Movement of Replicating DNA through a Stationary
Replisome
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Lemon, Katherine P, and Alan D Grossman. “Movement of
Replicating DNA through a Stationary Replisome.” Molecular Cell
6, no. 6 (December 2000): 1321-1330. Copyright © 2000 Cell
Press
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http://dx.doi.org/10.1016/S1097-2765(00)00130-1
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Molecular Cell, Vol. 6, 1321–1330, December, 2000, Copyright 2000 by Cell Press
Movement of Replicating DNA
through a Stationary Replisome
Katherine P. Lemon and Alan D. Grossman*
Department of Biology
Massachusetts Institute of Technology
Cambridge, Massachusetts 02139
Summary
We found that DNA is replicated at a central stationary
polymerase, and each replicated region moves away
from the replisome. In Bacillus subtilis, DNA polymerase is predominantly located at or near midcell. When
replication was blocked in a specific chromosomal
region, that region was centrally located with DNA
polymerase. Upon release of the block, each copy of
the duplicated region was located toward opposite cell
poles, away from the central replisome. In a roughly
synchronous population of cells, a region of chromosome between origin and terminus moved to the replisome prior to duplication. Thus, the polymerase at
the replication forks is stationary, and the template is
pulled in and released outward during duplication. We
propose that B. subtilis, and probably many bacteria,
harness energy released during nucleotide condensation by a stationary replisome to facilitate chromosome partitioning.
Introduction
The production of viable progeny depends on the faithful
replication and partitioning of genomic DNA. Bacteria
do not appear to have a eukaryotic-like mitotic apparatus. Like many bacteria, B. subtilis has a single circular
chromosome (4200 kb; Kunst et al., 1997) with a single
origin (oriC) from which bidirectional DNA replication
initiates (Kornberg and Baker, 1992). Separation of newly
duplicated chromosome regions occurs while DNA replication of the rest of the chromosome proceeds (Webb
et al., 1997; Teleman et al., 1998; Webb et al., 1998).
Recent work with B. subtilis, E. coli, and C. crescentus
demonstrates that the chromosome is oriented such
that for most of a cell cycle the two sister origins reside
on opposite sides of the cell, while the unduplicated
terminus region is centrally located (Glaser et al., 1997;
Gordon et al., 1997; Lewis and Errington, 1997; Lin et
al., 1997; Mohl and Gober, 1997; Webb et al., 1997; Niki
and Hiraga, 1998; Sharpe and Errington, 1998; Teleman
et al., 1998; Webb et al., 1998; Jensen and Shapiro,
1999; Niki et al., 2000). Regions located between the
origin and terminus on the circular chromosome appear
to be positioned between them in the cell (Teleman et al.,
1998; Niki et al., 2000). Efforts to associate chromosome
movement with the act of DNA replication are confounded by two factors. First, the rate of bacterial DNA
polymerization is estimated at 500–1000 nucleotides per
* To whom correspondence should be addressed (e-mail: adg@
mit.edu).
second (Baker and Bell, 1998). This is equivalent to moving of a single base pair a distance of ⵑ170–340 nanometers each second. Second, DNA inside cells is highly
compacted (reviewed in Trun and Marko, 1998). The
ⵑ4200 kilobase pair (kbp) circular genome of B. subtilis
(Kunst et al., 1997) is neatly organized in a rod-shaped
cell two to four microns in length and about half a micron
in diameter.
Here, we describe experiments that demonstrate that
DNA is duplicated at a centrally located, stationary replisome. Previously, we visualized the B. subtilis replicative
DNA polymerase using a functional fusion of the catalytic subunit, PolC, to green fluorescent protein (GFP)
(Lemon and Grossman, 1998). PolC–GFP localizes to
discrete positions predominantly at or near midcell or
sites that will become midcell, indicating that the replisome is stationary and centrally located. We reasoned
that if DNA replication is reversibly blocked at a specific
chromosomal region, then that region should be centrally located and coincident with the DNA polymerase.
When the replication block is relieved, that site should be
duplicated and no longer coincident with the stationary
replisome.
A reversible DNA replication arrest occurs in B. subtilis
during the stringent response (Levine et al., 1991, 1995;
Autret et al., 1999) induced by amino acid starvation.
Replication arrests in regions ⵑ100–130 kbp to the left
(LSTer or left stringent terminus region) and ⵑ150–200
kbp to the right (RSTer) of oriC (Levine et al., 1991, 1995;
Autret et al., 1999). DNA polymerase resumes chromosome synthesis in the STer regions when amino acids
are added back to the cells. Starvation for an amino
acid activates the production of the alarmone (p)ppGpp
by the relA gene product, which induces the pleiotropic
stringent response in many bacteria (Cashel et al., 1996;
Wendrich and Marahiel, 1997), though stringent arrest
of DNA replication at sites away from the origin has only
been characterized in B. subtilis.
We visualized the more precisely mapped LSTer region (Autret et al., 1999). During stringent arrest, the
LSTer region was located at or near midcell. Under these
conditions, the newly duplicated sister origins are already separated to opposite sides of the cell (Sharpe
and Errington, 1998). After release from replication arrest, the LSTer region was duplicated, and the two copies were positioned away from midcell in opposite
halves of the cell.
We also visualized a region of the chromosome (270⬚)
approximately midway between origin and terminus.
Just prior to a burst of duplication of the 270⬚ region in
a roughly synchronous population of cells, we observed
an increase in the occurrence of coincidence of the 270⬚
region and DNA polymerase. Taken together, our results
indicate that the stationary, centrally positioned DNA
polymerase does indeed represent the location of active
replication forks and suggest that the DNA is threaded
through the polymerase during duplication and then extruded from the polymerase after duplication.
Molecular Cell
1322
Table 1. Number of Foci of the LSTer Region per Cell before,
during, and after Stringent Arrest
Table 2. Number of Foci of the LSTer Region in Cells
during Growth after Synchronization
Percentage of Cells with
Indicated Number of Focib
Conditionsa (Number of Cells)
Asynchronous growth (728)
Stringent arrest (572)
Release from arrest (504)
1
2
42
77
28
56
22
64
a
Strain KPL549 [dnaB134(ts), hutM⫹-lacO, lacI-gfp] was grown in
defined minimal medium at 30⬚C, and the number of foci of the
LSTer region per cell was determined. Samples were taken (1) during
asynchronous exponential growth, (2) 30 min after induction of stringent arrest in a synchronized population, and (3) 30 or 35 min after
release from stringent arrest.
b
During asynchronous growth and stringent arrest, ⬍2.5% of cells
had three or four foci. After release from arrest, 6.9% of cells had
three foci and 1.4% had four foci.
Percentage of Cells with
Indicated Number of Focib
Timea
Number of Cells
Asynchronous
0
15
30
45
60
75
856
406
442
348
449
394
566
1
2
45.2
62.3
64.3
52
39.7
31.2
27.2
51.5
37.4
35.7
46.8
58.1
65.5
69.3
a
The replication cycle was synchronized by shifting cells to nonpermissive temperature (45⬚C) to block initiation of replication and to
allow most ongoing rounds to finish. Samples of strain KPL549
[dnaB134(ts), hutM⫹-lacO, lacI-gfp] were taken at the indicated
times (min) after shift back to permissive temperature, and the number of foci of the LSTer region per cell was determined.
b
At all times, less than 3.5% of cells had three or four foci.
Results
Visualization of the LSTer Region
We chose the more precisely defined LSTer region over
the RSTer region for analysis (Autret et al., 1999). The
LSTer region contains two approximately equivalent arrest sites, LSTer1 and LSTer2, separated by about 27
kbp (Autret et al., 1999). Stalling of DNA polymerase in
both the left and right STer regions is about 80% effective in a synchronized population of cells treated with
arginine hydroxymate (Levine et al., 1991, 1995; Autret
et al., 1999). We marked the LSTer region by inserting
a tandem array of E. coli lac operator sites adjacent to
hutM on the B. subtilis chromosome, which is ⵑ40 kbp
from LSTer2 and ⵑ67 kbp from LSTer1. We chose this
position for convenience and to minimize the possibility
that the array would disrupt function of the LSTer region.
The hutM-lacO array was visualized with a carboxyterminal fusion of Lac repressor to a variant of GFP
(LacI-GFP) (Cormack et al., 1996).
During exponential growth, most cells have a partly
duplicated chromosome. The LSTer region is within 130
kbp of the origin and is duplicated relatively early (Autret
et al., 1999). Indeed, during asynchronous exponential
growth, most cells had two well-separate foci of the
LSTer region (Table 1). The pattern of localization of the
sister LSTer regions appeared to be similar to that of
the origin region (Webb et al., 1997; Teleman et al., 1998;
Webb et al., 1998) and the chromosome partitioning
protein Spo0J (Glaser et al., 1997; Lewis and Errington,
1997; Lin et al., 1997; Sharpe and Errington, 1998; Teleman et al., 1998), which binds to sites (parS) in the originproximal 20% of the chromosome (Lin and Grossman,
1998).
Synchronization of DNA Replication in a Population
of B. subtilis
In order for most of the cells to undergo replication
arrest at a similar time, the population was first synchronized for DNA replication (Levine et al., 1991, 1995; Autret et al., 1999). A mutant that is temperature sensitive
for initiation of DNA replication, dnaB134 (Mendelson
and Gross, 1967; Karamata and Gross, 1970), was shifted
to nonpermissive temperature to allow most ongoing
rounds of replication to finish while preventing initiation
of new rounds of replication. In B. subtilis, the replication
initiation protein DnaB (not a helicase as is E. coli DnaB)
is membrane associated and is required for origin enrichment in membrane fractions (Winston and Sueoka,
1980). Immediately after shiftdown to permissive temperature, ⵑ62% of the population was synchronized as
judged by having a single focus of the LSTer chromosome region (Table 2), indicating a single chromosome
per cell. This method gives at least one roughly synchronous round of replication in a population (Dworkin et
al., 1972).
The LSTer Region Is Centrally Located during
Stringent Response–Induced
Replication Arrest
Stringent response was induced in a dnaB134(ts)-synchronized population of cells by addition of the amino
acid analog arginine hydroxymate (250–500 ␮g/ml) (Levine et al., 1991, 1995; Autret et al., 1999). Thirty minutes
after induction of stringent DNA replication arrest, 77%
of cells had a single, unduplicated focus of the LSTer
region (Table 1). In contrast, in the absence of stringent
arrest, the percent of cells (52%) (Table 2) with a single
focus 30 min after synchronization was similar to that
in a culture of asynchronously growing cells (45%) (Table
2). During stringent arrest, the single focus of LSTer was
located at or near midcell (Figure 1A).
DNA Polymerase Is Centrally Located during
Stringent Response–Induced
Replication Arrest
To be sure that the manipulations to synchronize and
induce stringent arrest did not affect DNA polymerase
position, we observed DNA polymerase in live cells using a functional fusion of the Tau subunit to GFP. A
dimer of Tau is the scaffolding that holds the leading
and lagging strand polymerases together (Kelman and
O’Donnell, 1995; Baker and Bell, 1998). The Tau-GFP
fusion produced a slightly stronger signal than the PolCGFP fusion, and previous work indicated that during
exponential growth, the position of Tau-GFP is similar
Movement of DNA through a Stationary Replisome
1323
Figure 1. Position of the LSTer Region and
DNA Polymerase during Replication Arrest Induced by the Stringent Response
During stringent arrest, most cells have a single centrally located focus of the LSTer region. After release, most cells have two wellseparated foci of the LSTer region.
(A) shows the position of the LSTer region
(hutM-lacO) in cells with a single focus during
stringent arrest (341 cells). In (B), the position
of DNA polymerase in cells with a single focus
of Tau-GFP during stringent arrest (402 cells)
is shown. (C) shows the position of the LSTer region (hutM-lacO) in cells with two foci after release from stringent arrest by nutrient addback
(114 cells). Position was determined by measuring the distance from midfocus to the cell pole. In cells with two foci, distance was measured
from the same cell pole. (A) and (C), strain KPL549 [dnaB134(ts), hutM⫹-lacO, lacI-gfp]; (B), strain KPL502 [dnaB134(ts), dnaX-gfp].
to that of catalytic subunit fusion, PolC-GFP (Lemon
and Grossman, 1998).
In cells with a single focus of Tau-GFP during stringent
arrest, the focus was located predominantly at or near
midcell (Figure 1B), similar to the LSTer region position
during stringent-induced DNA replication arrest (compare Figures 1A and 1B).
After Release from Replication Arrest, Most Cells
Had Two Well-Separated Foci
of the LSTer Region
DNA replication arrest was released by washing cells
(to remove arginine hydroxymate) and resuspending
them in a nutrient-rich medium. Within 30–35 min after
release, most cells (64%) had two foci of the LSTer
region (Table 1). Thus, a majority (67%) of the cells with
a single focus during arrest appeared to replicate the
LSTer region after release. Duplication of the LSTer region correlated with a resumption in growth as measured by an increase in the culture’s optical density.
The two newly duplicated copies of the LSTer regions
were well separated from each other and located at
ⵑ35% and ⵑ65% of the cell length (Figure 1C). In addition to the shift from one to two foci, after release there
was an increase in the proportion of cells with three and
four foci (from 1% to 8%).
Visualization of DNA Polymerase and LSTer
in the Same Cells
The experiments described above were done in live cells
from two different strains; one contained the LacI-GFP
fusion and the lac operator array near the LSTer region,
and the other contained a functional Tau-GFP fusion.
When DNA replication was stalled in the LSTer region,
the majority of cells had a single focus of both the LSTer
region and DNA polymerase at or near midcell (Table 1;
Figures 1A and 1B). To directly show that the LSTer
region and the polymerase colocalize during replication
arrest, we visualized the LSTer region and the replisome
in the same cells with and without the stringent arrest.
The development of variants of GFP with sufficiently
separable excitation and emission spectra permits the
observation of two separate fusion proteins in the same
cell (Tsien, 1998; Miller et al., 1999). DNA polymerase
was visualized using a functional fusion of Tau to a
yellow variant of GFP (YFP) (Ormo et al., 1996). The lacO
array near the LSTer region (hutM-lacO) was visualized
using a fusion of LacI to a cyan variant of GFP (CFP)
(Heim and Tsien, 1996).
During asynchronous growth, the majority of cells
(83%) had two foci of the LSTer region (Table 3, line
A). Immediately after synchronization of the replication
cycle, ⵑ43% of the cells had two foci of the LSTer region
and ⵑ55% had a single focus (Table 3, line B). One hour
later after growth at permissive temperature, most of
the cells (83%) had two foci of the LSTer region (Table
3, line C). In marked contrast, when stringent arrest was
induced, only ⵑ47% of the cells had two foci and 49%
had a single focus of the LSTer region (Table 3, line D).
When stringent arrest was released, cells began to grow,
and by thirty minutes a majority had duplicated and
separated the LSTer region (Table 3, line E). The increase
in cells with three and four foci after release (Table 3)
indicates the occurrence of multifork replication under
these growth conditions (see below).
A clear pattern emerged when we observed cells that
had a centrally located polymerase (either a single focus
or two closely juxtaposed foci [Lemon and Grossman,
1998]) and one or two foci of LSTer. During exponential
growth in an asynchronous culture, 86% of the cells had
two well-separated foci of the LSTer region, one on
either side of the central DNA polymerase (Figure 2).
This same pattern was observed in the majority of cells
(79%) that were growing without arrest 60 min after
synchronization (Figures 2, 3A–3C, and 4). In contrast,
when DNA replication arrest was induced (stringent response), there was a marked increase in the proportion
of cells in which a single focus of LacI-CFP bound near
the LSTer was either coincident with (31%) or touching
(18.5%) a focus of DNA polymerase (Tau-YFP) (Figures
2, 3D–3F, and 4). Likewise, there were many fewer cells,
only ⵑ36%, with the typical exponential growth pattern
(Figures 2 and 4). The lacO array is inserted ⵑ67 kbp
from the first arrest site (LSTer1) and ⵑ40 kbp from the
second site (LSTer2). LSTer1 and 2 are approximately
equivalent (Autret et al., 1999), so in about half the cells
the DNA replication fork is blocked ⵑ67 kbp away from
the lacO array that we visualized. It is likely that in cells
in which the LacI-CFP focus touches the Tau-YFP focus,
LSTer is coincident with the replication fork. Therefore,
we have combined these two classes in Figures 2 and 4.
When cells were released from stringent arrest, within
30 min the pattern shifted back toward that seen for
cells growing without arrest. Approximately 53% of cells
had two well-separated LSTer foci flanking a central
Molecular Cell
1324
Table 3. Number of Foci of the LSTer Region in Cells Containing Tau-YFP
Percentage of Cells with Indicated Number of Foci
a
Conditions (Number of Cells Counted)
(A) Asynchronous (623)
(B) Synchronous, 0 min (706)
(C) Synchronous, no arrest (600)
(D) Synchronous, stringent arrest (457)
(E) Synchronous, release from arrest (696)
1
2
5.6
54.8
12.7
49
20
83.3
42.5
83
47.3
66.4
3
4
4.7
2
2.7
2.8
9
6.4
0.7
1.7
0.9
4.6
a
Samples of strain KPL730 [dnaB134(ts), hutM⫹-lacO, lacl-cfp, dnaX-yfp] were taken under the indicated conditions, and the number of foci
of the LSTer region per cell was determined. (A), asynchronous growth; (B), synchronous cells immediately after shift to permissive temperature
after 45 min at 42⬚C (to prevent initiation of new rounds of replication and to allow most ongoing rounds to finish); (C), synchronous cells 60
min after shift to permissive temperature with no stringent arrest; (D), synchronous cells 60 min after shift to permissive temperature and
during stringent arrest; and (E), synchronous cells 30 min after addition of nutrients to release stringent arrest. Nutrients were added 60 min
after stringent arrest and shift to permissive temperature.
DNA polymerase (Figures 2, 3G–3I, and 4). The pattern
of localization of DNA polymerase and the LSTer region
described here strongly supports the model that the
DNA template is duplicated by a stationary, centrally
located DNA polymerase. After duplication, the two sister chromosome regions then move in opposite directions apart from each other and away from the replisome.
LSTer out toward each pole, both of which were either
adjacent to or coincident with a focus of DNA polymerase (Figures 3J–3O). In these cells, DNA replication has
probably reinitiated or is about to initiate at the two
sister origins prior to cell division. We speculate that
initiation occurs at the cell quarters, which are nascent
Multifork Replication
B. subtilis, like many other bacteria, is capable of doubling, under optimal growth conditions, in about a third
of the time it takes to duplicate the genome. This is
accomplished by reinitiating DNA replication from the
sister origins before cell division so that there are multiple bidirectional replication forks per cell, essentially
giving the replication cycle a headstart on the division
cycle. When cells were released from stringent arrest
by resuspension in a nutrient-rich medium, multifork
replication began to occur in about 20% of cells, as
indicated by cells with more than two LSTer foci (Table
3). After release, we observed cells that had a focus of
Figure 3. Visualization of DNA Polymerase and the LSTer Region
in the Same Cells
Figure 2. Relative Positions of the LSTer Region and DNA Polymerase in the Same Cells
Samples of strain KPL730 [dnaB134(ts), hutM⫹-lacO, lacI-cfp, dnaXyfp] were taken under the indicated conditions, and the relative
positions of the LSTer region (hutM-lacO; gray circles) and DNA
polymerase (triangles) were determined by fluorescence microscopy. Data are shown for cells with a central focus (or two closely
juxtaposed central foci) of Tau-YFP and one or two foci of the LSTer
region. The number of cells counted for each condition is indicated
in parenthesis. The coincident class includes cells in which the
LSTer and polymerase foci were coincident and those in which the
foci were touching (see Results).
Samples of strain KPL730 [dnaB134(ts), hutM⫹-lacO, lacI-cfp, dnaXyfp] were taken for microscopy various times after synchronization
of the replication cycle. (A–C) Sixty minutes after synchronization.
(D–F) Sixty minutes after synchronization and induction of the stringent response (replication arrest at LSTer). LSTer is coincident with
the replisome. (G–I) Thirty minutes after release from stringent response, two copies of LSTer away from the centrally located replisome. (J–L) A cell undergoing (or about to undergo) multifork replication 30 min after release from stringent replication arrest. The central
polymerase is from the initial round of replication, and additional
replisomes have assembled at approximately the cell quarter positions. (M–O) Replisomes approximately at the cell quarters in a cell
that seems to have finished the initial round (no replisome at midcell).
Left panels (A, D, G, J, and M) show foci of DNA polymerase (TauYFP; orange) and cell outline (FM 4-64; gray). Middle panels (B, E,
H, K, and N) show foci of LSTer (LacI-CFP; cyan) and cell outline
(gray). Right panels (C, F, I, L, and O) show merged images of the
left and middle panels. Scale bar is 1 ␮m.
Movement of DNA through a Stationary Replisome
1325
Figure 4. Analysis of the LSTer Region and DNA Polymerase
Samples of strain KPL730 [dnaB134(ts), hutM⫹-lacO, lacI-cfp, dnaXyfp] were taken 60 min after synchronization of the replication cycle
(no arrest), 60 min after synchronization and induction of the stringent response (arrest), and 30 min after release from the stringent
response (release). Dark bars indicate the percent of cells with a
single LSTer region that was coincident with a central DNA polymerase (as in Figure 3F). Light bars indicate the percent of cells with
two well-separated foci of the LSTer region and a centrally located
DNA polymerase (as in Figure 3I). Error bars indicate the 95% confidence interval.
Figure 5. Analysis of the 270⬚ Chromosomal Region and DNA Polymerase
midcell positions. Some of these cells also had a centrally located DNA polymerase that probably represents
an initial round of replication that was not yet complete
(Figures 3J–3L).
Visualization of DNA Polymerase and the 270ⴗ Region
of the Chromosome in Synchronized Cells
It appears that in B. subtilis, the origin region is located
at approximately midcell or a position that will become
midcell, prior to duplication (Lewis and Errington, 1997;
Teleman et al., 1998) (D. C.-H. Lin, P. S. Lee, and A. D. G.,
unpublished data). The LSTer region is close to oriC,
less than 150 kbp away, whereas the distance from oriC
to the terminus is about 2100 kbp. Thus, we could
demonstrate the central position of the LSTer region
during replication arrest and movement of the two sister
copies away from the center after duplication. It was
not possible to clearly demonstrate movement of the
LSTer region to the cell center prior to duplication. Nor,
given the size of the LSTer region, was it possible to
place this region at a more origin-distal chromosomal
site. Regions of the B. subtilis chromosome that fall
between the origin and the terminus of the circular map
are generally positioned between them in cells (Teleman
et al., 1998). The 270⬚ region is halfway between oriC
and the terminus. Sister origin regions are localized at
approximately the cell quarter positions (D. C.-H. Lin,
P. S. Lee, and A. D. G., unpublished data), while the
terminus is at approximately midcell (Webb et al., 1997;
Teleman et al., 1998; Webb et al., 1998). Thus, the 270⬚
region is usually positioned between the quarter and
midcell (Teleman et al., 1998). We visualized the 270⬚
region using a tandem array of lac operators adjacent
to cotS (Teleman et al., 1998) combined with our LacICFP fusion. DNA polymerase was again visualized with
Tau-YFP.
Immediately after synchronization (t ⫽ 0), most of the
cells (62%) had a single focus of the 270⬚ region (Figure
5A). Between 60 and 75 min after synchronization, there
was a marked increase in the number of cells with two
foci of the 270⬚ region (Figure 5A), indicating that in
Samples of strain KPL727 [dnaB134(ts), cotS⫹-lacO (270⬚), lacI-cfp,
dnaX-yfp] were taken at the indicated times after shiftdown to permissive temperature, and the foci of DNA polymerase (Tau-YFP)
and the 270⬚ region (LacI-CFP) were analyzed.
(A) The percentage of cells with one focus (squares) or two foci
(circles) of the 270⬚ region as a function of time after synchronization
is shown.
(B) The percentage of cells with the 270⬚ region coincident with
(squares) or separate from (circles) a focus of DNA polymerase
is shown. Only cells with a single focus of the 270⬚ region were
counted.
many cells the 270⬚ region was duplicated during this
interval. We observed three general patterns when we
compared the position of the 270⬚ region to that of DNA
polymerase over the time course in cells with a single
focus of 270⬚. (1) At all time points, 30%–35% of the
cells had the 270⬚ region adjacent to a focus of DNA
polymerase (Tau-YFP). This is not surprising, given that
270⬚ is usually positioned between the cell quarter and
midcell (Teleman et al., 1998), while the replisome is at
or near midcell (Lemon and Grossman, 1998). (2) We
also observed cells in which the 270⬚ region was clearly
separated from DNA polymerase and (3) cells in which
the 270⬚ region was coincident with a focus of DNA
polymerase. The percent of cells in which the 270⬚ region
was coincident with DNA polymerase increased between 45 and 60 min after synchronization (Figure 5B).
This increase occurred just prior to the shift from predominantly one focus of 270⬚ per cell to predominantly
two foci of 270⬚ per cell (Figure 5A). The timing of this
position change is consistent with movement of the 270⬚
region to the stationary DNA polymerase just prior to
duplication.
Discussion
A Centrally Located, Stationary Bacterial Replisome
Our results indicate that during replication in B. subtilis,
DNA moves through a stationary replisome located at
or near midcell. After a region of DNA is replicated, the
two duplicated regions move away from the replisome
Molecular Cell
1326
toward opposite ends of the cell. We propose that B.
subtilis and probably many bacteria harness the energy
released during replication by a stationary replisome
to facilitate chromosome movement. In this extrusioncapture model, the centrally located stationary replisome pulls the DNA template in, duplicates it, and releases the two products into opposite halves of the cell.
We speculate that once the origin regions are extruded
from midcell, they are captured and maintained at approximately cell quarter positions until DNA replication
reinitiates.
DNA and RNA Polymerases Are Motor Proteins
For years, people have thought of DNA polymerase as
propelling itself along the DNA template during replication. A stationary DNA polymerase would instead spool
the template through itself during replication. Measurements of the force generated by a single, stationary
RNA polymerase during transcription (25 picoNewtons)
(Gelles and Landick, 1998; Wang et al., 1998) indicate
that RNA polymerase is a more powerful motor than
either myosin or kinesin (for reviews see Block, 1998;
Goldman, 1998; Cook, 1999; Mehta et al., 1999; Vale
and Milligan, 2000). A replisome contains two types of
nucleoside triphosphate-dependent molecular motors,
the DNA polymerase and helicase. Given that the rate
of DNA synthesis is approximately ten times faster than
that of RNA synthesis, it seems likely that the replisome
is at least as powerful a motor as RNA polymerase.
Although the replisome has a mass of ⵑ1,000 kDa, the
mass of the chromosome dwarfs that of the replisome. A
stationary replisome would bind and release and move
the chromosome in single base pair increments of ⵑ660
Da and ⵑ0.34 nm (Kornberg and Baker, 1992; Cook,
1999). During chromosome duplication, the same amount
of energy is used whether the polymerase is stationary
or moving. Given the limits of our understanding of the
viscosity and crowding of the intracellular environment,
it has been difficult to estimate if spooling the chromosome through the replisome would be more energetically cost effective than moving the replisome along the
polymerase. We speculate that an added benefit of the
stationary polymerase, combined with rapid recompaction of the DNA after duplication, is the ability to rapidly
separate newly duplicated regions, thus limiting the possibility of sister chromosomes tangling in the crowded
nucleoid mass.
E. coli Probably Has a Stationary Replisome
There is increasing evidence that the gram-negative
bacterium E. coli also has a centrally located, stationary
replisome. In E. coli, newly replicated DNA detected by
pulse-labeling with [3H]thymidine, is centrally located in
most cells (Koppes et al., 1999). Previously, we speculated (Lemon and Grossman, 1998) that the location
of SeqA (Hiraga et al., 1998) also indicates that newly
replicated DNA is found at or near midcell in E. coli.
SeqA binds both the origin and newly replicated, hemimethylated DNA (Slater et al., 1995). Fluorescence microscopy indicates that the bulk of SeqA does not colocalize with oriC. Rather, SeqA localizes as discrete foci
predominantly at midcell or the cell quarters (Hiraga et
al., 1998; Onogi et al., 1999) in a pattern similar to that
of the B. subtilis replisome (Lemon and Grossman,
1998). The SeqA foci probably represent the majority of
SeqA bound to newly duplicated, hemimethylated DNA
adjacent to the replication forks. We suspect that the
extrusion-capture model for how DNA replication might
power, at least in part, chromosome partitioning will
apply to other bacteria.
Is the Replisome Anchored?
While there is no evidence to suggest that the replisome
is membrane bound, it seems reasonable to expect it
might be anchored in order to prevent movement during
DNA replication. In B. subtilis, one of the proteins (DnaB)
needed to initiate replication is a membrane protein and
might play a role in helping to anchor the replisome.
The bidirectional replication forks might also be maintained in place by the viscosity inside the cell. Recent
findings indicate that large protein complexes may not
diffuse freely throughout the cell (Elowitz et al., 1999).
Head-to-Head Replication Forks
In vitro data from E. coli replication indicates that the
helicase for each fork is loaded onto the origin bubble
in a such a way that they either must pass each other
or, if stationary, remain head-to-head as each spools
and unwinds DNA (Fang et al., 1999). Head-to-head stationary helicases spooling DNA might be envisioned in
a manner similar to models for how RuvA and RuvB
accomplish Holliday junction branch migration (Rafferty
et al., 1996) (see also http://www.sdsc.edu/journals/
mbb/ruva.html). When a model of head-to-head helicases is drawn (Fang et al., 1999), it suggests that the
leading and lagging strand DNA polymerase at each fork
might use the same template strand (Figure 6). In other
words, we suggest that each replication fork duplicates
one entire template strand (one fork for Watson and one
for Crick) instead of each fork duplicating both strands
from half of the chromosome. Head-to-head bidirectional forks might help to facilitate chromosome separation and also help to limit movement of the replisome.
The Replisome Is Probably Positioned
during Initiation of Replication
The simplest model is that DNA replication initiates centrally or at sites that will become central after cell division, and this establishes the position of the replisome.
This model is supported by the pattern of origin region
localization in both B. subtilis and E. coli which indicates
that replication initiates at or near midcell or at positions
that will become midcell. In E. coli, the origin region as
detected by fluorescence in situ hybridization (FISH)
appears to be centrally located in cells just prior to onset
of DNA replication (Roos et al., 1999; Niki et al., 2000).
B. subtilis origin-region DNA can be labeled with BrdU
when spores of a thymine auxotroph are germinated.
Initially, the BrdU-labeled origin regions form a single
focus near midcell (Lewis and Errington, 1997). Later,
cells have two well-separated origin regions. When the
B. subtilis origin region is labeled with the lacO-LacIGFP system, the origin is centrally located in cells with
a single focus (D. C.-H. Lin and A. D. G., unpublished
data). Spo0J, which is an excellent marker of origin region position in B. subtilis (Lewis and Errington, 1997;
Movement of DNA through a Stationary Replisome
1327
2000; Sawitzke and Austin, 2000). SMC may be loaded
onto newly replicated DNA to establish these constrained (⫹) supercoils and compact the DNA. In addition to SMC, Spo0J, a member of the ParB protein family,
is required for efficient partitioning (Ireton et al., 1994),
probably by functioning to organize the origin region
into a higher order structure (Glaser et al., 1997; Lin et
al., 1997; Britton et al., 1998; Lin and Grossman, 1998;
Britton and Grossman, 1999).
Figure 6. A Model of Two Head-to-Head, Opposing, Stationary Hexameric Helicases Spooling the DNA Template through as They Unwind the Double Helix
Adapted from Fang et al. (1999).
Lin et al., 1997; Teleman et al., 1998)), binds to eight
identified parS sites located in the origin-proximal 20%
of the chromosome (Lin and Grossman, 1998). During
exponential growth, most cells have two well-separated
foci of Spo0J. In cells with a single focus, Spo0J, and
hence the origin region, is predominantly at midcell (Teleman et al., 1998; P. S. Lee and A. D. G., unpublished
data). Most cells in which the initiation of DNA replication
is inhibited and old rounds have finished have a single
focus of Spo0J-GFP located at approximately midcell
(our unpublished data). Finally, our results with replication upon release from stringent arrest (Figures 3J–3O)
indicate that during multifork replication initiation occurs
at the cell quarters, positions that will become midcell
upon division.
Other Proteins Are Required for Efficient
Chromosome Partitioning
Although we have proposed that the replisome plays an
important role in chromosome partitioning, it is certainly
not alone. It is likely that proteins involved in chromosome organization, compaction, and supercoiling contribute to the orderly movement of sister DNA regions
away from the central replisome. We speculate that
SMC, which is clearly important for chromosome partitioning (Britton et al., 1998; Graumann et al., 1998; Moriya et al., 1998; Britton and Grossman, 1999; Hirano,
1999), may play such a role. It has recently been proposed that SMC and the E. coli analog MukB generate
higher order compaction of bacterial DNA by introducing
constrained positive supercoils (Holmes and Cozzarelli,
The Common Theme of Stationary Replisomes
A diverse array of organisms including prokaryotes,
archaea, and eukaryotes may have stationary replisomes. Many of the components required for DNA replication are functionally conserved and homologous in all
organisms (Baker and Bell, 1998). The term “replication
factory” was first applied to the multiple foci of replication proteins observed in the eukaryotic nucleus using
immunofluorescence microscopy (for review see Newport and Yan, 1996; Cook, 1999). The prevailing view
is that these factories represent a clustering of many
replicons that probably initiate replication synchronously during S phase (Newport and Yan, 1996). Recently, a fusion of GFP to the eukaryotic sliding clamp,
GFP-PCNA, has been observed using time-lapse microscopy in a stable tissue culture cell line (Leonhardt
et al., 2000). Replication foci (GFP-PCNA) assembled
and disassembled during S phase but did not show
directional motion (Leonhardt et al., 2000). This observation is consistent with earlier models suggesting that
these eukaryotic “replication factories” might represent
a conglomerate of stationary replisomes through which
the DNA template moves (Hozak et al., 1993).
Most eukaryotes use a mitotic spindle and mitotic
motors for the separation of sister chromosomes. These
components involved in chromosome partitioning have
not been found in prokaryotes. Perhaps the differences
in the distances needed to move chromosomes during
partitioning partly explain the dramatically different
mechanisms used by eukaryotes and prokaryotes.
Experimental Procedures
Growth Media and Antibiotics
Rich medium was Luria-Bertani broth (LB), and defined minimal
medium was S7 medium with 1% glucose and 0.1% glutamate (Vasantha and Freese, 1980; Jaacks et al., 1989) and required amino
acids (phenylalanine [40 ␮g/ml], tryptophan [40 ␮g/ml], and threonine [240 ␮g/ml]). Where needed, antibiotics were used at the indicated concentrations: chloramphenicol (cat), 5 ␮g/ml; erythromycin,
0.5 ␮g/ml and lincomycin, 12.5 ␮g/ml together (mls); and spectinomycin (spc), 100 ␮g/ml.
Strains and Alleles
All strains are derivatives of JH642, which is trpC2 pheA1 (Perego
et al., 1988). Relevant alleles that we constructed for this work are
described in the appropriate sections below. dnaB134, a temperature sensitive mutation that prevents initiation of replication at nonpermissive temperature (Levine et al., 1991, 1995; Autret et al., 1999),
was used to synchronize the replication cycle in a population of
cells (see below). cotS⫹:pAT25 (cotS⫹-lacO cat) is an array of Lac
operators (on pAT25) integrated by single crossover into cotS in the
270⬚ region of the chromosome (Teleman et al., 1998).
Construction of B. subtilis strains by transformation was by standard procedures (Harwood and Cutting, 1990). Strains (and relevant
genotypes) used in experiments include KPL502 [dnaB134(ts),
zhb83::Tn917(mls) (linked by transformation to dnaB), dnaX-
Molecular Cell
1328
gfp(mut2) spc]; KPL549 [dnaB134(ts), hutM⫹-lacO cat, thrC::(PpenlacI⌬11-gfp(mut2) mls), polC-myc spc]; KPL727 [dnaB134(ts),
cotS⫹-lacO cat (270⬚), thrC::(Ppen-lacI⌬11-cfp(W7) mls), dnaX-yfp(mut2) spc]; and KPL730 [dnaB134(ts), hutM⫹-lacO cat, thrC::(PpenlacI⌬11-cfp(W7) mls), dnaX-yfp(mut2) spc].
Lac Operator/Lac Repressor System for Visualizing Regions
of Chromosomal DNA
The Lac repressor/lac operator system for visualizing chromosome
regions was first developed for budding yeast and Chinese hamster
ovary cells (Robinett et al., 1996; Straight et al., 1996; Belmont and
Straight, 1998) and was adapted for use in B. subtilis and E. coli
(Gordon et al., 1997; Webb et al., 1997). To mark the LSTer region
for visualization, we inserted a tandem array of lac operator sites
next to hutM. pAT12 contains a tandem array of lac operators and
a chloramphenicol resistance (cat) cassette (Webb et al., 1997). A 3⬘
fragment of hutM extending past the stop codon was PCR amplified
using primers oKPL148 and 149, which added a 5⬘ AatII site and a
3⬘ BamHI site. The hutM fragment was digested with AatII and BamHI
and cloned between the AatII and BamHI sites of pAT12 to give
pKL167. pKL167 was integrated by single crossover onto the B.
subtilis chromosome by standard methods (Harwood and Cutting,
1990). The resulting strain has a tandem array of lac operators downstream of an intact hutM.
To mark the 270⬚ region, a tandem array of lac operators (lacO)
inserted by single crossover at cotS (Webb et al., 1997) was transformed into JH642 using chromosomal DNA from AT52 (Teleman et
al., 1998).
We found that a carboxy-terminal fusion of LacI (minus the last 11
codons to remove the tetramerization ability) to GFP, or its variants,
produces a signal that is significantly brighter during exponential
growth than the previously described amino-terminal fusion protein
(GFP-LacI) (Robinett et al., 1996; Webb et al., 1997). Here we describe the construction of both LacI-GFP and LacI-CFP. The 3⬘ end
of lacI, absent the last 11 codons, was PCR amplified from pDH88
(Henner, 1990) using primers (oKPL 134 and 135) that added an
EcoRI site at the 5⬘ end and an XhoI site at the 3⬘ end. The resultant
product was then fused in-frame to the 5⬘ end of gfpmut2 with a
five-codon linker (LEGSG) to give plasmid pKL153. pKL153 was
integrated into strain KPL417 by single crossover into a copy of lacI
(driven by the Ppen promoter; Yansura and Henner, 1984) that had
been introduced into the B. subtilis chromosome at the thrC locus
with pPL97 (P. Levin, J. Quisel, F. Aragoni, and A. D. G., unpublished
data) to give strain KPL420. The resulting Ppen-lacI(⌬11)gfpmut2
expression unit was PCR amplified using primers (oKPL145 and
147) that added EcoRI and BamHI to the 5⬘ and 3⬘ ends, respectively,
and subcloned into pDG795 (provided by P. Stragier) (Ireton et al.,
1994) to give pKL160 and into pUC9 to give pKL178. pKL160 was
integrated by double crossover at thrC on the B. subtilis chromosome, resulting in strain KPL471 that expresses LacI-GFP. A cyan
variant of gfp, cfpW7 (Heim and Tsien, 1996), was swapped for the
gfpmut2 of pKL178 using the XhoI and SphI sites yielding pKL189.
pKL189 was digested with EcoRI and BamHI, and the Ppen-lacIcfpW7 fragment was inserted into pDG795 to give pKL190. pKL190
was integrated by double crossover at thrC on the B. subtilis chromosome, resulting in strain KPL682 that expresses LacI-CFP. We
found that in B. subtilis, the W7 CFP variant (Heim and Tsien, 1996)
with the normal jellyfish codon usage produced a brighter fusion
protein than the ECFP variant that is optimized for mammalian codon
usage.
Tau-YFP
We constructed yfpmut2 by site-directed mutagenesis of gfpmut2
(S65A, V68L, S72A) (Cormack et al., 1996), changing amino acid 203
from threonine to tyrosine (Ormo et al., 1996). The yfpmut2 gene
was then subcloned in-frame behind a 3⬘ fragment of dnaX (Tau),
replacing gfpmut2 in pKL147 (Lemon and Grossman, 1998). The
resulting construct, pKL183, was integrated by single crossover
onto the B. subtilis chromosome, such that the fusion is the only
functional copy of the gene and is under control of the endogenous
promoter.
Stringent Response Experiments
Stringent response was induced similarly to previous descriptions
(Levine et al., 1991, 1995; Autret et al., 1999). All strains were temper-
ature-sensitive mutants (dnaB134) (Mendelson and Gross, 1967;
Karamata and Gross, 1970) for initiation of DNA replication. Cells
were grown at 30⬚C in defined minimal medium. To synchronize
the cells for DNA replication, an exponentially growing culture was
shifted to 45⬚C for 30 min (single-labeled strains) or 42⬚C for 45
min (double-labeled strains). Arginine hydroxymate (250–500 ␮g/ml)
(Sigma) was prepared and added immediately prior to shift back to
30⬚C. For experiments with the double-labeled strain, the culture
was split into two flasks just before shift back to 30⬚C, and arginine
hydroxymate was added to one but not the other.
To release cells from stringent response, cells were removed from
medium with arginine hydroxymate by pouring the culture onto a
sterile 0.2 ␮m Nalgene bottle top filter. Cells were washed with 2–3
volumes of medium and resuspended in fresh growth medium plus
the following additions: arginine (600 ␮g/ml), a standard mix of all
20 amino acids (Harwood and Cutting, 1990), adenosine (20 ␮g/ml),
guanosine (20 ␮g/ml), thymidine (50 ␮g/ml), and cytidine (50 ␮g/ml).
All data presented in the tables and figures are cumulative from
at least two experiments, with each experiment giving similar results.
Live Cell Microscopy
Slides of live cells were prepared essentially as described (Lemon
and Grossman, 1998). Briefly, an aliquot of cells was removed from
an exponentially growing culture and stained with the vital membrane stain FM4-64 (140–280 ng/ml) (Molecular Probes) prior to
placement on a pad of 0.5%–1% agarose in a solution of 1⫻ T⬘
base, 1 mM MgSO4 (Harwood and Cutting, 1990). The following
filter sets were used: a Cy3 filter set for FM4-64, Chroma filter set
41029 for YFP, and Chroma filter set 31044 for CFP. Images were
acquired using a Nikon E800 microscope equipped with a CCD
camera (Hamamatsu, model C4742-95) and Improvision OpenLab
software. Images were colorized and merged using Improvision
OpenLab software and cropped in Adobe Photoshop 3.05 or 5.5.
Acknowledgments
We thank A. Teleman, C. Webb, and R. Losick for providing the
lacO array plasmid, the cotS::lacO strain, and advice on their use.
We thank R. Tsien for the W7 variant of cfp. We thank P. Levin, J.
Quisel, and F. Aragoni for providing pPL97. We thank J. C. Lindow,
T. A. Baker, F. Solomon, S. Bell, R. Losick, and K. Bacon Schneider
for comments on the manuscript.
This work was supported by Public Health Services grant
GM41934 and by the Merck/MIT Collaborative Program.
Received August 2, 2000; revised October 4, 2000.
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