MolGenGenet (1981) 184:450-456

© Springer-Verlag 1981

Multiply Branched Replicative Intermediates in E. coli and Bacteriophage 2

Manuel S. Valenzuela and Ross B. Inman

Biophysics Laboratory and Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA

Summary. Multiple branched DNA fragments present in a fast sedimenting complex comprising a minute fraction of the E. coli genome have been isolated. Similar structures were also observed among bacteriophage ). DNA replicative intermediates after in- fection of synchronized E. coli cells. These structures were found to be associated with the amino acid and thymidine starvation steps required for synchronization and originate either by initia- tion from secondary sites or by snap-back of daughter strands containing substantial single stranded regions in the vicinity of the growing point.

Introduction

When gently lysed E. eoli cells are treated with a restriction enzyme, the bacterial DNA can be separated into two compo- nents after fractionation through a sucrose gradient. The major slow sedimenting component consists of short DNA fragments resulting from the action of the restriction enzyme. However, between 1-5% of the D N A sediments as a fast component be- cause of binding to membrane material. Inspection of these com- ponents in the electron microscope (after further purification of the DNA by detergent, pronase and CsC1 equilibrium centrifu- gation) shows that the fast sedimenting material is enriched for branched DNA fragments (Valenzuela and Inman 1978). About 55% of these molecules are single branched or fork structures, 40% are multiply branched and the remaining 5% are double branched or eye structures. We have recently presented evidence indicating that single branched fragments represent DNA grow- ing forks excised from replicative intermediates by the action of the restriction enzyme (Valenzuela et al. 1981). In this report we will examine several properties of the multiply branched struc- tures and determine what conditions are needed for their forma- tion. We will also show that under appropriate conditions similar complex branched molecules can be found among intracellular bacteriophage 2 replicative intermediates.

Materials and Methods

a) Bacterial and Phage Strains

E. coli K12 strain CR34 (thr, leu, thy) and its derivative CR34A1 (Valenzuela and Inman 1978) were used in this study. The phages 2cII6sCIII67 and 2vir gals 0ts were obtained from Dr. W. Dove and were propagated on strain CR34. 3H-labeled 2 DNA was

Offprint requests to ." M.S. Valenzuela

prepared by growing the phage in the presence of [3H-methyl]thy- midine. The radioactive phage was pelleted by centrifugation and resuspended in 0.01 M Tris, pH 7.3, 0.01 M MgSO~ in D20.

b) Isolation of E. coli DNA Fragments

The methodology of Valenzuela et al. (1981) was followed.

c) Isolation of lntracellular )~ DNA

This method differs from that previously employed (Schn6s and Inman 1970) in that the bacteria were starved for amino acids and thymidine over extensive periods prior to phage infection. E. coli CR34 was grown at 37 ° C in 80 ml of D-maltose medium (Ogawa et al. 1968) supplemented with leucine, threonine, B1 and thymidine (each at 10 gg/ml final concentration) and yeast extract (0.02% final concentration). At OD590=0.6, the cells were centrifuged, washed twice with 40 ml of 0.01 M Tris, pH 7.3, 0.01 M MgSO4 in D20 and resuspended in 40 ml of D-maltose medium plus B1 and thymidine. Cultures were incu- bated at 37°C for 3 h, cells pelleted, washed as above, and resuspended in 40 ml D-maltose medium which contained all the supplements minus thymidine (yeast concentration was re- duced to 0.01%). After 3 h at 37 ° C, cells were again pelleted, washed, resuspended in 3 ml 0.01 M Tris, pH 7.3, 0.01 M MgSO4 in D20 and infected with 3H 2 phage at a multiplicity of six. After adsorption at 37 ° C for 10 min, 1 ml was added to 10 ml of warmed D-glucose medium containing all supplements and incubated at 37°C for 5 min (sample 1). The remainder was added to 20 ml of D-glucose medium with all supplements minus thymidine (yeast concentration reduced to 0.01%) and incubated at 37 ° C for 27 min. At this point, an 11 ml aliquot was taken (sample 2). To the remainder, thymidine (10 gg/ml) was added and the yeast extract concentration increased to 0.02% and the culture further incubated for 5 min at 37°C (sample 3). Each of the three samples was then poured onto 12 ml of cold 0.05 M KCN, 0.05 M NAN3, 0.01 M EDTA and 8 g crushed ice. After 2 min at 4°C the cells were centrifuged and resuspended in 2 ml of 0.05 M KCN, 0.05 M NAN3, 0.01 M EDTA. Each sus- pension was then frozen and thawed three times. 30 gl of a 50 mg/ml solution of lysozyme in water was added and incubated at 4 ° C for 20 rain. Then 0.1 ml of 20 mg/ml pronase in 0.005 M EDTA was added and incubated at room temperature for 60 min, followed by incubations with 40 Itl of 30% sarkosyl (15 min at room temperature) and 0.1 ml pronase (2 h at 37 ° C). The lysate was then adjusted to a density of 1.67 with solid CsC1 and centrifuged at 30,000 rpm for 3 days at 9 ° C in a 60 Ti rotor. The gradient was fractionated from top to bottom, at

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a rate of 0.35-0.7 ml/min into 0.2 ml fractions. Fractions with densities between heavy-light and light-light were pooled, mixed with a CsC1 solution with a density of 1.67, centrifuged and fractionated a second time. The resulting D N A fractions with densities between heavy-light and light-light were inspected in the electron microscope.

d) Electron Microscopy and Computation of Electron Microscope Data

The methodology described by Valenzuela and Inman (1975) was followed.

Results

Experiments with E. coli

Multiple branched E. coli D N A fragments were isolated as de- scribed previously (Valenzuela et al. 1981). These molecules rep- resent about 40% of all branched structures observed and have the general configuration shown in Fig. 1 a; namely, a forked

(-'

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Fig. 1 a, b. Two possible configurations for a multiply branched struc- ture. The three arms of the fork should consist of two homologous segments (thin lines) and a non-homologous region (thick line). The eye can, therefore, lie on the non-homologous arm (as in a) or on one of the homologous segments (as in b)

molecule made of three arms with one arm containing an eye structure. In about 70% of these molecules only one eye is present, 20% show two or more eyes arranged in tandem, and the remaining 10% show a D-loop structure (an eye in which one of the branches is completely single stranded), or a fork instead of the eye.

Multiple branched D N A fragments were photographed at random and a collection of 30 such molecules that had been spread under neutral pH conditions was analyzed. The striking feature in all these molecules is that the eye appears to be located adjacent to the fork junction (Fig. 2). The D N A segment separat- ing them ranges from 0.02 to 1.0 microns, with an average length of 0.2+0.2 microns. The size of the eye is in most cases small; it ranges from 0.1 to 1.4 microns, with an average length of 0.5 _+0.4 microns. In the cases where more than one eye is pres- ent, they are always arranged in tandem on a single arm of the fork and found close to the fork junction. Close inspection of these molecules shows that in most cases, the fork is made up of three arms of unequal length and that one of the arms shows a single strand connection to the fork junction; moreover, this arm is never the one that contains the eye. Both branches that form the eye structure are of equal length, and are predomi- nantly double stranded but a single strand connection can often be seen at one of the junction points (Fig. 2).

The three arms of the fork in a multiply branched molecule should consist of one non-homologous and two homologous segments. Thus multiply branched structures can have one of the two configurations shown in Fig. 1. The eye could be situated on the non-homologous branch (Fig. 1 a) or on one of the homol- ogous segments (Fig. l b). We have analyzed 27 multiply branched fragments by partial denaturation mapping to answer two questions. First, we wished to be sure that branch points do, in fact, occur at the junction between one non-homologous and two homologous duplex strands. Second, we wished to deter- mine which of the two possibilities shown in Fig. 1 best charac- terized these complex branched structures.

Fig. 2. Electron micrograph of a multiply branched E. coli DNA fragment. The three arms of the fork are labelled (a, b and c) and arm (a) contains an eye structure. Branch points are indicated by the arrows and the bar represents 0.2

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Figure 3 shows the partial denatura t ion pat tern of 27 multiply branched fragments. The majori ty of these structures consist of a fork and an eye, however, 5 units contain two eyes (numbers 2, 15, 19, 20 and 25) and two have a D-loop rather than an eye (numbers 7 and 27). Inspection of the denatura t ion patterns of the individual units shown in the figure allow three conclusions. First, it is apparent that each fork consists of one non-homologous and two homologous arms. The paired homo- logus segments (identified by closely matching denatura t ion pat- terns) are drawn to the right of each branch point in the figure. Because of the small size of the eye structures, there are not enough denatured sites in these regions to confirm that they also consist of homologous sections, but we assume that this is true. Second, we can be sure (on the basis of the denatura t ion patterns), tha t eyes are, without exception, located on the non- homologous arms of forks. This is true even when two eyes

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Fig. 3. Diagrammatic representation of partially denatured multiply branched E. coli DNA fragments. The junction points present on each molecule are represented by vertical lines. All the molecules have been aligned with respect to the junction point of the fork for display purposes only. Denatured sites are represented by filled rectangles. The sites appear to match between the paired segments shown to the right of the junction point of each fork. Open boxes to the left of the fork represent eye structures. In some instances (molecules 7 and 27) the bottom part of the box is represented by broken lines. This indicates that one of the branches is fully single stranded. These probably represent D- or R-loop structures. Other single strand regions elsewhere on the molecule have been omitted for clarity. Each division on the scale represents 3 microns

are present. Of the two possible configurations shown in Fig. 1, we therefore know that the observed multiply branched struc- tures correspond to configurat ion (a) ra ther than (b). Third, on the basis of the denaturat ion patterns of the set of molecules shown in Fig. 3, we can be reasonably certain that the fragments do not arise from a unique section of the chromosome. Al though we see matching denatura t ion patterns between two arms of each unit, we do not see significant matching between different units. Different regions of the genome are therefore represented in this collection of molecules.

Al though the findings reported above come from synchron- ized E. eoli cells tha t have been allowed to replicate for about 3 rain in heavy medium, we have noticed that even after 60 rain replication in this medium one can still observe a high propor t ion of multiply branched DNA. The doubling time of the cells in heavy medium is about 2-3 h. These structures therefore do

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Fig. 4. a Electron micrograph of a multiply branched 2 replicative intermediate. This particular circular replicative intermediate contains two eyes and a tail. The five branch points are indicated by numbers and correspond to those shown in the simplified drawing (b). The circular component is 17 g long

not seem to be confined to the very early stages of a round of replication. Since the cells were starved for amino acids and thymidine in order to synchronize DNA replication, it is possible that the multiple branched fragments are caused by these starva- tion steps. Two independent observations suggest that this may be the case. First, DNA fragments isolated from exponentially growing cells which have not been starved for amino acids and thymidine show 6% branched structures but none are multiply branched, and second, multiple branched structures are also ab- sent in DNA fragments isolated from a dnaA mutant in which DNA synchronization has been achieved by temperature shift rather than by amino acid and thymidine starvation (Santanu Dasgupta, personal communication).

Experiments with Bacteriophage 2

E. coli CR34 cells were grown in heavy medium and starved in this medium for amino acids and thymidine respectively. Five

min after infection with bacteriophage 2cIIcIII in a thymidine containing medium, viral DNA was isolated and purified by CsC1 centrifugation. Electron microscopic analysis of fractions containing viral DNA, showed that 64% of all circular DNA molecules were either simple theta or sigma replicative intermedi- ates. In this respect the results are similar to earlier data (Schn6s and Inman 1970; Valenzuela et al. 1976). The origin of replica- tion, estimated from 36 bidirectional molecules, was positioned at 13.8 +0.4 I~ and is not significantly different from the earlier estimates. In addition 6% contained multiple branches. When similar experiments were carried out without extensive amino acid and thymidine starvation, multiply branched structures were not detected (Schn6s and Inman 1970; Valenzuela et al. 1976). Multiply branched structures consisted of a circle with two (or in some cases 3) eyes, or circles containing eye(s) and an addition- al tail. Figure 4 shows an example of the latter type of molecule. If the infected cells were incubated in the absence of thymidine for 28 min in heavy medium, replicative intermediates could still

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Table 1. Intracellular 2 DNA isolated after infection of synchronized E. coli cells

Treatment a Percent of circular structures

Simple replicative intermediates

Theta Sigma

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(1) 5 min incubation in presence of thymidine 46 18 6

(2) 28 rain incubation in absence of thymidine 8 18 1

(3) Same as (2) with an additional 5 min incubation in the presence of thymidine 8 23 11

CR34 was grown in D-medium and then starved for amino acids and thymidine respectively. Cells were then resuspended in adsorp- tion medium and phage added at a m.o.i, of 6 and incubated 10 rain at 37 ° C. Infected cells were then resuspended in D-medium as de- scribed in the Table

be isolated (26 %), but the propor t ion of multiple branched struc- tures dropped to about 1%. Presumably replication proceeded in this case because of low levels of thymidine present in the yeast extract. However, if thymidine was then added to the medi- um and further incubated for 5 minutes, the propor t ion of multi- ple branched molecules increased to 11% whereas normal repli- cative intermediates amounted to 31% (Table 1). The various t reatments shown in Table 1 also resulted in a changed ratio of simple theta and sigma replicative structures.

Figure 5 shows 26 multiply branched circular 2 structures which have been aligned on the basis of their partial denatura t ion patterns. Many of these molecules (numbers 1 18, 26) have eye or tail segments which span or partially span the origin of replica- t ion (13.7_+0.24 g), or are within twice the experimental error of this region (numbers 19-21); however, several (numbers 22 25) do not conform to this general pattern. Except for one mole- cule (number 26), the eye or tail segments do not overlap but occur at separate regions of the genome. Molecule 26 has an unusual disposition of branch points; more molecules of this type will have to be studied before conclusions can be drawn about the relevance of such a structure. Generally, therefore, we find that double or triple eye structures occur close together and it is likely that one of the eyes span the origin of replication.

Next, we decided to investigate the effect tha t a muta t ion in a gene known to be necessary for init iat ion of 2 D N A replica- t ion (gene O) would have on the format ion of multiple branched 2 D N A molecules. To this end, synchronized E. coli cells were infected with 2vir galaOt+ and D N A isolated following the same protocol used for AcIIcIII, except tha t whenever the infected cells were deprived of thymidine the temperature was also raised to 42 ° C. Multiply branched structures and normal replicative intermediates were not found at the non-permissive temperature even in the presence of thymidine. These results confirm earlier results by Inman and Schn6s (1973). If the infected cells were incubated at 42 ° C for 30 min in the absence of thymidine, then transferred to 32 ° C and thymidine added for 10 min, the propor- t ion of normal replicative intermediates was 15 % but no multiply branched D N A molecules were observed. The simplest conclu- sion we can draw from these experiments is tha t an active O gene is required to generate multiple branched 2 D N A molecules

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and that this activity has to be present at early times after infec- t ion of synchronized E. coli cells.

D i s c u s s i o n

When E. coli, which has been synchronized by successive amino acid and thymidine s tarvat ion steps (Louarn et al. 1974) is gently lysed, the resulting D N A sediments as a fast DNA-membrane material complex. After digestion with the restriction enzyme

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EcoR1, most of the D N A is released from the complex but the remaining fast sedimenting material is 'found, after further purification, to be enriched for branched fragments compared with the slowly sedimenting fragments released by the restriction enzyme (Valenzuela and Inman 1978; Valenzuela et al. 1981). A significant proportion of these branched fragments have an unexpected multiply branched appearance consisting of one or more eye structures on one arm of the fork (Figs. 1 and 2). Evidently these complex structures are associated with the amino acid and/or thymidine starvation steps because if the experiment is repeated without starvation or if synchronization is achieved by temperature shift of a dnaA mutant we do not observe multi- ply branched structures but rather single forked or eye fragments. Partial denaturation mapping shows that in the multiply branched structures the eyes are always located on the non- homologous arm of the fork. The other two arms are found to be homologous and never contain eye structures.

When ,I. replicative intermediates are isolated after infection ofunstarved cells one observes simple theta or sigma replicative intermediates. If, however, starved cells are infected, then a sig- nificant proportion of the intracellular phage D N A is found to also have a complex branched structure.

Two quite different preparative procedures led to the observa- tion that amino acid and thymidine starvation lead to multiply branched structures. In the case of the E. coli experiments, the fragments were isolated from a fast sedimenting membrane com- plex whereas for 2 they were isolated by virtue of a density shift. Evidently, extensive starvation of E. coli alters the mode of replication of the bacterial genome, and of infecting ), to produce multiply branched structures. Two models can be pro- posed to explain these observations.

The starvation conditions might lead to an over-abundance of the proteins needed for initiation of replication. When replica- tion is allowed, these proteins then cause initiation at secondary sites as well as at the normal position. According to this view, and the results shown in Figs. 3 and 5, the secondary sites are usually formed at positions close to already active growing points.

Possible activation of secondary origins due to changes in growth conditions have been observed in both procaryote and eucaryote systems. Perlman and Rownd (1976) have shown that under conditions of reduced thymine concentration, plasmid RF100.1 appears to show more than one active origin of replica- tion in a P. rnirabilis host, whereas other investigators have found that this plasmid shows one single origin in an E. coli host in which the cells were synchronized for D N A replication and a normal concentration of thymine was present during plasmid replication (Silver et al. 1977). When Chinese hamster cells are subjected to a prolonged treatment with fluorodeoxyuridine, there appears to be activation of secondary origins during repli- cation (Taylor 1970). Pritchard (1978) has suggested that there is an analogy between this result and the effect of reduced thy- mine concentration mentioned above. More recently, Harland and Laskey (1980) have indicated that certain DNA' s can repli- cate even if they do not contain their normal origins for replica- tion. In these experiments the D N A was injected into unfertilized eggs of Xenopus laevis using BrdUTP to follow incorporation into DNA.

Secondary sites appear to be also activated if the normal origin is absent or inactive. This effect has been demonstrated for a deleted origin mutant in T7 (Tamanoi t980) and for a temperature sensitive replication gene in SV40 (Martin and Set- low 1980). The multiply branched 2 replicative intermediates that we have isolated do not seem to have arisen through this

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Fig. 6a-d. Diagram showing how multiply branched structures could arise from a single growing point by snap-back. An unlikely snap-back event is shown in (b); here a normal growing point (a) snaps-back and the resulting single stranded material is then assumed to be de- graded to produce a multiply branched structure (c). In (d) we show a more likely possibility involving a growing point containing substan- tial single stranded regions. Such a structure could easily snap-back to yield a multiply branched structure (e)

mechanism, since the 2 Ors mutant does not show any replicative intermediates at a non-permissive temperature.

A quite different model could also explain our results. A normal growing point could, in principle, snap-back at one or more positions as shown in Fig. 6(b) and if the resulting single stranded material was degraded we would have a structure resem- bling the observed multiply branched units (Fig. 6c). We know that 2 infection of unstarved cells does not produce multiply branched structures (Schn6s and Inman 1970; Valenzuela et al. 1976), so we conclude that the reaction shown in Fig. 6(a, b) is unlikely. If, however, prior starvation of E. coli results in a mode of replication leading to extensive gaps in daughter strands then, depending on the match between these gaps, the snap-back reaction would be inevitable. Figure 6(d) shows an extreme view of this notion, where the gaps have been arranged for a perfect snap-back reaction. Such a snapback reaction would have to be quite extensive to explain several of the multiply branched ). molecules shown in Fig. 5.

On the basis of our present results, we cannot differentiate between the above two explanations for the formation of multi- ply branched replicative intermediates. Either prior starvation of E. coli results in initiation of secondary sites on the genome or in extensive gaps in D N A synthesis on daughter strands which result in snap-back and the consequent formation of apparent secondary initiation sites.

The possibility that SOS induction may occur under the con- ditions of our experiments (Lark and Lark 1978) and the role that RecA may play on the formation of multiply branched structures is currently under investigation.

Acknowledgments. The superb technical assistance of Ms. Selma Sachs is greatly appreciated. This work was supported by grants from the National Institutes of Health and the American Cancer Society.

References

Harland RM, Laskey RA (1980) Regulated replication of DNA micro- injected into eggs of Xenopus laevis. Cell 21:761-771

Inman RB, SchnSs M (1971) Structure of branch points in replicating DNA: Presence of single stranded connections in 2 DNA branch points. J Mol Biol 56:319-326

Inman RB, Schn6s M (I973) D-loops in intracellular 2 DNA. In: Wells RD and Inman RB (eds) DNA Synthesis in vitro. University Park Press, Baltimore, pp 437 449

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Lark KG, Lark CA (1978) recA-dependent DNA replication in the absence of protein synthesis: characteristics of a dominant lethal replication mutation, dnaT, and requirement for recA ÷ function. Cold Spring Harbor Syrup Quant Biol 43:537 549

Louarn J, Funderburg M, Bird RE (1974) More precise mapping of the replication origin in Escherichia coli K-12. J Bacteriol i20: 1-5

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Ogawa T, Tomizawa J, Fuke M (1968) Replication of bacteriophage DNA. II. Structure of replicating DNA of phage lambda. Proc Natl Acad Sci USA 60:861-865

Perlman D, Rownd RH (1976) Two origins of replication in composite R plasmid DNA. Nature 259:28][-284

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Schn6s M, Inman RB (1970) Starting point and direction of replication in P2 DNA. J Mol Biol 55:31-38

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Tamanoi F, Saito H, Richardson CC (1980) PhysicaI mapping of primary and secondary origins of bacteriophage T7 DNA replica- tion. Proc Natl Acad Sci USA 77:2656-2660

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Valenzuela MS, Aguinaga MDP, Inman RB (1981) Isolation of DNA fragments containing replicating growing forks from both E. coli and B. subtilis. Mol Gen Genet 181:241-247

Valenzuela MS, Inman RB (1975) Visualization of a novel junction in bacteriophage 2 DNA. Proc Natl Acad Sci USA 72:3024-3028

Valenzuela MS, Inman RB (1978) Restriction enzyme cleavage of DNA resulting from gently lysed Escherichia coli. Mol Gen Genet 166 : 245-249

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Communica ted by F. Stahl

Received April 24 / October 5, 1981