
Stationary overnight cultures grown at 42 °C of
W3110(pBADtopB) and
W3110parE10(pBADtopB) were diluted to an
A600 of 0.02 in fresh media, and the growth of
the cultures were followed at 42 °C (Fig.
1A). The wild-type culture
commenced growth immediately; on the other hand, the mutant culture
exhibited a pronounced lag but then grew at a rate that was roughly the
same as the wild-type culture. It should be noted that under the
conditions described, W3110parE10 itself will not grow at
all, all cells having reached a terminal phenotype during the original
overnight incubation at the non-permissive temperature. Furthermore,
examination of the cell and nucleoid morphology under conditions of
topB rescue demonstrated that Topo III was acting to
decatenate the daughter chromosomes (Fig. 1, B and
C).
Wild-type and mutant parE10 cells grown at 30 °C in the
presence of pBADtopB and arabinose gave essentially
indistinguishable morphologies with about 95% of the population
appearing as small cells with either two or four nucleoids. The other
5% of the population was made up of an assortment of abnormally
appearing cells (Fig. 1, B and C). When these two
strains were grown at the non-permissive temperature as described
above, the fraction of wild-type appearing cells in the
W3110(pBADtopB) culture decreased to 83%, and there was a
significant fraction of small cells with condensed, non-separated nucleoids (type 2, Fig. 1C). In addition,
filaments could be observed that contained nucleoids that either were
condensed (type 6, Fig. 1C) or appeared normal
(type 5, Fig. 1C); anucleates were observed as
well (type 3, Fig. 1C). Interestingly, the
largest population of abnormally appearing cells were small cells
clearly containing two nucleoids that were linked together by DNA
(type 4, Fig. 1C), suggesting that either
chromosome decatenation or segregation was delayed at 42 °C compared
with 30 °C. On the other hand, whereas the culture of
W3110parE10(pBADtopB) grown at the non-permissive temperature contained a significant fraction of wild-type appearing cells (43%, Fig. 1C), the proportion of type 2 and type 3 cells was elevated, suggesting that the rescue by overexpression of Topo III was not completely penetrant. However, when compared with the cellular morphologies present in a culture of
W3110parE10 grown at the non-permissive temperature, it was
clear that overexpression of Topo III resulted in rescue of the Topo IV
defect and effected decatenation of the daughter chromosomes.
W3110par10(pBR-kan-inc3) cells grown overnight at 30 °C,
diluted by 100-fold, and then shifted to 42 °C for 2 h
exhibited a classic par phenotype (Fig. 1B). Half
the cells in the population were long filaments with either a central
very large, dense nucleoid or two symmetrically placed, somewhat
smaller dense nucleoids. The other half of the population consisted of
anucleate cells (Fig. 1C). No wild-type cells were observed
at all.
pBR-suppressor-6 and pBADtopB exhibited different
efficiencies of rescue of C600parC1215, suggesting that
there was a certain minimal level of Topo III needed to effect
chromosome decatenation. We therefore examined the levels of Topo III
produced from these plasmids in W3110parE10 growing at
42 °C (Fig. 2). We used the parE10 strain, rather than C600parC1215, because
in the former strain both plasmids rescued to the same extent,
suggesting that we would obtain an unbiased value for the extent of
overproduction. The pBRsuppressor-6 plasmid produced about 60-fold more
Topo III at 42 °C than was present in W3110parE10 at
30 °C, whereas the pBADtopB overproduced Topo III by
about 180-fold. The level of Topo III in wild-type cells is very low,
having been estimated at about 10 copies per cell. Our current estimate
for Topo IV is about 1000 copies per cell (33); thus, Topo III works
effectively as the cellular decatenase when it is present at roughly
the same concentration as Topo IV.
Topo III Is Substituting for the Decatenation Activity of Topo
IV--
If the observed rescue by Topo III was a result of it
substituting completely for the activity of Topo IV, then
overexpression of Topo III should also rescue E. coli in the
complete absence of Topo IV. To assess this issue, we constructed a
conditional parE null strain.
W3110
To address this possibility, we constructed a modified topB
expression vector that could produce higher levels of Topo III than
pBADtopB; however, we found that even modest increases in the levels of Topo III expression over those produced by
pBADtopB were toxic to wild-type cells. Thus, we probed the
nature of the Topo IV activity present in W3110parE10 by
purifying the mutant protein (the amino acid substitution is C316Y) and
assaying its activity in vitro (Fig.
3). Both the superhelical DNA relaxation and the decatenation activity of ParEC316Y Topo IV was exquisitely temperature-sensitive compared with the wild-type protein, which showed
only minimal heat inactivation even after 20 min at 50 °C. It thus
seems unlikely that there is any residual available topoisomerase
activity of Topo IV under the conditions of Topo III rescue of the
temperature-sensitive strains described above. We also note that
W3110parE10 was completely rescued by the pBR-suppressor-6 plasmid. pBADtopB produced 3-fold more Topo III than
pBR-suppressor-6, a relationship that was maintained in
W3110
The second possibility is that
W3110
A third possibility is that the failure of Topo III overexpression to
rescue the parE deletion strain reflects a physical requirement for ParE at some stage of the cell cycle and that this
requirement is distinct from the requirement for ParE to form Topo IV
that is active for decatenation.
Topo III Is Very Efficient at Removing Precatenanes--
Under
most circumstances, Topo III will be a very poor superhelical DNA
relaxing enzyme. This is because of its strong dependence on
single-stranded DNA for activity and an apparently poor ability to bind
and partially denature double-stranded DNA to create its substrate.
Thus, maximal relaxation activity in vitro requires elevated
temperature to increase the single-stranded character of negatively
supercoiled substrates (8). Consequently, the enzyme is essentially
inactive in removing positive supercoils (36). How then can Topo III
operate on the topology of replicating chromosomes if during DNA
replication the only possible position for Topo III action is behind
the replication fork (where nicks and gaps presumably exist because of
the process of sealing Okazaki fragments)? The most likely answer to
this question is that Topo III works by removing precatenanes, an
alternative form that can be taken by the positive linkages that arise
between the daughter chromosomes during replication.
As noted originally by Champoux and Been (37), the positive linkages
between replicating daughter chromosomes that arise as a result of
unwinding of the parental duplex can distribute either ahead of the
replication fork as positive supercoils or behind the replication fork
as precatenanes, torus-like topological linkages that, if replication
were completed, would become catenanes. This equilibrium is governed by
the extent of DNA that is replicated: with positive supercoils
predominating when the majority of the template is unreplicated and
precatenanes predominating when the majority of the template is
replicated (38). The differential action of Topo IV and DNA gyrase in
the cell can be explained, at least in part, by their preference for
positive supercoils or catenanes as substrates (36, 39). We
demonstrated previously that Topo III can support both nascent strand
progression (40) and daughter chromosome decatenation (22) in
vitro. In addition, we have argued, based on indirect evidence,
that Topo III probably functions by removing precatenanes (36). Given
the observations reported here, we developed an assay to explore this
issue directly.
A replication system reconstituted with purified proteins was used to
replicate a plasmid DNA template carrying oriC (30). The
template DNA, which was 6 kb long, also carried replication terminator
Ter sites 2 kb clockwise and 3 kb counterclockwise from the
origin. Both Ter sites were oriented to stop replication fork progression. Thus, when the replication reaction is performed in
the presence of Tus, the forks formed at oriC will be
terminated and complete replication of the plasmid prevented. We showed
previously, using neutral agarose gel electrophoresis and electron
microscopy, that the partially replicated daughter duplexes in these
molecules are intertwined in precatenanes. The precatenanes take the
form of a ladder of bands during neutral agarose gel electrophoresis, the mobility and spacing of which almost precisely matches that of
complete catenanes (32). The steps in the ladder differ by one
precatenane. Such a ladder can be observed in the partially replicated
DNA that we prepared (Fig. 4A,
lane 1). Removal of the precatenanes will yield a classic
The precatenated DNA substrate was treated with either DNA gyrase, Topo
III, or Topo IV (Fig. 4A). Both Topo III and Topo IV could
clearly remove the precatenanes, as evinced by the disappearance of the
DNA ladder and the accumulation of LRI DNA. On the other hand, gyrase
was very inefficient at precatenane removal, even at concentrations
25-fold greater than those at which Topo III and Topo IV were
effective. These data are consistent with previous observations that
address the substrate specificity of Topo IV and gyrase (36, 39).
Topo III and Topo IV were powerful precatenane-removing enzymes, with
activity detectable at sub-nanomolar concentrations (Fig. 4,
B and C). The specific activities of the two
enzymes in this reaction were comparable (Fig. 4C). The DNA
replication reaction used to prepare the precatenated substrate lacked
DNA ligase; thus there are nicks present between the Okazaki fragments that could represent the sites of Topo III action. To assess if this
was the case, the precatenated substrate was treated with a combination
of DNA polymerase I, DNA ligase, and RNase H to remove the RNA primers
and seal the Okazaki fragments, resulting in the conversion of all the
nascent DNA to a long linear strand equal in length to the distance
between the two Ter sites (Fig. 5A). Interestingly, this
treatment had no effect on the ability of Topo III to remove the
precatenanes (Fig. 5B). However, this process does not
eliminate all the single-stranded gaps on the lagging strand. We showed
previously that when a replication fork collides with the
Tus-Ter complex, the site of leading-strand arrest is within
a nucleotide or two of the edge of Ter site, whereas the
site of the last primer on the lagging strand is about 60 nucleotides
upstream, leaving a gap at the fork in the partially replicated
molecule (41). This arrangement represents a snapshot of the
disposition of the nascent strands at the growing point of a
replication fork. This gap cannot be sealed by the treatment described
above and thus represents a probable position for the site of Topo III
action.
These studies demonstrate directly that Topo III can remove
precatenanes. It is thus likely that the ability demonstrated here of
Topo III to substitute for the decatenation activity of Topo IV
in vivo is a manifestation of this capacity.
Although the biochemical properties of Topo III are well
established, the manner in which these activities are expressed in the
cell are far from clear. The purified protein is capable of relaxing
supercoiled DNA and decatenating linked DNA circles (8). Its activity
is strongly dependent on single-stranded DNA to provide a productive
binding site on the substrate. Thus, negatively supercoiled DNA serves
as a substrate because of its underwound character, but positively
supercoiled DNA, with its overwound character, is not a substrate.
Similarly, Topo III-catalyzed decatenation requires that the DNA rings
contain discontinuities in the strands and is stimulated when actual
gaps are provided (8). We have shown that Topo III can support both
nascent strand progression (40) and daughter chromosome decatenation
(22) in oriC replication systems in vitro.
However, deletion of topB is of little consequence to the
cell (8).
Here we report a genetic system where survival of E. coli is
dependent on overexpression of Topo III. Increasing the level of Topo
III in the cell by about 70-fold was sufficient to allow cells with
temperature-sensitive mutations in either parC or
parE, the genes encoding the subunits of Topo IV, to grow at
the nonpermissive temperature. Although, as indicated by the cellular
and nucleoid morphologies, rescue was not complete, it was clear that
Topo III was decatenating the replicating daughter chromosomes,
allowing them to be partitioned and thus permitting successful cytokinesis.
There are two possible modes of strand unlinking during Where is Topo III acting in vivo? In support of the previous
observations, we have demonstrated directly in this report that Topo
III and Topo IV, but not gyrase, can efficiently remove precatenanes. We therefore think it likely that the observed ability of Topo III to
support daughter chromosome decatenation arises as a result of it
working behind the fork to unlink precatenanes. However, the actual
sites on the chromosome that can be occupied successfully by Topo III
to accomplish this task is limited because of its requirement for
single-stranded DNA. Topo III could be acting in one of two general
locations: at gaps produced on the lagging strand during the process of
sealing of Okazaki fragments or at the replication fork itself, in the
gap on the lagging strand between the 3'-end of the nascent leading
strand and the 5'-end of the last primer for Okazaki fragment
synthesis. We favor the latter as the likely preferred location of Topo
III because chromosome segregation requires that the topological
linkage between the daughters be reduced to zero. Thus, in order for
Topo III to support daughter chromosome unlinking, it must be able to
act after the last parental duplex turn is unwound and before all gaps
in the daughters are sealed. The gap on the lagging strand at the forks will persist after complete unwinding of the parental duplex until the
leading strand from the clockwise-moving fork meets up with the 5'-end
of the last Okazaki fragment from the counterclockwise-moving fork, for
example (Fig. 6). The gaps between
Okazaki fragments may be too transient to provide a site at which Topo
III can complete its task. This competition between sealing of gaps in
the daughter chromosomes and productive Topo III binding provides a
possible explanation for the incomplete penetrance of the rescue by
Topo III, because once all the gaps are sealed, Topo III will not be able to work.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
sequences (the long terminal repeats of the Ty
retrotransposon) (12), encoded a protein that had significant
similarity to E. coli Topo I and therefore renamed the gene
TOP3. TOP3 was actually more homologous to E. coli Topo III than to E. coli Topo I and proved to be
the first eukaryotic member of the Topo III family identified.
(18) and
(19).
Deletion of the former causes embryonic lethality in mice (20). Many of
these effects are thought to be manifested via interaction, or the lack
thereof, of Top3 with members of the RecQ-like family of DNA
helicases (21).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
strain as described
(22). ParE and ParC were purified as described by Peng and Marians
(23). DNA gyrase was prepared as described (9). Polyclonal antisera
against Topo III was raised in rabbits. Goat anti-rabbit IgG conjugated
to horseradish peroxidase was from Bio-Rad.
, which was used to prepare all
plasmid DNAs, was from Invitrogen. Cultures were grown in Luria broth
or on Luria agar plates. Antibiotics, when added, were at the following
concentrations: 100 µg/ml ampicillin and 50 µg/ml kanamycin.
Competent cells were prepared by CaCl2 treatment.
topB) was constructed by digesting pBR-suppressor-6 with HpaI, which cuts the plasmid once in
topB about 800 bp downstream of the start codon, and with
ScaI, which cuts the plasmid once about 900 bp upstream of
the start codon of topB, within the gene encoding
-lactamase, gel purifying the larger of the two DNA fragments
produced, and religating it. pBADtopB was constructed by
inserting an NaeI-SmaI DNA fragment from pTBE302 (the gift of R. DiGate, University of Maryland) that contained araC as well as the topB ORF under the control of
the ara promoter into the EcoRV site of
pBR-kan-inc3. pLEXparE has been described previously
(27).
parE(pLEXparE)--
The technique of
Link et al. (28) was used. A DNA fragment was constructed by
overlap PCR such that 33 random, coding bp were inserted in-frame
between the ATG and TAA codons of parE flanked by about 700 bp of upstream and downstream genomic sequence. The extreme outside
primers also contained a BamHI site. The DNA was digested
with BamHI and introduced into the BamHI site of pKO3, which carries cat and sacB, and is
replicated via a temperature-sensitive pSC101 origin, to give
pKO3parEk/o. This plasmid was transformed into W3110, and
the transformants were plated at 42 °C. A chloramphenicol-resistant colony was used to grow cells for transformation with
pLEXparE (27) at 42 °C. A single
chloramphenicol-resistant, ampicillin-resistant (because of
pLEXparE) colony was diluted in media and plated at 30 °C
on plates containing 80 µM IPTG, 5% sucrose, and
ampicillin. These colonies were then replica-plated in the presence and
absence of IPTG and also checked for chloramphenicol sensitivity. A
chloramphenicol sensitive-, IPTG-dependent colony was then
checked by PCR for the presence of the genomic parE deletion
that has the sequence Met-His-Ala-Ile-Thr-Leu-Thr-Leu-Gln-Ile-Tyr-Asn
replacing the parE ORF.
-1 plasmid DNA (31) as a template. This pBR322-based
plasmid carries oriC and two Ter sites oriented
to block replication forks coming from oriC, one 2 kb
downstream from oriC in the counterclockwise direction and
the other 3 kb downstream from oriC in the clockwise
direction. Thus, when Tus is included in the replication reaction
mixture, replication is terminated at the Ter sites, leaving
1 kb of unreplicated DNA. A significant fraction of the excess positive
linkages generated during replication persist as precatenanes in this
replication product (32). The precatenated DNA was treated with SDS and proteinase K, isolated by gel filtration through Bio-Gel A-5M, and
concentrated by lyophilization before use. Standard precatenane unlinking reaction mixtures (5 µl) containing 50 mM
HEPES-KOH (pH 8.0), 10 mM MgOAC, 10 mM
dithiothreitol, 2 mM ATP, 100 µg/ml bovine serum albumin,
2.8 nM precatenated DNA, and the indicated concentrations
of the different topoisomerases were incubated at 37 °C for 5 min.
Reactions were terminated by the addition of EDTA to 20 mM,
and the DNA products were analyzed by electrophoresis at 1.5 V/cm for
15 h through vertical 1% agarose gels using 50 mM
Tris-HCl (pH 7.8 at 23 °C), 40 mM NaOAC, and 1 mM EDTA as the buffer. The gels were dried, exposed to a
PhosphorImager screen, and autoradiographed.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
subunits of the DNA polymerase III holoenzyme. Further investigation of
this observation led to the discovery that the Topo IV activity was
temporally regulated during the cell cycle (33). Here we conducted a
screen for high copy suppressors of the conditional lethality of
C600parC1215 (24). A plasmid library (26) was generated by
inserting size-selected E. coli genomic DNA into a pBR322
vector (pBR322-kan-inc3) that carried a mutation giving it a
slightly higher copy number than normal. All E. coli genes
present in this library were expressed from their natural promoters.
Library DNA was transformed into C600parC1215, and about
80,000 transformants were plated at 42 °C. Of the 13 colonies that
grew, 6 regrew at both the permissive and nonpermissive temperatures.
Plasmid DNA was isolated from these six clones and retransformed into
C600parC1215. Plasmid DNA from all six potential suppressor
clones conferred to C600parC1215 the ability to grow at
42 °C (Table I). This screen yielded
four different genes that functioned as high copy suppressors of the temperature sensitivity of parC1215: parC (two
clones), as expected; topB (two clones), as discussed in
this report; dnaN (one clone), encoding the
subunit of
the DNA polymerase III holoenzyme; and spcA
(suppressor of parC A) (one clone), an
integral membrane protein that links chromosome segregation to cellular
architecture. The interaction of the proteins encoded by these latter
two genes with Topo IV will be the subject of a separate report.
Rescue of Topo IV temperature-sensitive strains by overexpression
of Topo III
topB). HpaI cuts the suppressor clone once
within topB about 800 bp downstream from the start of the
gene and ScaI cuts once about 900 bp upstream of the 5'-end
of the genomic insert, within the previously disrupted gene encoding
-lactamase. pBR-suppressor-6(
topB) failed to rescue the temperature-sensitive phenotype of C600parC1215 (Table
I), supporting the argument that topB was the suppressor
gene. To confirm that this was the case, the topB ORF was
introduced to the suppressor vector under the control of an
arabinose-inducible promoter, giving plasmid pBADtopB.
Interestingly, in the presence of 30 mM arabinose, this
plasmid effected a better rescue of the parC1215 allele at
42 °C than the original suppressor clone (Table I). This observation
suggested that the efficiency of rescue by Topo III was
dose-dependent, an issue that is explored below.
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Fig. 1.
Characterization of rescue of
W3110parE10 by overexpression of
topB. A, growth curves of
W3110(pBADtopB) and
W3110parE10(pBADtopB) at 42 °C. Overnight
cultures grown in rich medium were diluted to an
A600 of 0.02, and their growth in rich medium
was followed at 42 °C. B, cell and nucleoid morphologies
of W3110(pBADtopB),
W3110parE10(pBADtopB), and
W3110parE10(pBR-kan-inc3) growing at 30 and 42 °C.
Samples were taken from cultures of the former two strains at an
A600 of 0.7. The latter strain was grown as an
overnight culture at 30 °C; fresh media were inoculated to an
A600 of 0.02, and the culture was then grown for
2 h at 42 °C. Preparation of cells and fluorescence microscopy
were as described under "Experimental Procedures." C,
comparison of the types of cells present in the three different
cultures at 42 °C. Roughly 300-400 cells from each culture were
examined at random and assigned, based on cell and nucleoid morphology,
to the six cell types illustrated in the figure. Type 1 cells were wild
type in appearance. Type 2 cells were small but contained condensed
nucleoids. Type 3 cells were anucleates. Type 4 cells were normal-sized
but had nucleoids that appeared connected by DNA. Type 5 cells were
filaments with non-condensed nucleoids, whereas type 6 cells were
filaments with condensed nucleoids.
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Fig. 2.
Levels of expression of Topo III from the
different suppressor plasmids in W3110parE10 and
W3110 parE(pLEX-parE).
Cultures of W3110parE10 growing at 30 °C,
W3110parE10(pBR-suppressor-6) growing at 42 °C,
W3110parE10(pBADtopB) growing at 42 °C in the
presence of 15 mM arabinose,
W3110
parE(pLEX-parE) growing at 37 °C
in the presence of IPTG,
W3110
parE(pLEX-parE, pBR-suppressor-6)
growing at 37 °C in the presence of IPTG, and
W3110
parE(pLEX-parE, pBADtopB)
growing at 37 °C in the presence of IPTG and 15 mM
arabinose were sampled at an A600 of 1.0. The
cells were pelleted, resuspended in loading dye for SDS-PAGE, and
heated at 100 °C, and the resulting extract was electrophoresed
through a 10% SDS-PAGE. The gel was then Western-blotted using
polyclonal antibody to Topo III. Each lane contains the equivalent of
37.5 µl of culture.
parE(pLEXparE) carries a precise deletion of the chromosomal parE covered by an expression plasmid
(pLEX5BA (35)) where the parE ORF is under the tightly
regulated control of a modified lac operator. Consequently,
growth of this strain at any temperature is dependent on the presence
of IPTG to induce ParE expression. pBADtopB could not rescue
growth of this strain at 37 °C in the presence of arabinose when
IPTG was omitted from the culture medium. In the presence and absence
of arabinose to induce Topo III expression, the plating efficiency of
W3110
parE(pLEXparE, pBADtopB)
was 0.96 (with arabinose/without arabinose) when IPTG was also present.
In the absence of IPTG, no growth was observed at all in either the
presence or absence of arabinose. We considered three explanations for
the lack of rescue of the parE deletion strain. The first
relates to the possibility that the observed rescue by overexpression
of Topo III in the temperature-sensitive parE and
parC strains was dependent on a partial level of Topo IV
decatenation activity, and therefore, because the ParE deletion strain
had no Topo IV, higher levels of Topo III were required to effect rescue.
parE(pLEXparE) (Fig. 2), yet still could
not rescue the null strain. We therefore conclude that overexpression
of Topo III to levels roughly comparable with those of Topo IV in
wild-type cells allow it to substitute completely for the chromosome
decatenation activity of the latter enzyme.
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Fig. 3.
ParEC316Y Topo IV is
temperature-sensitive. ParEC316Y Topo IV was overexpressed and
purified as described under "Experimental Procedures." The
temperature sensitivity of the ParEC316Y and wild-type Topo IV was
compared. The enzymes were first heated at 50 °C for the time
indicated and then chilled on ice. The activity of the heat-treated
enzyme was then assessed in standard assays for either superhelical DNA
relaxation (A) or decatenation of kinetoplast DNA
(B) as described under "Experimental Procedures."
parE(pLEXparE) cannot be rescued in the
absence of IPTG because the presence of ParC in the cell unfettered by
association with ParE is toxic. This possibility is difficult to
discount; however, we note that overexpression of Topo III did, in
fact, rescue the temperature-sensitive parE strain, where,
presumably, because the ParEC316Y mutation is likely to cause folding
defects, at the nonpermissive temperature the same situation holds,
i.e. there is a surfeit of free ParC in the cell. On the
other hand, our experience with various parC expression
constructs is consistent with high level, overexpression of ParC being
toxic to wild-type cells.
-type late replication intermediate (LRI) where the two partially
replicated daughters are held together by the region of unreplicated
parental DNA. This species has a very low mobility during agarose gel
electrophoresis (9).
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Fig. 4.
Topo III and Topo IV, but not gyrase, can
remove precatenanes. A, standard precatenane
unlinking reaction mixtures containing the indicated concentrations of
either DNA gyrase (Gyr) (lanes 2-4), Topo III
(lanes 5-7), Topo IV (lanes 8-10), or no
topoisomerase (lane 1) were incubated and analyzed as
described under "Experimental Procedures." B and
C, comparison of the precatenane-removing activities of Topo
III and Topo IV. B, standard precatenane unlinking reaction
mixtures containing the indicated concentrations of either Topo III
(lanes 2-7), Topo IV (lanes 8-13), or no
topoisomerase (lane 1) were incubated and analyzed as
described under "Experimental Procedures." C, the extent
of precatenane unlinking in the reactions displayed on the gel in
B was quantitated by using a PhosphorImager to determine the
amount of radioactivity present on the gel as LRI.
LRI, late replication intermediate; precats,
precatenanes; PSL, photo-stimulated luminescence.
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Fig. 5.
The precatenane unlinking activity of Topo
III does not require gaps between Okazaki fragments in the nascent
daughter chromosomes. A, treatment of the precatenated
DNA with RNase H, DNA ligase, and DNA polymerase I (RLP)
seals all the Okazaki fragments. The denaturing alkaline-agarose gel
shows the nascent DNA present in the precatenated DNA before
(lane 1) and after (lane 2) treatment with RLP.
Because the plasmid replicates bidirectionally, when sealed, all the
Okazaki fragments synthesized by the clockwise-moving fork will be
joined to the leading strand synthesized by the counterclockwise-moving
fork and vice versa, resulting in a continuous linear nascent DNA
product that spans the distance between the two Ter sites (5 kb). B, treatment of the precatenated DNA with RLP does not
inhibit precatenane removal by Topo III. Precatenane unlinking by 3.2 nM Topo III was compared using precatenated substrate that
either had (lanes 3 and 4) or had not
(lanes 1 and 2) been treated with RLP.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-type DNA
replication (36): removal of positive supercoils ahead of the
replication fork and removal of positive precatenanes behind the fork.
Execution of the latter mode is ultimately required for complete
removal of all topological linkages between daughter chromosomes. The
biochemical properties of Topo III suggested that it likely acts behind
the fork, removing precatenanes. In support of this, we demonstrated
that the rate of Topo III-supported fork progression from early
replication intermediates where replication forks had stalled because
of accumulation of positive supercoils actually increased as the extent
of the template replicated increased. In contrast, the rate of
gyrase-supported fork progression decreased as the extent of the
template replicated increased. We interpreted these observations to
mean that at the beginning of the reaction, when only about 20% of the
DNA was replicated, most of the positive windings existed as positive
supercoils, substrates for DNA gyrase, not Topo III. As replication
proceeded, precatenanes, substrates for Topo III and not gyrase, accumulated.
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Fig. 6.
Lagging strand gaps can persist at the
replication fork after complete unwinding of the parental template at
the termination of replication. (i) Two replication
forks approaching each other at the termination of replication. Gaps
exist between the 3'-end of the leading strand and the 5'-end of the
last Okazaki fragment on the lagging strand at each replication fork.
(ii) The gaps on the lagging strand persist as the parental
template becomes completely unwound. (iii) The gaps are
sealed only after complete unwinding of the parental template, when the
leading strand from one direction extends to the 5'-end of the last
Okazaki fragment from the opposite fork. (iv) Completely
replicated sisters. Arrowheads correspond to 3'-ends.
Topo III could not, when expression of the ParE was shut down, rescue
growth of a strain that had been deleted for parE and was
dependent on expression of ParE from an IPTG-inducible promoter. We do
not think that this observation reflects the fact that some residual
Topo IV activity was required for the rescue of the
temperature-sensitive parE and parC strains by
Topo III. At least in the case of the parE strain, we could
demonstrate that the endogenous Topo IV was clearly
temperature-sensitive for both its superhelical DNA relaxation and
decatenation activities, and the expression of a 3-fold greater level
of Topo III than was sufficient to rescue the temperature-sensitive
parE strain was still insufficient to rescue the
parE strain in the absence of IPTG. The simplest
explanation for the lack of rescue of the null is that free ParC is
toxic to the cell. ParC must be complexed with ParE for its DNA
cleavage and religation activity to be manifested; however, ParC alone is a DNA-binding protein, and its indiscriminate binding to the chromosome could interfere with processes such as replication and
transcription. Although this is the most likely possibility, it is
interesting to note that both the
parE strain in the
absence of IPTG and the temperature-sensitive parE strain at
the nonpermissive temperature presumably both have free ParC, yet Topo
III overexpression rescued the latter and not the former. This
observation suggests another, admittedly speculative, possibility that
ParE, and possibly Topo IV, has two distinct roles in the cell, only
one of which (decatenating the daughter chromosomes) depends on the
formation of catalytically active Topo IV. What this second task could
be is obscure. Additional studies are required to resolve this issue.
In eukaryotic cells, Topo III and the RecQ-like DNA helicases appear to
cooperate to preserve genomic integrity, perhaps by processing some
types of recombination intermediates that can form at stalled
replication forks (21). Little evidence of such cooperation exists in
E. coli, although our observation that Topo III can remove
precatenanes in vivo supports its potential involvement in
such reactions. Harmon et al. (42) have reported that the combination of E. coli RecQ and Topo III can catenate
covalently closed, superhelical DNAs in vitro. These authors
proposed that a functional interaction between these two proteins
allowed Topo III to execute the required two sequential strand passage
events by exploiting the ability of RecQ to unwind the superhelical
DNA. The existence of such a mechanism in E. coli would
obviate the issue discussed above with respect to the requirement for a
single-stranded gap for Topo III action in decatenating the replicating
daughter chromosomes. Accordingly, we asked whether disruption of
recQ affected the ability of Topo III to rescue the
parE temperature-sensitive strain. Interestingly, it did not
(data not shown). Of course, this experiment may not be an accurate
test of the hypothesis given the requirement for overproduction of Topo
III to effect rescue of the parE strain.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM34558.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Molecular Biology
Program, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New
York, NY 10021. Tel.: 212-639-5890; Fax: 212-717-3627; E-mail: k-marians@ski.mskcc.org.
Published, JBC Papers in Press, December 31, 2002, DOI 10.1074/jbc.M211211200
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ABBREVIATIONS |
---|
The abbreviations used are:
Topo, topoisomerase;
IPTG, isopropyl-1-thio--D-galactopyranoside;
ORF, open
reading frame;
LRI, late replication intermediates.
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