From the Department of Molecular and Cell Biology,
University of California, Berkeley, California 94720 and the
Department of Chemistry, New York University,
New York, New York 10003
Received for publication, July 27, 2000, and in revised form, October 27, 2000
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ABSTRACT |
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The advance of a DNA replication fork requires an
unwinding of the parental double helix. This in turn creates a positive superhelical stress, a (+)- Unwinding of the parental strands by helicases during replication
allows DNA polymerases access to their template for the synthesis of
complementary strands. This unwinding behind the replication fork will
cause an overwinding of the parental duplex in front of the fork. The
paths of the DNA strands are best described in terms of the concept of
linking number (Lk).1 Lk is a
measure of the net number of crossings of the two strands of a
topologically closed molecule of DNA. It is the sum of twist (Tw), or
crossings in the double helix itself, and writhe (Wr), which results
from one section of double helix crossing over another section of the
same molecule (1). It can only be changed by breaking and resealing DNA
strands. Determination of the conformation of replication intermediates in
response to a (+)- The conformation of partially replicated molecules with a (+)- Plasmid DNAs--
Plasmids pREP83 and pREP48 have been described
previously (5), and their names indicate the extent of replication
allowed by the placement of Ter sites: for example, 83% of
pREP83 will replicate. pREP83 was replicated bidirectionally in
vitro and contains the E. coli origin of replication,
oriC, and Ter sites to block each fork. The
5.8-kb, pBR322-based pREP48 was used to generate intermediates in
vivo, and contains the unidirectional pUC origin of replication
followed by one Ter site after 2.8 kb. pTus (5) is a plasmid
which expresses the E. coli Tus protein under the control of
the arabinose promoter. This protein blocks replication forks at
Ter sites. In vitro and in vivo
replication was as described previously (5, 21).
Preparation and Purification of in Vivo Replication
Intermediates--
E. coli harboring both pREP48 and pTus
were used to generate partially replicated pREP48 molecules in
vivo (5). Tus protein was induced in exponential phase and cells
were allowed to grow for 1.5 h. Plasmid DNA was extracted from
cells using a variation of the neutral extraction method (22). The
partially replicated plasmid DNA was purified by gel electrophoresis,
followed by electroelution, phenol extraction, and ethanol precipitation.
Gel Electrophoresis in the Presence of
Intercalators--
One-dimensional 0.9% agarose gels were run in TAE
buffer with or without 5 µg/ml chloroquine to compare the ( Enzymatic Reactions--
DNA was relaxed with 20 units of wheat
germ topoisomerase I (23) per 0.2-1 µg of partially replicated
plasmid DNA in 50 mM Tris-HCl (pH 8), 2.5 mM
EDTA, 50 mM NaCl, and 2 mM potassium phosphate
(pH 7). DNA was nicked with DNase I in a final concentration of 20 mM Tris-HCl, 50 mM NaCl, 10 mM
MgCl2, and 360 µg/ml ethidium bromide.
In restriction endonuclease reactions, 20-50 ng of DNA was
preincubated in 10 mM Tris-HCl with or without 5 µM ethidium bromide for 15 min at 37 °C. Reactions
(New England Biolabs), containing 3 units of SapI or 15 units of PvuII in a total volume of 20 µl, were for 10 min
at 37 °C.
Denaturing Agarose Gels--
DNA bands were excised from a 1%
agarose gel in TAE and extracted using the Qiagen gel extraction kit
(Qiagen). The DNA was precipitated with ethanol, resuspended in 50 mN
NaOH and 1 mM EDTA, and run in a 1% agarose gel in 50 mN
NaOH, 1 mM EDTA at 4 °C at 23 V h/cm.
Scanning Force Microscopy (SFM)--
Partially replicated
plasmid DNA (2.5-3 ng) in 5 µM ethidium bromide was
incubated for 10 min at room temperature. It was then brought to 2 mM MgCl2, 10 mM NaCl, and 4 mM HEPES (pH 7.4), and the DNA was deposited onto freshly
cleaved mica (24). After 2-5 min, it was washed with 3-5 ml of EM
grade distilled water. The samples were then briefly dried with
nitrogen prior to imaging.
All SFM images were obtained in air at room temperature with a
Nanoscope III microscope (Digital Instruments Inc., Santa Barbara, CA)
operating in the tapping mode. Commercial diving board silicon tips
(Nanosensor, Digital Instruments) with a force constant of 40 nN/nm and
a 250-300 kHz resonance frequency were used. An E type scanner
(12 × 12 µm) was used for all imaging. Images were collected
with a scan size between 1 and 4 µm at a scan rate of 1.9 Hz. Images
were processed with a standard flatten filter using Nanoscope software.
Partially Replicated Plasmids with a (+)-
Our first experiments used pREP83 DNA replicated in vitro in
the presence of Tus protein to halt synthesis at the Ter
sites (25) (Fig. 2). The control
reactions contained gyrase, which is known to introduce a (
A definitive test of this conclusion employed two-dimensional agarose
gel electrophoresis, in which the second dimension contained chloroquine (Fig. 3). Plasmid DNA, as
opposed to partially replicated DNA, with a (
The two-dimensional gel of pREP83 partially replicated in
vitro with DNA gyrase (Fig. 3B) has a strikingly
different pattern. Fourteen topoisomers are resolved and have a
(
We obtained the same results with a partially replicated plasmid
generated in vivo. pREP48 replicated from the unidirectional pUC origin of replication toward a single Ter site was run
on a two-dimensional gel (Fig. 3C). These molecules have a
( A (+)-
Two restriction enzymes were used: PvuII, which cuts 366 base pairs (bp) from the Ter site in the replicated region,
and SapI, which cleaves 184 bp from the pUC19 origin of
replication in the replicated region. In this way, we studied the
structure at the terminus and origin independently. From the
unwinding angle of ethidium bromide (27) and an average
After cleavage with either restriction enzyme, two closely spaced
low-mobility bands were detected (Fig. 4B). The bottom of these two bands predominated in the presence of ethidium bromide, while
the top band predominated in its absence.
It seemed likely that the bottom band is the single-forked structure
predicted by the chickenfoot model, and that the top is the
double-forked structure derived from the usual three-way replication
fork. Two experiments proved these assignations. First, we extracted
these two bands from PvuII-treated, relaxed replication intermediates and analyzed them on an alkaline denaturing agarose gel
(Fig. 4C). The bottom band (B) yielded only the
two single-stranded products expected from the single-forked structure
generated from the chickenfoot-containing molecule, corresponding to
the 2.4-kb daughter strand from the long arm and the 5.8-kb parental
strand. As predicted if the top band (T) is the double forked structure (see Fig. 4A), three fragments were detected corresponding
to the 366 base daughter strand of the short arm, the 2.4-kb daughter strand of the long arm and the 5.8-kb parental strand.
Second, the small fragment released from the middle toe of the
chickenfoot by restriction digestion was visible on a 2% agarose gel
run for 24 h (data not shown). Its intensity correlated with the
amount of the bottom band; i.e. it predominated when
ethidium bromide was present. As expected, this high-mobility fragment comigrated with a 200-bp marker when the partially replicated DNA was
cleaved with SapI and with a 370-bp marker when cleaved with
PvuII.
Because the same amount, about 50%, of each partially replicated DNA
was cleaved to form the bottom band indicative of the chickenfoot (Fig.
4D), we conclude that a chickenfoot was equally likely to be
at the origin as at the terminus. Relaxation did not change the
results, implying that the chickenfoot distribution did not change with
increased middle toe length.
To make sure that the effect of ethidium bromide was due to an increase
in Visualization of the Chickenfoot--
We visualized the
chickenfoot directly by SFM. Partially replicated pREP48 molecules were
incubated with ethidium bromide, deposited onto cleaved mica, and
imaged by SFM using the tapping mode in air. Typical molecules are
shown in Fig. 5, A-F. For 70% of topologically closed molecules, a chickenfoot was clearly evidenced by one or two long linear duplexes emerging from a three-way junction. This linear duplex does not appear without incubation in ethidium bromide, nor does it appear in plasmid DNA incubated in ethidium bromide (data not shown).
The mean total middle toe length per molecule is 472 bp (Table
I). We expect the middle toes to equal
470 bp. This quantitative agreement is probably fortuitous, because
there is a large variation in middle toe lengths as deposition of the
molecules onto mica traps them in a single conformation of a dynamic
structure.
The duplex emerges from either one or both of the three-way junctions.
This confirms the restriction results that the chickenfoot can form at
either the origin or the terminus.
Unexpectedly, the molecules with chickenfeet appear supercoiled.
Because the electrophoresis results showed clearly that the partially
replicated molecules are not supercoiled, we believe this is an
artifact of the deposition procedure for SFM. It is possible that the
magnesium necessary for deposition on the mica displaced ethidium ions
from the DNA (28). The chickenfoot may not have been re-absorbed
because of the slow rate of branch migration in ethidium and magnesium
(26, 29), but the DNA could have become supercoiled before deposition.
We showed that a (+)- Thermodynamics and Kinetics of Chickenfoot Formation under
Superhelical Stress--
Formation of alternative DNA structures under
the action of a (
The chickenfoot has a junction of four double helices, and thus
resembles the cruciform structure, which is formed in palindromes of
(
Our SFM and enzymatic results show that either one or two chickenfeet
can form on a partially replicated plasmid. A single chickenfoot at
either the origin or the terminus is enthalpically favored because
there is one rather than two junction penalties. A chickenfoot at each
junction, on the other hand, would be entropically favored by the
increased number of possible conformations. There is an additional
factor that influences whether one or two chickenfeet are formed. In
molecules with fully ligated daughter strands, two chickenfeet would
result in the replication bubble becoming a second topological domain,
that would become (+)-supercoiled as the first domain, composed of
parental strands, relaxed.
The cleavage data indicate that there is no preference for chickenfoot
formation at the origin or terminus. This is interesting because the
two three-way junctions have different properties, due to potentially
incomplete Okazaki fragments near the terminus and RNA primers at the origin.
Physiological Implications of the Chickenfoot--
The chickenfoot
was first postulated in 1976 by Higgins et al. (14) who were
studying human cells treated with methyl methanesulfonate. These
authors proposed that the four-way junction allowed the repair of a
methyl methanesulfonate-induced lesion on the leading strand template,
as shown in Fig. 6A. Soon
afterward, other researchers found similar evidence for mutagen-induced
nascent-nascent duplex at replication forks (12, 13). Chickenfeet have
also been observed in replicating DNA isolated from the eggs of
Drosophila melanogaster (36).
It has more recently been proposed that the chickenfoot may play a role
any time the replication fork stalls. In E. coli cells, the
chickenfoot may emerge at replication forks stalled at the replication
terminus (16), at a stalled RNA polymerase (17), or due to a mutant
replicative helicase (15, 19). In addition, it has been postulated that
the chickenfoot forms in yeast cells at replication fork blocks in
ribosomal DNA (37).
Several ways that a stalled fork can be restarted are illustrated in
Fig. 6B. It is possible that the three-way junction itself breaks without the intermediary of a chickenfoot, creating a substrate for RecBCD, the recombinogenic exonuclease (38), as shown in Fig.
6B, left. Recombination ensues with the sister arm, and a new replication fork is formed. In Fig. 6B, middle, is shown
the regression of the replication fork to form the chickenfoot. This nascent-nascent duplex itself can be chewed back entirely by RecBCD, resulting in a reformed replication fork (15). Formation of a four-way
junction at replication forks may be a method by which the cells create
a recombinogenic end from a three-way junction, because a four-way
junction is a natural intermediate in recombination. The four-way
junction of the chickenfoot may be resolved by the Holliday junction
processing proteins RuvA, -B, and -C (15) (Fig. 6B, right).
This cleavage will result in a severed replicated arm, which can then
become a substrate for recombination and replication restart much like
the example on the far left. The role of recombination in replication
restart has recently been extensively reviewed (20, 39).
This paper demonstrates that the chickenfoot forms spontaneously in DNA
free of proteins. We find that it is stabilized by a (+)- Positive Supercoiling and Stalled Forks in the Cell--
DNA
replication causes a (+)-
(+)-Lk, that must be relaxed by
topoisomerases for replication to proceed. Surprisingly, partially
replicated plasmids with a (+)-
Lk were not supercoiled nor were the
replicated arms interwound in precatenanes. The electrophoretic
mobility of these molecules indicated that they have no net writhe.
Instead, the (+)-
Lk is absorbed by a regression of the replication
fork. As the parental DNA strands re-anneal, the resultant displaced daughter strands base pair to each other to form a four-way junction at
the replication fork, which is locally identical to a Holliday junction
in recombination. We showed by restriction endonuclease digestion that
the junction can form at either the terminus or the origin of
replication and we visualized the structure with scanning force
microscopy. We discuss possible physiological implications of the
junction for stalled replication in vivo.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Lk is the difference between the Lk of a molecule and that
of the same molecule in an unconstrained, relaxed state
(Lk0).
Lk can be either (+) or (
). During replication,
Lk increases even though Lk remains the same, because separation of
the parental strands lowers the value of Lk0. The
Lk of replication increases by about one for every 10 base pairs of
DNA that are replicated. The Tw of the DNA is converted to Wr when the
strands are separated, which in turn must be removed to relax the DNA. Topoisomerases relieve this strain by catalyzing DNA passages, allowing
the fork to move unhindered. In Escherichia coli, the most
important such topoisomerase is DNA gyrase, which removes (+)-
Lk by
introducing (
)-
LK (2-4). The new winding of the parental and
daughter strands introduced during replication do not contribute to DNA
topology because the daughter strands are not topologically closed.
Lk is crucial to understanding DNA unlinking during replication. These conformations dictate the actual substrates for topoisomerases in replication. In a previous study of replication intermediates, we used partially replicated E. coli plasmids
that had been stalled at a termination site, Ter (5). These
DNA molecules have a homogeneous structure, are relatively easy to prepare, and model a replicating chromosomal domain. When these intermediates are isolated from cells or are formed in an in
vitro replication reaction containing DNA gyrase, they have a
(
)-
Lk due to the activity of gyrase, perhaps, after replication
halted. The (
)-
Lk equilibrates between two forms. One has (
)
supercoils ahead of the replication fork. The other, called
precatenanes, has (
) crossings between the replicated DNA arms (Fig.
1A). Precatenanes were originally postulated by Champoux and
Been (6) and were subsequently analyzed theoretically (7). Additional
studies have also presented evidence that precatenanes are a
potentially important structure during replication (8-11).
Lk had
not yet been examined, although this is thought to be the
physiologically important form. We anticipated that partially replicated molecules with a (+)-
Lk would look just like those with a
(
)-
Lk, except that the supercoils and precatenanes would have the
opposite handedness (Fig. 1B). To generate a (+)-
Lk replication intermediate, we added intercalating agents to unwind the
parental strands of the partially replicated molecules described above.
To our surprise, they contained no supercoils or precatenanes. Instead,
the (+)-topological stress is relieved exclusively by a retreat of the
replication fork and reannealing of parental strands. Any (+)-supercoil
and precatenane links are thereby converted into an increased Tw of the
parental strands. The displaced nascent strands subsequently base pair
to form a four-way junction at the replication fork, producing the
structure shown in Fig. 1C. We call this structure the
"chickenfoot." Such a structure had been proposed by several
authors to explain findings of daughter-daughter duplex DNA as detected
by bromodeoxyuridine incorporation in eukaryotes (12-14), as well as
double-strand breaks and homologous recombination at stalled
replication forks in prokaryotes (15-17). This four-way junction
previously has been called a reversed replication fork (18, 19). We
provide proof of the structure of this intermediate, and that it is
promoted at a replication fork by a (+)-
Lk. Our structural
evidence combined with these in vivo studies suggests that the chickenfoot structure may be important when replication forks
pause. This conclusion gains added significance from the recent
realization that replication forks stall normally in aerobically growing cells (20), presumably from damage to the chromosomes.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
)-
Lk
partially replicated molecules to those with a (+)-
Lk. For
two-dimensional gels, DNA was first run in 0.9 or 1% agarose gels in
TAE buffer at 79 V h/cm. The gels were soaked for 3 h in TAE plus
10 or 11 µg/ml chloroquine, turned 90°, and run in the second
dimension in buffer containing chloroquine at an additional 113 V h/cm. The gels were then Southern blotted and probed with pBR322.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Lk Have the Same
Electrophoretic Mobility as Those That Are Relaxed--
We showed
previously that partially replicated plasmids with a (
)-
Lk have
both a (
)-supercoiled unreplicated region and a (
)-precatenated
replicated region as diagrammed in Fig.
1A (5). However, the actual
substrate for topoisomerases during replication is thought to have (+),
not (
),
Lk. We prepared a (+)-
Lk intermediate by adding
intercalating dyes such as chloroquine or ethidium bromide to
(
)-
Lk forms. These dyes increase
Lk by unwinding the parental
strands, increasing Wr. Our first experiments used electrophoretic
mobility to study the structure of the replication intermediates.
Because Wr, composed of supercoiling and precatenanes, compacts DNA
molecules, DNA with a higher Wr runs faster on an agarose gel than
DNA with a lower Wr .
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Fig. 1.
Possible conformations of replication
intermediates. Throughout this paper, black
lines denote parental strands, and red lines denote
nascent strands. A, partially replicated DNA with a
( )-
Lk contains (
)-plectonemic supercoils in the unreplicated
region and (
)-precatenanes, or intertwinings of the replicated arms.
B, partially replicated plasmids with a (+)-
Lk were
expected to have both (+)-supercoils in the unreplicated region and
(+)-precatenanes in the replicated region. C, we find
instead that a (+)-
Lk is taken up by a re-annealing of parental
strands and the displaced nascent strands base pair to each other,
forming a four-way junction at the replication fork we call the
chickenfoot. The end result is a molecule without Wr and with nascent
strands paired at one or, as shown, at both forks.
)-
Lk in
the replication intermediates (lanes 1 and 3).
The gyrase reaction gave the expected distribution of replication
intermediate topoisomers, with different numbers of supercoils and
precatenanes (see bands marked RI, lane 1). A second set of
reactions contained topoisomerase (Topo) IV as the sole topoisomerase
(lanes 2 and 4). Because this enzyme removes both
(+)- and (
)-supercoils, we expected the replication intermediates to
be relaxed. The Topo IV reaction, however, yielded a single band
(labeled RI) that comigrated with nicked replication
intermediates rather than the expected ladder of relaxed
topoisomers (lane 2). In an attempt to resolve these
topoisomers, we increased the
Lk of these DNA molecules by running
the reaction mixtures on a gel containing 5 µg/ml chloroquine
(lanes 3 and 4). This amount of chloroquine
reduced the (
)-
Lk of the partially replicated molecules from the
gyrase reaction and thereby their electrophoretic mobility (lane
3), but the intermediates from the Topo IV replication reaction (lane 4) remained unresolved and migrated at the same rate
as in the absence of chloroquine (lane 2). The unexpected
electrophoretic mobility of the intermediates is not due to nicking by
Topo IV because unreplicated plasmid DNA in these reactions ran as the covalently closed relaxed topoisomers (bands marked RC in lane 4). We concluded that the (+)-
Lk intermediates exist in some form other than supercoiled topoisomers or precatenanes.
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Fig. 2.
One-dimensional gel electrophoresis of
partially replicated DNA shows an absence of (+)-supercoiling and
precatenation in the presence of chloroquine. pREP83 is
schematized on top with a double-headed arrow symbolizing
oriC, and the two Ter sites are indicated with
black flag symbols. pREP83 was replicated in
vitro as described (41) in the presence of Tus protein and either
gyrase (lanes 1 and 3) or Topo IV (lanes
2 and 4). These reactions were run on 0.9% TAE gels in
the absence (lanes 1 and 2) or presence
(lanes 3 and 4) of 5 µg/ml chloroquine and
Southern blotted. The replication intermediates relaxed with Topo IV
comigrate with nicked replication intermediates in lanes 2 and 4. RI, replication intermediate;
NRI, nicked replication intermediate; NC, nicked
unreplicated circle; SC, supercoiled unreplicated circle;
RC, unreplicated relaxed circle topoisomers.
)-
Lk forms an arc on
the two-dimensional gel, as diagrammed in Fig. 3A. In the
first dimension, run in the absence of chloroquine, topoisomers with a
more negative
Lk migrate faster. Chloroquine, in the second
dimension, introduces a (+)-
Lk. Topoisomers that still have a
(
)-
Lk (labeled "(
)" in Fig. 3A) form the lower
portion of the arc. Topoisomers relaxed in chloroquine migrate at the
center of the arc (Rel). Topoisomers with less (
)-
Lk than the
relaxed topoisomers become (+)-supercoiled by chloroquine (labeled
"(+)" in Fig. 3A) and form the upper part of the
arc.
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Fig. 3.
Two-dimensional gel electrophoresis of
partially replicated DNA. A, the expected result for
plasmid DNA analyzed by two-dimensional gel electrophoresis, in which
the second dimension, but not the first, contains chloroquine. Negative
topoisomers are resolved in the first dimension. Topoisomers that still
have a ( )-
Lk in the second dimension form the bottom portion of an
arc. The middle topoisomer of the distribution takes up just enough
chloroquine to be relaxed in the second dimension. The topoisomers with
less (
)-
Lk than the relaxed topoisomer become (+) supercoiled in
the second dimension, forming the top part of the arc. (+), (
), and
Rel. refer to whether the molecules have a (+)-
Lk, a
(
)-
Lk, or are relaxed, respectively, in the second dimension.
Two-dimensional gels of partially replicated pREP83 (B) and
the in vivo replication intermediate of pREP48
(C) were run. Because replication intermediates do not adopt
(+)-Wr, they do not form the top of the arc. Unidirectional and
bidirectional origins of replication are indicated with
arrows, and Ter sites are indicated with
black flag symbols.
)-
Lk in the first dimension. The chloroquine in the second
dimension is sufficient to relax the sixth slowest topoisomer. The
(
)-
Lk arc is clearly present, but a straight line with the
mobility of the relaxed topoisomer replaces the (+)-
Lk arc.
)-
Lk to begin. Once again, the (
)-
Lk arc is present, but the
(+)-
Lk topoisomers migrate with the same mobility as relaxed
partially replicated DNA. We conclude that partially replicated
molecules produced both in vitro from a bidirectional origin
and in vivo from a unidirectional origin do not have (+)-Wr
even though they have a (+)-
Lk.
Lk Causes a Four-way Junction to Form at the Terminus and
the Origin of Partially Replicated Plasmids--
Since Wr = 0, partially replicated molecules with a (+)-
Lk must have a (+)-
Tw.
The most likely cause of the (+)-
Tw is that the molecules compensate
for the (+)-
Lk by rewinding the parental strands. This necessarily
requires a concomitant unwinding of the most recently replicated DNA.
The energy of base pairing would be conserved, however, if the
displaced nascent strands of the unwound replicated DNA anneal to each
other to generate a four-way junction at the fork (Fig. 1C).
We named this four-way junction the chickenfoot, because of an
obvious resemblance to the fowl appendage. If the chickenfoot model is
correct, increase in
Lk from intercalating dyes will cause a linear
duplex, resulting from the nascent-nascent pairing, to extrude from the
replication fork. Cleavage with a restriction enzyme of a site in the
replicated region provides a strong test of this model, as diagrammed
in Fig. 4A. Because the site
is in the replicated region, two double-strand cuts will be made. If no
chickenfoot forms, these cleavages will result in a single product: a
molecule with two forks of different sizes (Fig. 4A, left
side). The pair of cleavages of a molecule with a chickenfoot will
result in two products: a short linear duplex, the amputated middle toe
of the chickenfoot, and, after branch migration, a molecule with a
single fork (Fig. 4A, right side). Because the second
cleavage in the parental-parental duplex removes the topological
constraint in these molecules, the chickenfoot is no longer the lowest
energy form of the molecule. Branch migration of the forks occurs
readily in the absence of magnesium (26), and this will cause
absorption of the middle toe. Once this three-way junction is formed it
will be a branch migration "sink," and reformation of the four-way
junction will be unfavorable. A possible path is shown in Fig.
4A. The double-forked structure should migrate more slowly
on an agarose gel than the single-forked structure.
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Fig. 4.
Cleavage of the chickenfoot by restriction
endonucleases. A, a unique restriction site close to
the fork of a partially replicated molecule will reside either in the
replicated arms (left) or in the extruded middle toe of the
chickenfoot and in the re-annealed parental strands (right).
If no chickenfoot forms, cleavage will result in a molecule with two
three-way junctions (left). If a chickenfoot is formed by
intercalation of ethidium bromide (EtBr), a middle toe will
be cleaved. The resulting structure forms a single-forked molecule and
a short linear duplex after branch migration (right).
B, partially replicated pREP48 molecules were cleaved
directly (Form I), or first relaxed with wheat germ Topo I (Form IV) or
nicked with DNase I (Form II). Molecules were cleaved either with
SapI, which cuts 184 bp from the origin of replication, or
with PvuII, which cleaves 366 bp from the Ter
site. DNA was run in 1% agarose gels and Southern blotted. The two
major bands correspond to single-forked or double-forked structures.
C, partially replicated relaxed pREP48 was incubated with or
without ethidium bromide, cleaved with PvuII and run on an
agarose gel as in B. The top and bottom
bands were excised, run on an alkaline agarose gel and Southern
blotted. S, uncut sample DNA; T, top band DNA;
B, bottom band DNA. The markings on the left
denote the migration of linear fragments in kilobases. As expected, the
bands in the lanes marked T run at 5.8 kb, 2.4 and .4 kb,
and those in the lanes marked B run at 5.8 and 2.4 kb.
D, the top and bottom bands shown in B were
quantified using a PhosphorImager. Error bars indicate one
S.D.
Lk for
pREP48 replication intermediates of
17 (Form I) (5), we expect the
average length of the extruded toe to be about 470 bp/molecule, which
could potentially come from a single middle toe or two per molecule. We
also relaxed the partially replicated molecules with wheat germ Topo I
before adding dye (Form IV), which should increase the toe length per molecule to about 700 bp.
Lk rather than unrelated chemical effects of the dye, we
performed a control using nicked replication intermediates (Form II).
There was no difference between restriction reactions with and without
ethidium bromide for these molecules (Fig. 4B).
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Fig. 5.
Scanning force microscopy of replication
intermediates. Partially replicated pREP48 molecules were
incubated in 5 µM ethidium bromide for 5-10 min, brought
to 5 mM MgCl2, 10 mM NaCl, 4 mM HEPES, then deposited onto mica and imaged by SFM.
Linear duplexes emerging from three-way junctions are clearly visible
in the molecules, and have been marked with white arrows.
Molecules with one chickenfoot (B) or two chickenfeet
(A, C, D, E, and
F) are shown. Bar, 100 nm.
SFM studies on chickenfeet. Middle toe lengths and total lengths, in
bp, of 14 molecules with one or two chickenfeet were averaged
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Lk provokes the formation of a four-way
junction at a replication fork in vitro. The result is a
molecule without Wr, in which the (+)-
Lk is taken up by an unwinding
of parental DNA and a concomitant formation of a fourth arm of the junction. The evidence is: 1) gel electrophoresis indicates that partially replicated plasmids with a (+)-
Lk are neither
(+)-supercoiled nor precatenated and comigrate with relaxed replication
intermediates; 2) a DNA duplex can be extruded from a junction by
addition of ethidium bromide and detected by restriction enzyme
digestion; and 3) the four-way junctions can be visualized by scanning
force microscopy. This is the first definite proof of the structure of
a (+)-
Lk replication intermediate. We discuss next the energetics of
chickenfoot formation, previous evidence for its occurrence in
vivo, and the circumstances whereby a (+)-
Lk may build up around a replication fork and favor chickenfoot formation.
)-
Lk is a well known phenomenon (30). These
alternative structures have a higher free energy than the regular
double helix, but their formation reduces the total free energy of
supercoiled DNA by diminishing superhelical stress (see Ref. 31, for
example). Chickenfoot formation in (+)-
Lk replication intermediates
is no exception.
)-supercoiled DNA, but it should form much more readily. Hairpin
loops with unpaired bases in the cruciform contribute to the large free
energy difference between the cruciform and linear structures. These
hairpin loops, however, are absent in the chickenfoot structure.
Another difference is that a cruciform forms from a linear DNA, whereas
the chickenfoot is formed from a three-arm junction. Thus, very little
additional irregularity is associated with chickenfoot formation, as
opposed to cruciform extrusion. For cruciform extrusion the free energy
cost is 20-28 kcal/mol (32), depending on ionic conditions. We expect
that chickenfoot formation would be associated with only about 5 kcal/mol free energy change and thus can be formed at relatively low
torsional stress in comparison with cruciform extrusion, as observed.
Moreover, cruciforms form slowly due to the necessity of forming a
large open region in the double helix as an intermediate (33-35).
Nothing like this is needed to form the chickenfoot, and thus there is a much smaller kinetic barrier to the transition. Indeed, only a
Lk
of (+)-1 extrudes the chickenfoot (see Fig. 3). The ease of chickenfoot
formation is probably due to the entropic gain resulting from the
increased number of possible conformations of the four-way junction.
View larger version (20K):
[in a new window]
Fig. 6.
Possible models for the emergence,
processing, and function of chickenfeet in vivo.
The 3' end of a DNA strand is denoted by an arrow.
A, a role of the chickenfoot in repair proposed by Higgins
et al. (14). 1) A lesion on the leading strand
template (black box) inhibits replication progression on the
leading strand, but not on the lagging strand. 2) Branch
migration at the replication fork allows the annealing of the nascent
leading strand to the lagging strand. 3) The lagging strand
becomes a template for leading strand synthesis (dotted
line), and the leading strand eventually bypasses the lesion on
its original template. 4) Regression of the chickenfoot
reforms the three-way junction, and replication resumes. This process
gives the cell time to repair the lesion on the parental DNA, now
behind the replication fork. B, models for restart of
blocked replication forks. When replication forks stall in the absence
of damage, the three-way junction may become cleaved (left).
This cleavage presents a double-stranded end that will be a substrate
for RecBCD (white Pac-Man)-mediated degradation. Processing by RecBCD
allows for homologous recombination and resolution by the Holliday
junction endonuclease RuvC (white arrowheads). The end
result is a re-formed replication fork. Alternatively, a stalled
replication fork can form a chickenfoot, either using the energy of a
(+)- Lk or with the help of RuvAB-mediated branch migration
(middle). The chickenfoot itself can be a substrate for
RecBCD, which can degrade the middle toe completely, resulting in a
reformed three-way junction. In addition, the four-way junction of the
chickenfoot itself may be cleaved by RuvC (right). Cleavage
will result in a broken chromosomal arm that will become a substrate
for RecBCD-mediated homologous recombination. C, two
possible scenarios for the buildup of (+)-superhelical strain during
replication. First, two converging replication forks build up (+)-
Lk
between them. Second, RNA (blue) transcription in a
direction opposite to the moving replication fork will cause (+)-
Lk
to build up.
Lk, but
will also form at a low level in the absence of positive superhelical
strain. About 10% of partially replicated molecules with a (
)-
Lk
formed a chickenfoot, and this number increased slightly if the
molecules were nicked (Fig. 4B). Low levels of chickenfoot
formation due to branch migration in vitro have been seen in
molecules without a (+)-
Lk (18).
Lk in the domain surrounding the
replication fork. If this (+)-
Lk remains after a replication fork
pauses, the chickenfoot would be a favored outcome. We believe that the
domain surrounding the fork will have a (+)-
Lk in all organisms, but
certainly in eukaryotes and archaea which have no DNA gyrase. Thus,
there will be constant pressure to form a chickenfoot when replication
stops. In addition, (+)-
Lk will build up fastest when two
replication forks move toward each other (Fig. 6C, top), as
at the terminus of chromosomal replication. Louarn et al.
(16) have suggested that superhelical stress at the terminus may force
a four-way junction to form at one of the replication forks. In
eukaryotes, replication forks move toward each other much more
frequently, whenever two adjacent domains of replication are completed.
Lk may also build up if a replication fork is moving in the
direction opposite to an RNA polymerase. Simultaneous and oppositely
oriented replication and transcription leads to pausing of the
replication fork (40), and these pauses may allow for chickenfoot
formation (Fig. 6C, bottom). Thus, chickenfoot formation due
to a (+)-
Lk may be important during replication in the cell.
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ACKNOWLEDGEMENTS |
---|
We thank K. Marians and H. Hiasa for sharing in vitro partially replicated plasmids, and B. Peter for experimental help. We also thank S. Wickner, J. Mitchell, and C. T. Fink for helpful discussions, and N. P. Higgins, K. Kreuzer, and M. Cox for comments on the manuscript.
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FOOTNOTES |
---|
* This work was supported in part by grants from the National Institutes of Health (to N. R. C.).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.
§ Supported in part by a NIEHS National Institutes of Health training grant.
¶ Present address: Dept. of Neuroscience, University of New Mexico, Albuquerque, NM 87131.
** To whom correspondence should be addressed.
Published, JBC Papers in Press, October 30, 2000, DOI 10.1074/jbc.M006736200
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ABBREVIATIONS |
---|
The abbreviations used are: Lk, linking; Tw, twist; Wr, writhe; kb, kilobase(s); SFM, scanning force microscopy; bp, base pair(s).
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