Positive Torsional Strain Causes the Formation of a Four-way Junction at Replication Forks*

Lisa PostowDagger §, Chris UllspergerDagger , Rebecca W. KellerDagger , Carlos BustamanteDagger , Alexander V. Vologodskii||, and Nicholas R. CozzarelliDagger **

From the Dagger  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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The advance of a DNA replication fork requires an unwinding of the parental double helix. This in turn creates a positive superhelical stress, a (+)-Delta Lk, that must be relaxed by topoisomerases for replication to proceed. Surprisingly, partially replicated plasmids with a (+)-Delta 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 (+)-Delta 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

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. Delta Lk is the difference between the Lk of a molecule and that of the same molecule in an unconstrained, relaxed state (Lk0). Delta Lk can be either (+) or (-). During replication, Delta Lk increases even though Lk remains the same, because separation of the parental strands lowers the value of Lk0. The Delta 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 (+)-Delta Lk by introducing (-)-Delta 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.

Determination of the conformation of replication intermediates in response to a (+)-Delta 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 (-)-Delta Lk due to the activity of gyrase, perhaps, after replication halted. The (-)-Delta 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).

The conformation of partially replicated molecules with a (+)-Delta Lk had not yet been examined, although this is thought to be the physiologically important form. We anticipated that partially replicated molecules with a (+)-Delta Lk would look just like those with a (-)-Delta Lk, except that the supercoils and precatenanes would have the opposite handedness (Fig. 1B). To generate a (+)-Delta 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 (+)-Delta 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (-)-Delta Lk partially replicated molecules to those with a (+)-Delta 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.

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Partially Replicated Plasmids with a (+)-Delta Lk Have the Same Electrophoretic Mobility as Those That Are Relaxed-- We showed previously that partially replicated plasmids with a (-)-Delta 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 (-), Delta Lk. We prepared a (+)-Delta Lk intermediate by adding intercalating dyes such as chloroquine or ethidium bromide to (-)-Delta Lk forms. These dyes increase Delta 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 .



View larger version (20K):
[in this window]
[in a new window]
 
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 (-)-Delta Lk contains (-)-plectonemic supercoils in the unreplicated region and (-)-precatenanes, or intertwinings of the replicated arms. B, partially replicated plasmids with a (+)-Delta Lk were expected to have both (+)-supercoils in the unreplicated region and (+)-precatenanes in the replicated region. C, we find instead that a (+)-Delta 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.

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 (-)-Delta 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 Delta 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 (-)-Delta 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 (+)-Delta Lk intermediates exist in some form other than supercoiled topoisomers or precatenanes.



View larger version (24K):
[in this window]
[in a new window]
 
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.

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 (-)-Delta 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 Delta Lk migrate faster. Chloroquine, in the second dimension, introduces a (+)-Delta Lk. Topoisomers that still have a (-)-Delta 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 (-)-Delta Lk than the relaxed topoisomers become (+)-supercoiled by chloroquine (labeled "(+)" in Fig. 3A) and form the upper part of the arc.



View larger version (32K):
[in this window]
[in a new window]
 
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 (-)-Delta 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 (-)-Delta 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 (+)-Delta Lk, a (-)-Delta 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.

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 (-)-Delta Lk in the first dimension. The chloroquine in the second dimension is sufficient to relax the sixth slowest topoisomer. The (-)-Delta Lk arc is clearly present, but a straight line with the mobility of the relaxed topoisomer replaces the (+)-Delta Lk arc.

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 (-)-Delta Lk to begin. Once again, the (-)-Delta Lk arc is present, but the (+)-Delta 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 (+)-Delta Lk.

A (+)-Delta 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 (+)-Delta Lk must have a (+)-Delta Tw. The most likely cause of the (+)-Delta Tw is that the molecules compensate for the (+)-Delta 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 Delta 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.



View larger version (41K):
[in this window]
[in a new window]
 
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.

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 Delta 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.

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 Delta 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).

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).



View larger version (150K):
[in this window]
[in a new window]
 
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.

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.


                              
View this table:
[in this window]
[in a new window]
 
Table I
SFM studies on chickenfeet. Middle toe lengths and total lengths, in bp, of 14 molecules with one or two chickenfeet were averaged

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We showed that a (+)-Delta 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 (+)-Delta 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 (+)-Delta 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 (+)-Delta Lk replication intermediate. We discuss next the energetics of chickenfoot formation, previous evidence for its occurrence in vivo, and the circumstances whereby a (+)-Delta Lk may build up around a replication fork and favor chickenfoot formation.

Thermodynamics and Kinetics of Chickenfoot Formation under Superhelical Stress-- Formation of alternative DNA structures under the action of a (-)-Delta 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 (+)-Delta Lk replication intermediates is no exception.

The chickenfoot has a junction of four double helices, and thus resembles the cruciform structure, which is formed in palindromes of (-)-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 Delta 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.

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).



View larger version (20K):
[in this window]
[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 (+)-Delta 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 (+)-Delta Lk between them. Second, RNA (blue) transcription in a direction opposite to the moving replication fork will cause (+)-Delta Lk to build up.

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 (+)-Delta Lk, but will also form at a low level in the absence of positive superhelical strain. About 10% of partially replicated molecules with a (-)-Delta 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 (+)-Delta Lk (18).

Positive Supercoiling and Stalled Forks in the Cell-- DNA replication causes a (+)-Delta Lk in the domain surrounding the replication fork. If this (+)-Delta 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 (+)-Delta 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, (+)-Delta 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.

(+)-Delta 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 (+)-Delta Lk may be important during replication in the cell.


    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.


    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


    ABBREVIATIONS

The abbreviations used are: Lk, linking; Tw, twist; Wr, writhe; kb, kilobase(s); SFM, scanning force microscopy; bp, base pair(s).


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Cozzarelli, N. R., Boles, T. C., and White, J. H. (1990) in DNA Topology and Its Biological Effects (Cozzarelli, N. R. , and Wang, J. C., eds) , pp. 139-184, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
2. Marians, K. J., Ikeda, J. E., Schlagman, S., and Hurwitz, J. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 1965-1968[Abstract]
3. Gellert, M., Mizuuchi, K., O'Dea, M. H., and Nash, H. A. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 3872-3876[Abstract]
4. Kreuzer, K. N., and Cozzarelli, N. R. (1979) J. Bacteriol. 140, 424-435[Medline] [Order article via Infotrieve]
5. Peter, B. J., Ullsperger, C., Hiasa, H., Marians, K. J., and Cozzarelli, N. R. (1998) Cell 94, 819-827[Medline] [Order article via Infotrieve]
6. Champoux, J. J., and Been, M. D. (1980) in Mechanistic Studies of DNA Replication and Genetic Recombination: ICN-UCLA Symposia on Molecular and Cellular Biology (Alberts, B., ed), Vol. XIX , pp. 809-815, Academic Press, Inc., New York
7. Ullsperger, C. J. V., Vologodskii, A. V., and Cozzarelli, N. R. (1995) Nucleic Acids Mol. Biol. 9, 115-142
8. Hiasa, H., Digate, R. J., and Marians, K. J. (1994) J. Biol. Chem. 269, 2093-2099[Abstract/Free Full Text]
9. Hiasa, H., and Marians, K. J. (1994) J. Biol. Chem. 269, 32655-32659[Abstract/Free Full Text]
10. Sogo, J. M., Stasiak, A. M., Martinez-Robles, M. L., Krimer, D. B., Hernandez, P., and Schvartzman, J. B. (1999) J. Mol. Biol. 286, 637-643[CrossRef][Medline] [Order article via Infotrieve]
11. Postow, L., Peter, B. J., and Cozzarelli, N. R. (1999) BioEssays 21, 805-808[CrossRef][Medline] [Order article via Infotrieve]
12. Wanka, F., Brouns, R. M., Aelen, J. M., Eygensteyn, A., and Eygensteyn, J. (1977) Nucleic Acids Res. 4, 2083-2097[Abstract]
13. Nilsen, T., and Baglioni, C. (1979) J. Mol. Biol. 133, 319-338[Medline] [Order article via Infotrieve]
14. Higgins, N. P., Kato, K., and Strauss, B. (1976) J. Mol. Biol. 101, 417-425[Medline] [Order article via Infotrieve]
15. Seigneur, M., Bidnenko, V., Ehrlich, S. D., and Michel, B. (1998) Cell 95, 419-430[Medline] [Order article via Infotrieve]
16. Louarn, J. M., Louarn, J., Francois, V., and Patte, J. (1991) J. Bacteriol. 173, 5097-5104[Medline] [Order article via Infotrieve]
17. McGlynn, P., and Lloyd, R. G. (2000) Cell 101, 35-45[Medline] [Order article via Infotrieve]
18. Viguera, E., Hernandez, P., Krimer, D. B., Lurz, R., and Schvartzman, J. B. (2000) Nucleic Acids Res. 28, 498-503[Abstract/Free Full Text]
19. Michel, B. (2000) Trends Biochem. Sci. 25, 173-178[CrossRef][Medline] [Order article via Infotrieve]
20. Cox, M., Goodman, M., Kreuzer, K., Sherratt, D., Sandler, S., and Marians, K. (2000) Nature 404, 37-41[CrossRef][Medline] [Order article via Infotrieve]
21. Hiasa, H., and Marians, K. J. (1994) J. Biol. Chem. 269, 26959-26968[Abstract/Free Full Text]
22. Horiuchi, T., Hidaka, M., Akiyama, M., Nishitani, H., and Sekiguchi, M. (1987) Mol. Gen. Genet. 210, 394-398[Medline] [Order article via Infotrieve]
23. Dynan, W. S., Jendrisak, J. J., Hager, D. A., and Burgess, R. R. (1981) J. Biol. Chem. 256, 5860-5865[Abstract/Free Full Text]
24. Rivetti, C., Guthold, M., and Bustamante, C. (1996) J. Mol. Biol. 264, 919-932[CrossRef][Medline] [Order article via Infotrieve]
25. Hill, T. M., and Marians, K. J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2481-2485[Abstract]
26. Panyutin, I. G., and Hsieh, P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2021-2025[Abstract]
27. Wang, J. C. (1974) J. Mol. Biol. 89, 783-801[Medline] [Order article via Infotrieve]
28. Waring, M. J. (1965) J. Mol. Biol. 13, 269-282[Medline] [Order article via Infotrieve]
29. Mulrooney, S. B., Fishel, R. A., Hejna, J. A., and Warner, R. C. (1996) J. Biol. Chem. 271, 9648-9659[Abstract/Free Full Text]
30. Lilley, D. M. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 6468-6472[Abstract]
31. Wang, J. C. (1986) in Cyclic Polymers (Semlyen, J. A., ed) , pp. 225-260, Elsevier Applied Science Publishers Ltd., Essex, England
32. Vologodskaia, M. Y., and Vologodskii, A. V. (1999) J. Mol. Biol. 289, 851-859[CrossRef][Medline] [Order article via Infotrieve]
33. Courey, A. J., and Wang, J. C. (1983) Cell 33, 817-829[Medline] [Order article via Infotrieve]
34. Gellert, M., O'Dea, M. H., and Mizuuchi, K. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 5545-5549[Abstract]
35. Panyutin, I., Klishko, V., and Lyamichev, V. (1984) J. Biomol. Struct. Dyn. 1, 1311-1324[Medline] [Order article via Infotrieve]
36. Inman, R. B. (1984) Biochim. Biophys. Acta 783, 205-215[Medline] [Order article via Infotrieve]
37. Defossez, P. A., Prusty, R., Kaeberlein, M., Lin, S. J., Ferrigno, P., Silver, P. A., Keil, R. L., and Guarente, L. (1999) Mol. Cell 3, 447-455[Medline] [Order article via Infotrieve]
38. Horiuchi, T., and Fujimura, Y. (1995) J. Bacteriol. 177, 783-791[Abstract]
39. Cox, M. M. (1998) Genes Cells 3, 65-78[Abstract/Free Full Text]
40. Liu, B., and Alberts, B. M. (1995) Science 267, 1131-1137[Medline] [Order article via Infotrieve]
41. Hiasa, H., and Marians, K. J. (1994) J. Biol. Chem. 269, 6058-6063[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.