J. Mol. Biol. (1991) 220, 8433853

Unidirectional Replication as Visualized by Two-dimensional Agarose Gel Electrophoresis Luis Martin-Parras,

Pablo HernAndez, Maria Luisa Martinez-Robles and Jorge B. Schvartzmant

Centro de Investigaciones Bioldgicas (CIB) Consejo Superior de Investigaciones Cientificas (CSIC) Veldzquez 144, Madrid 28006, Spain (Received 29 January

1991; accepted 26 April

1991)

Two-dimensional (2D) agarose gel electrophoresis is progressively replacing electron microscopy as the technique of choice to map the initiation and termination sites for DNA replication. Two different versions were originally developed to analyze the replication of the yeast 2 pm plasmid. Neutral/Neutral (N/N) 2D agarose gel electrophoresis has subsequently been used to study the replication of other eukaryotic plasmids, viruses and chromosomal DNAs. In some cases, however, the results do not conform to the expected 2D gel patterns. In order to better understand this technique, we employed it to study the replication of the coZEl-like plasmid, pBR322. This was the first time replicative intermediates from a unidirectionally replicated plasmid have been analyzed by means of N/N 2D agarose gel electrophoresis. The patterns obtained were significantly different from those obtained in the case of bidirectional replication. We showed that identification of a complete arc corresponding to molecules containing an internal bubble is not sufficient to distinguish a symmetrically located bidirectional origin from an asymmetrically located unidirectional origin. We also showed that unidirectionally replicated fragments containing a stalled fork can produce a pattern with an inflection point. Finally, replication appeared to initiate at only some of the potential origins in each multimer of pBR322 DNA. Keywords:

pBR322; replication origins; replication termini; replicative two-dimensional agarose gel electrophoresis

1. Introduction

(Liu-Yang

virus

(EBV)

plasmid

maintenance

sequence,

oriP, contains both the initiation and termination sites for DNA replication (Gahn & Schildkraut, 1989) and to show that replication initiates at only some of the potential origins in each oligomeric form of the bovine papillomavirus (BPV) type 1 DNA

1977). It has been shown by electron microscopy that colEl-like plasmids replicate both in vivo and in vitro in a unidirectional manner (Tnselburg, 1974: Lovett et al.. 1974: Tomizawa et al.. 1974). The 843

$03.00/O

et al..

shows extensive functional and structural homologies with coEE1 (Balbis et al., 1986; Bolivar et al..

t Author to whom all correspondence should be addressed. $ Abbreviations used: 2D, two-dimensional; EBV, Epstein-Barr virus: BPV, bovine papillomavirus; N/N, Neutral/Pu’eutral; bp, base-pair(s); kb, lo3 base-pairs. 00~~~836/~1/1~io843--11

1990: Schvart,zman

1990). When Neutral/Neutral (N/N) 2D agarose gel electrophoresis (Brewer & Fangman, 1987) was used to study the replication of other eukaryotic chromosomal DNAs, however, the results obtained were sometimes difficult to interpret and did not conform to the expected 2D gel patterns (Delidakis & Kafatos, 1989; Heck & Spradling, 1990; Krysan & Calos, 1991; Linskens $ Huberman, 1990, Vaughn et al., 1990). In an attempt to solve these apparent ambiguities, to identify any possible artifact that might occur when using this technique a.nd to better understand it, we employed N/N 2D agarose gel electrophoresis (Brewer & Fangman, 1987) to analyze the mode of replication of the rolEI-like plasmid, pBR322. This plasmid vector received its replication origin from the plasmid pMB1 that

Two different two-dimensional (2D$) agarose gel electrophoresis techniques have been recently developed to analyze DNA replication in higher eukaryotes (Brewer & Fangman, 1987; Huberman et al., 1987). These techniques have been successfully used to map the initiation and termination sites for DNA replication in the yeast 2 pm plasmid (Brewer & Fangman, 1987; Huberman et al., 1987), to identify a replication fork barrier in the yeast chromosomal rDNA (Brewer & Fangman, 1988; Linskens & Huberman, 1988), to demonstrate that the EpsteinBarr

& Bot.chan,

intermediates:

0

1991

Academic Press Limit&

844

L. Ma&n-Parras

initiation site has been mapped and sequenced (Selzer et al., 1983; Veltkamp & Stuitje, 1981) and a lot of progress has been made in understanding the mechanism involved in the initiation of DNA replication (Staudenbauer, 1978; Kolter 6 Helinski, 1979; Dasgupta et al., 1987). We have analyzed the replication intermediates of pBR322 DNA, since no plasmid DNA replicating in a unidirectional manner has been studied, so far to our knowledge, using N/N 2D agarose gel electrophoresis. The results confirmed that pBR322 DNA replicates in a unidirectional manner from an origin located between positions 2400 and 2600 on the genetic map. The patterns obtained were significantly different from those obtained in the case of bidirectionally replicated fragments (Brewer & Fangman, 1987). The differences could ‘all be accounted for by the unidirectional manner of pBR322 DNA replication. Recombinants and multimerit forms were also identified. It appeared that only some of the potential origins were utilized in the multimers.

et al.

pBR322 (4363 bp)

. . . ...66.)

s+y1(‘36’

(a 1 A/wNI

AIwNI

I L

AP

ORI

2. Rationale The ciqcular map of pBR322 DNA and the different linear forms obtained with several restriction endonucleases that cleave the plasmid DNA only once are shown in Figure 1. The restriction endonucleases chosen were AZwNI, PstI, .EcoRI, SalI, Sty1 and PvuII. The replication origin would be located at different relative positions in each linear form. Since pBR322 replicates in a unidirectional manner, termination is also expected to take place at this site when the replicating fork approaches the origin. The distance from the left end to the replication origin as a percentage of the length of pBR322 DNA was 8.0% for AZwNI, 246% for P&I, 41.8% for EcoRI, 568% for SalI, 73.3% for Sty1 and 892 y. for PvuII (Fig. 1). The expected replication intermediates produced by progression of the replication fork along the linearized genome of pBR322 DNA in each case are shown in Figure 2. Progression of the replication fork in a unidirectional rightward manner would produce a bubble expanding towards the right end of the linearized plasmid. As soon as the replicating fork reaches the right end of the molecule the bubble would open up leading to a simple branched or simple “Y” form. But as this happens the active fork would appear at the opposite end moving rightward from the left end of the molecule towards the termination site. The emergence of this second branched structure would transform the topology of the replicative intermediate from a simple Y to a double Y. Termination would occur when this fork meets the initiation site.

PstI

PstI

Eco RI

EcoRI

So/I

So/I

sty1

sty1 L ORI

PvuIl

PVUII

I

L

AP

ORI

I

0

I

IO

I

20

I

I

I

I

I

I

I

1

30

40

50

60

70

80

90

100

Percentage of the length of the molecule

(b) Figure 1. Circular and linear genetic maps of pBR322 DNA. (a) The cleavage sites for a number of restriction endonucleases that cut the plasmid only once are shown on the circular map. (b) The different linear forms obtained with each of these restriction endonucleaees. The

position of the replication origin (designated ORI) and the direction of fork progression are indicated by filled black arrows. The position and direction of transcription of the ampicillin and tetracycline resistance genes are indicated by stippled arrow boxes.

Unidirectional

Replication

845

PHI

3IwNI AIwNI IL

EcoRI Psf I

AIwNI

PHI

EcoRI

EcoRI L

----(

ORI

ORI 1.0x

1.0X

Tc

ORI 1.0x

1.6X

I .7x

1.6~

1.7x

I ,9 x 1.9x

;ol I

sty I so/ I

SOlI

Pvull

Tc H

Ap

L

Tc

ORI

ORI I-Ox

PVUII

SfyI

sty1

L

1.0x

H

Ap

PVUI L 1 ORI

I.Ox

Figure 2. Progression of the replication fork along the genome of pBR322 as expected after linearization with different restriction endonucleases. The restriction endonuclease used is indicated at the top of each panel followed by the resulting linear map. The mass of unreplicated and almost completely replicated forms are shown on the left as well as the mass achieved when the fork reaches the right end of the molecule. The continuous black lines represent unreplicated DNA, while thick stippled lines represent replicated segments.

Initiation of DNA replication in the AlwNI linear form, for instance, would lead to an asymmetrically located bubble expanding rightward (Fig. 2). At the time the fork reaches the right end of the molecule, the replicative intermediate would have a mass equivalent to 1.9 x the mass of unreplicated forms, indicating that most of the plasmid has already been replicated.

A similar pattern would be expected for the rep!ication intermediates cleaved with the other five restriction endonucleases (Fig. 2). The main difference among them would be the position along the length of the linear form where initiation of DNA replication takes place. As a consequence, the mass of the replication intermediate at the time the growing fork reaches the right end of the linearized

L. Martin-Parras

846

molecule, referred to as the “switch” point, would also vary. The switch point would occur at a mass equivalent to 1.9 x the mass of unreplicated forms in the case of AZwNI, 1.7 x for PstI, 1.6 x for EcoRI, 1.4 x for SalI, 1.3 x for Sty1 and 1.1 x for PVUII. Simple and double-branched forms as well as molecules containing an internal bubble, can be readily identified using N/N 2D agarose gel electrophoresis (Brewer & Fangman, 1987). In short, DNA molecules are separated predominantly according to their mass in the first dimension, which is run in a low percentage agarose gel at low voltage. The second dimension maximizes the effect of retardation caused by the molecular topology of replicative intermediates. This second dimension is run in a high percentage agarose gel at high voltage in the presence of ethidium bromide (Brewer & Fangman, 1987). According to the diagrams shown in Figure 2, replicative intermediates would increase in mass as molecules containing an internal bubble until the switch point is reached. From this point on and until replication is completed, replicative intermediates would increase in mass as double-branched or double Y forms. Therefore, only two different patterns would be expected in the case of pBR322 DNA digested with restriction endonucleases that produce single cuts. Recombination of pBR322 molecules, however, can occur in some cases (Bedbrook et al., 1979). Digestion of recombination intermediates with restriction endonucleases that cut once per element is expected to generate X-shaped molecules. These X-shaped recombinant forms produce a unique pattern when analyzed by electrophoresis in N/N 2D agarose gels (Bell & Byers, 1983; Brewer et al., 1988). Recombination may lead to a series of oligomeric forms. There is evidence suggesting that multimeric forms of circular plasmids initiate replication at only some of the potential origins (Brewer & Fangman, 1987; Liu-Yang & Botchan, 1990; Nawotka & Huberman, 1988; Schvartzman et al., 1990; Waldeck et al., 1984). As a consequence, after digestion with restriction endonucleases that produce single cuts, those elements of the multimers replicated from an origin initiated elsewhere would lead to a population of simple Y replicative intermediates. If recombination occurs, another two patterns would be expected besides those corresponding to molecules containing an internal bubble and double Ys. These patterns would correspond to non-replicative X-shaped replicative recombinants and simple Y intermediates.

3. Materials and Methods (a) Bacterial

strains

and culture

medium

The Escherichia coli strain used in this study was RYClOO6 (kindly provided by F. Moreno) transformed with pBR322 DNA. Cells were grown at 37°C in LB medium containing 50 pg ampicillin/ml and 125 pg tetracycline/ml.

et al.

(b) Isolation

ofplasmid

DNA

Plasmid DNA isolation was adapted from the method of Clewell & Helinski (1969). Cells from overnight cultures were diluted 40-fold into fresh LB medium, grown at 37°C to exponential phase (A,,, = 94 to 96), quickly chilled and centrifuged. Cells were washed with 20 ml of 99% (w/v) NaCl, harvested by centrifugation and resuspended in 5 ml of 25% (w/v) sucrose, 925 M-Tris.HCl (pH 80). Lysozyme (10 mg/ml) and RNase A (61 mg/ml) were added and the suspension was maintained on ice for 5 min. Afterwards, 2 ml of 0.25 M-EDTA (pH 80) were added and the suspension was kept on ice for another 5 min. Cell lysis was achieved by adding 8 ml of lysis buffer (1% (v/v) Brij-58, 0.4% (w/v) sodium deoxycholate, 9063 M-EDTA (pH 80), 50 mm-Tris.HCl (pH 80)). The lysate was centrifuged at 20,000 g for 60 min in order to pellet the cell DNA and other bacterial debris. Plasmid DNA was recovered from the supernatant and precipitated by adding 2 or 3 volumes of 25% (w/v) polyethylene glycol 6000 and 1.5 M-NaCl. After centrifugation the DNA in the pellet was dissolved in 5 ml of TE buffer (0.01 M-Tris.HCl (pH 80) and 0901 M-EDTA (pB 8.0)) and digested with Proteinase K (100 pg/ml) in 1 M-NaCl, 10 mM-Tris. HCl (pH 90), 1 mM-EDTA and 61 y. (w/v) sodium dodecyl sulphate (SDS), at 65°C for 20 min. Proteins were extracted with 10 mM-Tris. HCl (pH 80). equilibrated phenol, phenol/chloroform/isoamyl alcohol (25 : 24 : 1, v/v) and chloroform/isoamyl alcohol (24 : 1, v/v). The DNA was precipitated with 95% (v/v) ethanol and resuspended in TE buffer. The DNA was digested with restriction endonucleases (Boehringer-Mannheim) as recommended by the manufacturer in the presence of 100 pg RNase A/ml (Boehringer-Mannheim) and 100 U RNase Tl/ml (Pharmacia). (c) Two-dimensional

agarose gel electrophoresis

The 1st dimension was electrophoresed in a 64% (w/v) agarose gel in TBE buffer (0.089 M-Tris-borate, 0902 MEDTA) at 66 V/cm at room temperature for 34 h. The lane containing the lambda DNA/HandIII marker sizes was excised, stained with 0.5 pg ethidium bromide/ml and photographed. The 2nd dimension was electrophoresed in a 1 o/o agarose gel in TBE buffer containing 65 pg ethidium bromide/ml at a 90” angle with respect to the 1st dimension. The dissolved agarose was poured around the excised lane from the 1st dimension and the electrophoresis was carried out at 5 V/cm in a 4°C cold-room using recirculating buffer. (d) Southern

transfer

and hybridization

Gels were twice washed for 15 min in 905 M-HCl, then twice for another 15 min in 0.5 M-NaOH containing 1 ivr-NaCl, followed by another 60 min wash in 1 MTris. HCl (pH 8.0) with 1.5 M-NaCl. The DNA was transferred to Optibind@ Nitrocellulose-supported membranes (Schleicher and Schuell, Inc.) in 10 x SSC (SSC is 915 MNaCl, 0015 M-sodium citrate) for 16 to 18 h and the membranes were baked at 80°C for 2 h. Prehybridization was carried out in 50% (w/v) formamide, 5 x SSC, 5 x Denhardt’s solution (100 x Denhardt’s contains 2% (w/v) bovine serum albumin. 2% (w/v) Ficoll and 2% (w/v) polyvinylpyrrolidone) , 6 1 “/o SDS and 250 pg sonicated salmon testes DNA/ml at 42°C for 16 to 18 h. Membranes were hybridized in 50% formamide, 5 x SSC, 5 x Denhardt’s solution, 250 pg sonicated salmon testes DNA/ml and 10% (w/v) dextran sulphate with 10’ cts

Unidirectional A/W NI

PSlI

Replication

x47 EcoRI

Figure 3. Replicative intermediates of pBR322 DNA as visualized by 2D agarose gel electrophoresis. Plasmid DKA was isolated from exponentially growing bacteria, digested with the indicated restriction endonuclease and analyzed by 2D agarose gel electrophoresis. Afterwards, the DNA in the gels was transferred to an Optibindm nitrocellulose supported membrane and probed with radioactively labeled pBR322 DNA as described in Materials and Methods. per min of probe-DNA labeled with [32P]dCTP by random priming, at 42°C for 24 to 48 h. After hybridization, the membranes were twice washed for 15 min in 2 x SSC and @I y!J SDS at room temperature followed by 2 or 3 washes in til x SSC and @l To SDS at 55°C for 30 min each time. Exposure of XAR-5 films (Kodak) was carried out at -80°C with 2 intensifying screens for 1 to 3 days.

4. Results Plasmid DNA was isolated from exponentially growing bacteria, digested with the restriction endonucleases listed earlier and analyzed by N/N 2D agarose gel electrophoresis (Fig. 3). Precise identification of all the different signals described herein was possible only after examining several autoradiograms exposed for different lengths of time. For convenience, however, a single autoradiogram corresponding to each of the different digestions is shown in Figure 3. The diagrams presented in Figure 4 summarize all the observations. Two different patterns were observed regardless of the restriction endonuclease that was used (Figs 3 and 4). One of these patterns (designated Simple Ys in the EcoRI panel in Fig. 4) corresponded to a population of DNA molecules whose migration in the second dimension was increasingly retarded as they increased in mass until the molecules were 1.5 x the mass of unreplicated forms. Subsequent

increases in mass led to a decrease in retardation and the signal returned to the arc of linear forms with a mass equivalent to double that of unreplicated forms. This pattern corresponded to simplebranched or simple Y forms produced by a single fork traversing the fragment from one end to the other (Brewer & Fangman, 1987). The second pattern (designated X-shaped recombinants in the EcoRI panel in Fig. 4) consisted of a straight line extending nearly upright in a diagonal fashion from a site, on the arc of linear forms with a size of 8.7 kb, double the size of unreplicated linear fragments (Figs 3 and 4). This pattern corresponded to non-replicative X-shaped recombinant, forms where the crossover occurred at different sites along the molecules (Bell & Byers, 1983; Brewer et aE., 1988). Digestion with the restriction endonucleases was not complete and the spot observed in almost every autoradiogram above the arc of linear forms close to the bottom right corner corresponded to open circles (Fig. 3). The three classes of non-linear forms aforementioned: open circles, simple Ys and X-shaped recombinants occurred regardless of the restriction endonuclease that was used. They all served as internal standards against which the variable novel replicating forms can be compared. The maximally retarded arc arising from the 4.3 kb linear spot (designated bubbles in the EcoRI panel in Fig. 4) was due to a population of DKA

L. Martin-Parras st. dimension

et al.

1st. dimension

AlwNI

PstI

.I

X ret

Sol 1

Switch= I-9x Distance to ter ~8.0%

2!5i5r

= 1st. dimension

PVU z.‘IT3 :0Er

Switch=l*7x Distance to ter = 24.6%

Switch = 1.6~ Distance to ter =41*8%

1

1st. dimension

1st. dimension

4.3 kb \ Switch= 1.4~ Distance toter

1

Switch-1*3x Oistonce tc tar-73.2%

~56,8%

Switch-l-lx Distance to ier =89*2%

Figure 4. Diagrammatic interpretation of the autoradiograms. The diagonal arc running across each diagram (marked linear forms in the EcoRI panel) represents linear DNA molecules. Drawn on these arcs as filled circles are the 4.3 kb (1.0 x or unreplicated) and the 87 kb (2.0 x or almost completely replicated) linear forms of the pBR322 DNA molecule. Black continuous thick lines represent molecules containing an internal bubble, horizontally hatched lines indicate simple Y forms, stippled lines indicate double Y forms and black broken lines represent non-replicative X-shaped recombinants. All these forms are specifically marked in the EcoRI panel. The thickness of the lines does not indicate the relative abundance of each particular kind of molecules in the autoradiograms. Those forms occurring regardless of the restriction endonuclease that was used are represented by thin lines. Thicker lines, on the other hand, represent forms that changed depending on the restriction endonuclease that was used to linearize the plasmid DNA. The mass of the replicative intermediate at the switch point (indicated switch) and the distance from the left end to the site where termination takes place as a percentage of the length of the linearized plasmid (indicated distance to ter) are shown at the bottom left corner in each frame. molecules whose migration in the second dimension retarded as they increased in size.

was increasingly This maximally

retarded

arc did not return

to the

curve of linear forms but ended at different retarded positions depending on the restriction endonuclease that

was

used

(Figs

3 and

4). For

instance,

it

extended maximally in the case of plasmid DNA linearized with AlwNI and the length of the signal decreased as the restriction site was displaced clockwise (Fig. 1). Two different features strongly support that this maximally retarded arc corresponded to molecules containing an internal bubble. First, it has been shown that molecules with an internal bubble migrate more slowly in the second dimension of a N/N 2D agarose gel system than either the simple Y or the double Y forms (Brewer et al., 1988). Second, the length of this maximally

retarded

arc diminished

as the restriction

site was

moved clockwise from the AlwNI site (Figs 3 and 4). The diagrams shown in Figure 2 predict that this is precisely the pattern expected for replicative intermediates that initiate DNA replication at different where the replication fork relative positions progresses rightward in a unidirectional manner. Another signal (designated Double Ys in the EcoRI panel in Fig. 4) also varied depending on the restriction endonuclease that was used (Figs 3 and 4). It was not visible in the case of AlwNI-digested DNA, but it became increasingly prominent as the restriction site was moved clockwise (Fig. 1). This signal started at different positions on the arc of simple Y forms and extended leftward as a straight line in a diagonal fashion in P&I, EcoRI and SalIdigested DNAs. In those cases where Sty1 or PvuII

Unidirectional

was used to linearize the plasmid, however, the signal also started as a straight line, but it bent down at a certain point towards the arc of linear forms. This was particularly evident in the case of PvuII-digested DNA. Not only did these signals start at different positions on the arc of simple Y forms, but they all extended up to the line of X-shaped recombinants. The position along the line of recombinants where these signals ended also varied depending on the restriction endonuclease that was used. For PvuII-linearized plasmid DNA the signal finished very close to the arc of linear forms. For EcoRI and MI-digested samples, it ended almost at, the top of the line of recombinants, where the maximally retarded X-shaped forms had migrated. Finally, for PstI and StyI-digested samples, t,he signal ended approximately in the middle of the line of recombinants. These patterns were interpreted as due to a population of doublebranched or double Y forms. All of them showed an interesting common feature, they started at a specific spot on the curve of simple Y forms and the intensity of the signal increased progressively at the other end. Identification of recombinant forms strongly suggested that multimeric forms of the plasmid could exist, in these E. coli transformed cells. This possibility was tested by analyzing undigested plasmid DNA by means of N/N 2D agarose gel electrophoresis. As already shown for the yeast 2 pm plasmid (Brewer & Fangman, 1987; Brewer et aE., 1988) and for BPV-1 DNA (Schvartzman et aE., 1990), N/PI’ 20 agarose gel electrophoresis can also be used to identify covalently closed circles (CCC), open circles (OC) and linear forms (L) as well as different forms of catenanes. The results obtained (data not shown) indicated that pBR322 DNA occurred, indeed, as monomers, dimers and trimers. RYCZOOO bacteria are recA -. We have used a recA - strain to limit’ the frequency of recombination. The presence of some recombinant intermediates and oligomeric forms, however, was not completely unexpected as other general recombination systems are known to be operative in E. coli (hells (Weinstock, 1987).

5. Discussion The principal aim of the present work was to study the different patterns observed when replicative intermediates from a unidirectionally replicated plasmid were analyzed by means of N/N 2D agarose gel electrophoresis. The results obtained are in good agreement with what is already known about the replication of pBR322 DNA (Dasgupta et al., 1987; Inselburg, 1974; Kolter & Helinski, 1979; Lovett et al., 1974; Selzer et al., 1983; Staudenbauer, 1978; Tomizawa et al., 1974; Veltkamp & Stuitje, 1981). In addition, several new important observations were made concerning the behaviour of DNA replicative intermediates during N/N 2D agarose gel electrophoresis as well as in relation to the replication mode of pBR322 DNA.

Replication

(a) The different with pBR322

unidirectional approximately

849

patterns obtained were consistent DNA being replicated in a manner from an origin located between positions 2400 and 2600

The patterns obtained after digestion with restriction endonucleases that cleaved the plasmid DNA only once at different positions around the molecule (Fig. 1) allowed us to map the location of the site where initiation and termination of pBR322 DNA replication takes place. As the arc corresponding to molecules containing an internal bubble became shorter, the signal corresponding t,o doublebranched forms increased in length. This was particularly evident in the top panels shown in Figure 3. In the other three cases, this correlation could be detected only after shorter exposures (data not shown). This correlation was similar to the case of molecules containing an asymmetrically located origin where replication progresses in a bidirectional manner (Brewer & Fangman, 1987). In that case, molecules containing an internal bubble are converted to simple Y forms when one fork reaches the end of the molecule. The position of the transition or switch point in the autoradiograms can actually be used to estimate the location of the site where initiation of Dr\iA replication took place (Brewer et al., 1988). Precise determination of the mass of the replicating molecules at this switch point is difficult, however, as even in low percentage agarose gels electrophoresed at very low voltages, migration of DNA molecules is not strictly proportional to mass. As a consequence, linear and nonlinear forms of the same mass can migrate to different extents. As already pointed out by Brewer and co-workers (Brewer et al., 1988), this effect’ of molecular topology on migration in the first dimension of an N/PI; 2D agarose gel system can be clearly noted in the pattern generated by X-shaped recombinant forms. The deviation from perpendicularity is due to the slight retardation caused by molecular topology during the first dimension (Hell & Bpers. 1983; Brewer et al.. 1988). Nonetheless, the switch point, could be identified in some of the autoradiograms shown in Figure 3. For instance, in PstI-linearized plasmid DNA. this switch point occurred when the mass of the replicating molecules was approximately 1.75 x that of unreplicated molecules. In EcoRI-digested samples, the switch point occurred when the mass of the replicative intermediates was about 160 x that of unreplicated forms (Figs 3 and 4). These relative masses were calculated by comparison wit’h the distance migrated by molecular weight standards that were run in a parallel lane during the first dimension. In order to achieve these masses, the fork would have moved 3.2 kb in the case of PstI and 2.6 kb in the case of EeoRT-digested samples. Therefore, initiation of DNA replication occurred somewhere between positions 2400 and 2600 on the genetic map. These results are consistent with electron microscopy, genetic and DNA sequence studies indicating that initiation of DNA replication in

850

L. Martin-Parras

et al.

pBR322 DNA takes place close to position 2535 on the genetic map (Bolivar et al., 1977). (b) Identi$cation of a complete arc produced by molecules containing an internal bubble is not sujkient to distinguish a symmetrically located bidirectional origin from an asymmetrically located unidirectional origin In the case of AlwNI-digested DNA, the pattern obtained for molecules containing an internal bubble (Figs 3 and 4) was not significantly different from that generated by molecules containing a symmetrically located origin where replication proceeds in a bidirectional manner (Brewer & Fangman, 1987). This result means that molecules of identical intermediate masses containing an internal bubble migrate in a very similar fashion regardless of the position of the bubble along the molecule. This observation pointed out that identification of a complete arc produced by molecules containing an internal bubble is not sufficient to infer the presence of a symmetrically or asymmetrically located origin of replication. Additional experiments are required to demonstrate whether replication proceeds from this origin in a unidirectional or bidirectional manner.

(0) AIwNI ORI

AIwNI AIWNI

AIwNI Tc

H

AP

ORI 1.0x

(c) Initiation of DNA replication does not appear to take place in all the origins of each oligomeric form of pBR322 DNA Two patterns were common to all the autoradiograms regardless of the restriction enzyme used to linearize the plasmid. These patterns corresponded to non-replicative X-shaped recombinants and simple Y replicative intermediates (Figs 3 and 4). Simple Y forms are generated by a single fork traversing a DNA molecule from one end to the other (Brewer & Fangman, 1987). The presence of simple Y forms indicated that some pBR322 DNA molecules, 4.3 kb long, were replicated by a single fork initiated elsewhere. There is evidence suggesting that multimeric forms of circular plasmids initiate replication at only some of the potential origins (Brewer & Fangman, 1987; Liu-Yang & Botchan, 1990; Nawotka & Huberman, 1988; Schvartzman et al., 1990; Waldeck et al., 1984). We detected covalently closed circles corresponding to dimeric and trimeric forms of pBR322 DNA in undigested samples of the DNA used in this study (data not shown). If only one origin was used in each dimeric form, each element of the dimer would replicate in a unique way (Fig. 5). One element would produce a pattern identical to that generated by the monomers, while the other element would always be replicated by a single fork moving across the molecule from one end to the other. Replication of this second element would generate a population of simple Y forms regardless of the restriction enzyme used to linearize the plasmid. Simple Y forms with the mass achieved at the switch point would be accumulated in each case, while the fork is

1*9x 1.9x

2.0x

(b) Figure 5. A possible mode for the replication of oligomerit forms of pBR322 DNA. (a) The circular genetic map of a pBR322 DNA dimer organized in a head-to-tail configuration. The cleavage sites for the same restriction endonucleases that cut the monomer DNA only once are indicated. (b) The diagram shows the progression of the replication fork along this dimeric form as expected after digestion with AZwNI and assuming that initiation of DNA replication took place at only 1 of the 2 potential origins.

replicating the second element of the dimer (Fig. 5). The signal corresponding to double-branched forms started always on the curve of simple Y forms at a prominent spot (Figs 3 and 4). The presence of such spots, readily identified in the PstI panel in Figure 3

Unidirectional and only after lower exposures in the other cases, and the fact that their relative position changed depending on the restriction endonuclease used to linearize the plasmid, indicated that molecules with the size and topology achieved at the switch point were preferentially accumulated in every case. These observations strongly suggest that DNA replication initiates at only some of the potential origins in each oligomeric form of pBR322 DNA. The intensity of the signal corresponding to molecules containing an internal bubble was significantly weaker than that produced by simple branched forms (Fig. 3). There are several possible explanations for this difference. It is possible that multimerit forms were preferentially replicated. This is unlikely, though, as it would have led to the disappearance of monomeric forms. Another possibility is that molecules containing an internal bubble were preferentially degraded either before or during electrophoresis. Due to the partial single-stranded nature of the DNA at the forks, the occurrence of otherwise undetectable nicks would completely change the topology of replicative intermediates (Linskens & Huberman, 1990). Double-branched forms and molecules containing an internal bubble are expected to be twice as sensitive as simple Ys to these nicks. Nicking of these replicative intermediates at one fork would lead to a population of simple branched forms. Finally, nicking of the recombinant intermediates at the recombination site could also lead to a population of simple Y forms that would be indistinguishable from simple branched replicative intermediates. (d) The pattern generated by double-branched forms ended at different positions along the signal of recomb&ants depending on the location of the termination site along the molecule The mobility of X-shaped recombinant forms progressively decreases as the crossover site is displaced from either end to the center of the molecules (Bell & Byers, 1983; Brewer et al., 1988). The recombinant form with the slowest mobility contains a crossover precisely at the midpoint, causing this molecule to have four arms of equal size. The topology of replicative intermediates that have almost terminated DNA replication, but where the sister molecules are still held together, should be similar to those of X-shaped recombinant forms. Their mobility, hence, should also change depending on the site along the molecule where termination of DNA replication takes place. Our data revealed that the pattern corresponding to double-branched forms ended at, different positions along the line of X-shaped recombinants depending on the restriction endonuclease used to linearize the plasmid DNA (Figs 3 and 4). In the case of AlwNI and PvuII-digested samples, termination of DNA replication was expected to occur at a site very close to either end of the molecule (Figs 1 and 2). The percentage of the length of the molecule, from left to right, where termination would have occurred, was

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CkOy0 in the case of AlwNI and 892 y0 in the case of PvuII. No signal corresponding to double Y forms was identified in the case of plasmid DNA digested with AlwNI (Figs 3 and 4). This was probably due to the coincidence of several different signals in the region of the autoradiogram where this pattern should have been detected. In the case of PvuIIdigested DNA, the signal corresponding to double Y forms started at a very low mass and finished very close to the site where linear molecules with a mass equivalent to 2.0 x that of unreplicated molecules had migrated (Figs 3 and 4). When the plasmid DNA was digested with P&I or StyI, this pattern ended approximately at the mid-portion of the line of X-shaped recombinants. In these cases termination was expected to occur at a position located 24.6 ye and 73.2 ye of the length of the molecule from left to right, respectively (Figs 1 and 2). In those cases where plasmid DNA was digested with EcoRI or SalI, the signal corresponding to double Y forms ended at or very close to the site where the maximally retarded recombinants had migrated (Figs 3 and 4). Termination of DNA replication in these cases was expected to occur at a site located 41.8% and 568% of the length of the molecule from left> to right, respectively (Figs 1 and 2). The intensity of the signal corresponding to molecules containing an internal bubble, in the case of AlwNI-digested DNA and double Y forms in all the other cases, was progressively stronger at the end (Figs 3 and 4). This observation suggests that the rate of fork progression slowed down as the fork approached the termination site. This slowdown would cause a relative accumulation of replicative intermediates with masses close to 2.0 x that of unreplicated forms. (e) The pattern generated by DNA molecules containing an asymmetrically located terminus in a unidirectionally replicated plasmid can have an in.ection point In those cases in which plasmid DNA was digested with Sty1 or PvuII, as the double-branched forms increased in mass their mobility decreased until an intermediate point in which this relationship was reversed. This caused an inflection in the signal. In both cases, the double-branched molecule with minimum mobility appeared to have a mass between 1.5 x and 2.0 x that of unreplicated molecules. Moreover, both the mass and complexity of the molecule with minimum mobility appeared to be higher in Sty1 than in PvuII-digested samples. Molecules that are already half-replicated at the time their switch point is reached could only increase their topological complexity as the moving fork enters the other end. On the contrary, molecules that are less than half-replicated when the switch point is reached could still go through a minimum mobility during completion of replication. The mass of the molecule with minimum mobility would depend upon the position of the replication origin with respect to their switch point. As the

L. Martin-Parras moving fork appears at the left end moving rightward it is essentially a modified simple Y. The mass of the molecule at the inflection point is now influenced by the constant size of the branch. The larger the branch, the more displaced the inflection point would be toward the 2.0 x position. Minimum mobility would be achieved when the moving fork reaches the midpoint of the molecule. In the case of an almost linear fragment, minimum mobility would be achieved when the mass of the replicative intermediate is slightly higher than 1.5 x that of the unreplicated form. If the stationary branch is located at the midpoint, on the other hand, the replicative intermediate with minimum mobility would have a mass of 2-Ox that of unreplicated forms. In this instance, in the case of StyI-digested DNA, the mass of the molecule with minimum mobility would be approximately 1.7 x that of unreplicated molecule, while it would be 1.6 x when PvuII was used to linearize the plasmid. Bending of the signal corresponding to double Y forms towards the arc of linear molecules was also observed by others (Khatri et al., 1989; Krysan & Calos, 1991). Khatri and co-workers (1989) used N/N 2D agarose gel electrophoresis to study the progression of replication forks in vitro around two recombinant plasmids carrying the E. coli terminator sequence, tus. The TER protein specifically binds to the tus DNA sequence, thereby impeding further unwinding of the template DNA in a polar dependent manner (Khatri et al., 1989; Kuempel et al., 1989). It was found that due to the stalling of one fork at the tu.s sequence, progression of the other fork around the plasmids leads to a hookshaped arc of double-branched forms similar to that observed in the present study, in the case of Styl-

digested pBR322 DNA (Fig. 3). We are grateful to Bonita Brewer, Veena Dhar, Joel Huberman and Carl Schildkraut for helpful discussions and comments on the manuscript. This work was supported by grant no. PB87-9468 from the Comision Interministerial de Ciencia y Tecnologia (CICYT). L.M.P. is the recipient of a predoctoral fellowship from the Ministerio de Education y Ciencia (MEC). This work was initiated while J.B.S. was a grantee under the Fullbright/ Spanish Ministry of Education and Science (MEC) program at the Albert Einstein College of Medicine, New York, U.S.A. References Balbas, P., Soberon, X., Merino, E., Zurita, M., Lomelli, H., Valle, F., Flores, N. & Bolivar, F. (1986). Plasmid pBR322 and its special-purpose vector derivatives-a review. Gene, 50, 3-46. Bedbrook, J. R., Lehrach, H. & Ausubel, F. M. (1979). Directive segregation is the basis of coZE1 plasmid incompatibility. Nature (London), 281, 447-452. Bell, L. & Byers, B. (1983). Separation of branched from linear DNA by two-dimensional gel electrophoresis. Anal. Biochem. 130, 527-535. Bolivar, F., Betlach, M. C., Heyneker, H. L., Shine, J., Rodriguez, R. L. & Boyer, H. W. (1977). Origin of replication of pBR345 plasmid DNA. Proc. Nut.

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Vaughn, J. P., Dijkwell, P. A. & Hamlin, J. L. (1990). Initiation of DNA replication occurs in a broad zone in the DHFR gene locus. Cell, 61, 1075-1087. Veltkamp, E. BE Stuitje, A. R. (1981). Replication and structure of the bacteriocinogenic plasmids Clo DF13 and colE1. Plasmid, 5, 76-99. Waldeck, W., Rosi, F. & Zentgraf, H. (1984). Origin of replication in episomal bovine papilloma virus type 1 DNA isolated from transformed cells. EMBO J. 3, 2173-2178. Weinstock, G. M. (1987). General recombination in Escherichia coli. In Escherichia coli and Salmonella and Molecular Biology typhimurium Cellular (Neidhardt, F. C., Ingraham, J. L., Low, K. B.. Magasanik B., Schaechler, M. & Umbarger, H. E., American Society for pp. 1034-1043, eda), Microbiology, Washington, DC.

by K. R. Yamamoto