Structure of protein-containing replicative intermediates of Bacillus subtilis phage φ29 DNA

VIROLOGY116,

l-18

Structure

(1982)

of Protein-Containing Bacillus subtilis

JOSfi M. SOGO, JUAN AND

Centro de Biologia Molecular

Replicative Phage 429

A. GARCIA, MARGARITA

(CSIC-UAM), Universidad

Intermediates DNA

MIGUEL SALAS Authnna,

Received July 14, 1981; accepted August

of

A. PENALVA, Canto 20,

Blanco, Madrid-.%& Spain

1981

Protein-containing 629 DNA replicative intermediates were isolated from extracts of phage-infected Bacillus subtilis. After allowing protein-protein interaction in vitro, analysis with the electron microscope showed the presence of circular type I molecules (unitlength duplex DNA with one single-stranded branch at a random position) and circular type II molecules (unit-length DNA with one double-stranded region and a singlestranded region extending a variable distance from one end). The circular replicative intermediates obtained are consistent with the presence of the terminal protein at the ends of the parental and daughter DNA strands, both in type I and type II molecules. The presence of protein in the replicating molecules was confirmed by retention of the labeled DNA on cellulose nitrate filters. By polyacrylamide gel electrophoresis, the protein bound to the DNA molecules was shown to be the terminal protein, p3. The results obtained support a model in which protein p3 acts as a primer in the initiation of $29 DNA replication.

molecule of the protein will react with the 5’ end nucleotide, dATP, and form a protein-dAMP covalent linkage, thus providing the required free 3’OH group needed for DNA elongation (Inciarte et al., 1980; Mellado et al., 1980). A similar mechanism was proposed for the initiation of adenovirus DNA replication which also has a protein covalently linked to the two 5’ termini (Rekosh et al., 1977; Carusi, 1977) and replicates by a similar strand displacement mechanism (Lechner and Kelly, 1977; Winnacker, 1978). It is known that protein p3, linked to the ends of $29 DNA, interacts in vitro with itself giving rise to the formation of circular structures and concatemers (Salas et aE., 1978). If the above model for the initiation of replication of 429 DNA were correct, one should find that all the replicative intermediates would contain the protein at the ends of both the parental and daughter DNA strands giving rise to different circular and concatemeric forms after in vitro protein-protein interaction as has been found in the case of adenovirus replication (Kelly and Lechner, 1979). In

INTRODUCTION

Phage 429 from Bacillus subtilis contains a linear, double-stranded DNA of molecular weight 11.8 X lo6 (Sogo et al., 1979) with a protein covalently linked to the two 5’ termini (Salas et al., 1978; Harding et al., 1978; Yehle, 1978; Ito, 1978) by a phosphoester bond between the hydroxy group of a serine residue and 5’dAMP (Hermoso and Salas, 1980). The DNAlinked protein, p3, is the product of cistron 3 (Salas et al., 1978) and is needed for the initiation of 429 DNA replication (Mellado et al., 1980). Replication of 429 DNA is initiated at both ends of the DNA, nonsimultaneously, and occurs by a mechanism of strand displacement (Inciarte et al., 1980; Harding and Ito, 1980). An inverted terminal repetition six nucleotides long exists at the ends of $29 DNA (Escarmis and Salas, 1981; Yoshikawa et al., 1981). A possible role as a primer for protein p3 in the initiation of $29 DNA replication has been proposed: a newly synthesized 1 To whom

reprint

requests

should

be addressed. 1

0042-6822/82/010001-18$02.00/O Copyright All rights

0 1982 by Academic Press, Inc. of reproduction in any form reserved.

2

SOGO

this paper we show an analysis, by electron microscopy, of 429 replicating molecules isolated without treatment with proteolytic enzymes. The results strongly suggest the presence of protein p3 at the ends of both the parental and daughter DNA strands. MATERIALS

AND

METHODS

(a) Bacteria and phage. Host bacteria were B. subtilis 1lONA try- spoA- su- and B. subtilis MO-99 (met- thr-)+ spoA- SU+~ (Moreno et al., 1974). Mutant sue14 (1242) that produces a normal burst and delayed lysis of the bacteria infected under restrictive conditions (Carrascosa et al., 1976) was used as wild-type phage. (b) Reagents and enzymes. [methyl3H]thymidine, [14C]uracil, and [35S]sulfate, carrier-free, were obtained from the Radiochemical Centre, Amersham. Sarkosyl NL97 was a gift from Geigy Chemical Company and p-(hydroxyphenylazo)uracil was a gift from Imperial Chemical Industries. Guanidinium chloride was obtained from Schwarz-Mann, glyoxal from Aldrich, B grade Pronase free of nucleases was from Calbiochem, and chromatographically purified fungal proteinase K, was from Merck. Benzyldimethylalkylammonium chloride (BAC) was kindly provided by Bayer. All other reagents were analytical grade, obtained from Merck. Proteinase K-treated 429 DNA, labeled with [14C]uracil, was prepared as described (Mellado et al., 1980). (c) Isolation of replicating $29 DNA molecules. B. subtilis 1lONA su- was grown at 30” in defined medium (Carrascosa et al., 1973) up to a concentration of 1 X lo8 cells/ ml. Fifteen milliliters of a bacterial culture was concentrated five-fold in defined medium containing amino acids at a final concentration of 0.5 mM, uridine (200 pg/ ml), and p-(hydroxyphenylazo)uracil (100 pg/ml) to inhibit host DNA replication (Brown, 1970) and infected with mutant sus14(1242) at a multiplicity of 10. After 50 min at 30”, [3H]thymidine (100 &i/ml; 46 Ci/mmol) was added and, 1.5 min later, the incorporation was terminated by addition of 25 mM sodium azide and by plac-

ET AL.

ing the cell culture in an ice-water bath. The cells were centrifuged at 4’ and the pellet was resuspended in 0.5 ml of a buffer containing 10 mM HEPES (N-2-hydroxyethyl-piperazine-N’-2-ethanesulfonic acid), pH 7, 0.2 mM EDTA, and lysozyme (2.5 mg/ml) and incubated for 5 min at 37” to allow the lysis of the infected bacteria. Buffer (0.5 ml) containing 2 mM HEPES, pH 7, 0.2 mM EDTA, and 2% sodium dodecyl sulfate was added and the mixture was incubated for 1 hr at 37” to release the DNA from the bacterial membrane (R. P. Mellado, and M. Salas, unpublished results). When indicated, glyoxal, to a final concentration of 2.6%, was also added. The sample was layered onto a 39-ml linear 5-20% sucrose gradient in 1 mM HEPES, pH 7,0.2 mM EDTA, 0.1% sodium dodecyl sulfate, and centrifuged for 17 hr at 18,000 rpm at 20” in a SW27 rotor. Fractions of about 1 ml were collected from about 1 cm from the bottom of the tube to eliminate the high-molecular-weight bacterial DNA, and the acid-insoluble, radioactive material was determined in a sample of each fraction. The fractions containing both mature and replicating DNA molecules (peak A; see Results) were pooled and precipitated with 2.5 vol of ethanol in the presence of 0.3 M sodium acetate, pH 6. The samples were kept overnight at -20” and the DNA was collected by centrifugation at 4” for 45 min at 30,000 rpm in a SW40 rotor. After washing with 80% ethanol, the DNA pellet was resuspended in 0.2 ml of 1 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.5% sodium dodecyl sulfate, layered onto a 5-ml 5-20% sucrose gradient in 10 mM Tris-HCl, pH 7.4, 0.1 M NaCl, 1 mM EDTA as described by Kelly and Lechner (1979) and centrifuged at 20” for 2.5 hr at 50,000 rpm in a SW65 rotor. Fractions were collected from the bottom of the tube and the acid-insoluble radioactivity was determined in a sample of each fraction. The fractions containing the replicating molecules and mature $29 DNA, to be used for electron microscopy, were pooled and dialyzed against 50 mM TrisHCl, pH 7.5,5 mMEDTA. When indicated, after incubation for 1 hr at 37” in the presence of 1% sodium dodecyl sulfate, the ly-

PROTEIN-CONTAINING

sates were further heated for 10 min at 60” and centrifuged in a sucrose gradient in the presence of sodium dodecyl sulfate as described above. In the latter case, after ethanol precipitation, the samples were suspended in 50 mM Tris-HCl, pH 7.8, 10 mM EDTA, 0.1 M NaCl, 1% (w/v) sarkosyl, and heated for 5 min at 65”. After cooling to room temperature, 1 vol of 8 M guanidinium chloride in 10 mM Tris-HCl, pH 7.8,1 mM EDTA was added and the mixture was incubated for 1 hr at 37” as described (Salas et al., 1978). The mixture was layered on the top of a lo-ml linear CsCl gradient of densities 1.45 to 1.55 g cm-3 in 50 mM Tris-HCl, pH 7.8, 10 mM EDTA, 0.1 M NaCl, and sedimented by centrifugation for 17 hr at 35,000 rpm and 10” in a SW40 rotor. Fractions of about 0.4 ml were collected from the bottom of the tube and the acid-insoluble radioactivity was determined. The fractions containing the DNA were pooled and dialyzed against 50 mM Tris-HCl, pH 7.5, 5 mM EDTA. (d) Labeling of 429 DNA molecules with [355’Jsulfate. B. subtilis 11ONA su- was grown at 30” in defined medium (Carrascosa et al., 1973) with the sulfate salts reduced lo-fold and with amino acids at a final concentration of 0.1 mM except methionine and cysteine. When the cell concentration reached 5 X 107/ml, 15 ml of culture was concentrated fivefold in defined medium with the sulfate salts reduced loo-fold and with amino acids at a final concentration of 0.5 mM except methionine and cysteine, in the presence of uridine (200 pg/ml) and p-(hydroxyphenylazo)uracil (100 pg/ml), infected with mutant sus14(1242) at a multiplicity of 20 and labeled by addition of carrier-free r5S]sulfate (300 &i/ml). After 50 min at 30”, [3H]thymidine (100 &i/ml; 46 Ci/ mmol) was added and, 1.5 min later, the incorporation was terminated as indicated before. The cells were lysed, incubated in the presence of 1% sodium dodecyl sulfate, first for 1 hr at 37” and then for 10 min at 60” as indicated under (c) and the lysates were centrifuged in a 12-ml linear 5-20% sucrose gradient containing 1 mM HEPES, pH 7,0.2 mMEDTA, 0.1% sodium

REPLICATIVE

3

629 DNA

dodecyl sulfate at 20” for 16 hr at 20,500 rpm in a SW40 rotor. Fractions of about 0.4 ml were collected from about 1 cm from the bottom of the tube and the acid-insoluble radioactivity was determined in a sample of each fraction. The fractions containing the replicating molecules and mature 429 DNA were pooled, precipitated with ethanol, and the sediment suspended in 0.3 ml of a buffer containing 50 mM Tris-HCl, pH 7.8, 10 mM EDTA, 0.1 M NaCl, and 1% (w/v) sarkosyl and heated for 5 min at 65”. One volume of 8 M guanidinium chloride was added as indicated under (c) and, after incubation for 1 hr at 3’7”, the mixture was centrifuged in a CsCl gradient as indicated. Fractions of about 0.4 ml were collected from the bottom of the tube and the acid-insoluble radioactivity was determined in a sample of each fraction. The fractions containing the DNA-protein complex were pooled and dialyzed against 0.05X SSC (SSC is 0.15 M NaCl, 0.015 M sodium citrate). (e) Polyacrylamide

gel electrophoresis.

The samples for electrophoresis were dissolved in 0.06 M Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate, 5% 2-mercaptoethanol, and 6 M urea and heated for 5 min in a bath of boiling water. Electrophoresis was carried out in 10 to 20% acrylamide gradient slab gels (Carrascosa et al., 1976). (fl Binding of protein-containing @?9 DNA to cellulose nitrate Jilters. To avoid

retention of single-stranded DNA, the cellulose nitrate filters were boiled for 30 min in distilled water and then incubated for at least 30 min at room temperature in 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.2 M NaCl (TES) (Tsai and Green, 1973). The samples in TES buffer were passed slowly through the filters and then rinsed with 10 ml of TES buffer. The filters were dried at 80” and the radioactivity counted. (g) Electron microscopy. The BAC protein-free spreading technique (Vollenweider et al., 1975; Sogo et al., 1979) was used with 25 mM Tris-HCl, pH 7.5, 2 mM EDTA, 37% (v/v) formamide, and 1.4 X 10w3% (w/v) BAC in the spreading solution and quartz bidistilled water in the hypophase. The DNA-BAC film was picked up with carbon-coated grids pretreated

4

SOGO

ET AL.

a SAC Digitizer. The data were processed in a PDP 11145 minicomputer with a DOS/ BATCH operating system and the histograms were displayed on a Model 31 Varian/Statos Plotter. RESULTS

(a) Sedimentation

Replicating Treatment

c 10

20 Fraction

30 number

FIG. 1. Neutral sucrose gradient of extracts from @9-infected B. subtilis, pulse-labeled with rH]thymidine. B. subtilis was infected with 429 and, after 50 min, labeled with [3H]thymidine for 1.5 min. The cells were incubated with lysozyme as described in the text and then sodium dodecyl sulfate was added to a final concentration of 1% and the lysates incubated for 1 hr at 37” in the absence (A) or presence (B) of 2.6% glyoxal. 14C-Labeled $29 DNA (6500 cpm) was added to each sample and they were sedimented in a 12-ml neutral 5 to 20% sucrose gradient as described under Materials and Methods in a SW40 rotor for 16 hr at 26,000 rpm and 20’. Fractions were collected from the bottom of the gradient and the trichloroaeetic acid-insoluble radioactivity was determined. A control of uninfected cells was labeled under the same conditions as the infected B. subtilis. l , 3H radioactivity, infected cells; A, 3H radioactivity, uninfected cells; 0, 14C radioactivity.

with ethidium bromide, stained with uranyl acetate, and rotary shadowed with platinum-carbon. Micrographs were taken in a Jeol 1OOB electron microscope at 80 kv and at a magnification of 15,000~. The magnification was determined with a carbon grating replica of 2160 lines/mm from Balzers Union. After a fivefold enlargement of the electron micrographs, the contour lengths of the DNA molecules were measured with--

Analysis of $29 DNA Molecules Isolated without with Proteolytic Enzymes

Figure 1A shows the sedimentation, in a neutral sucrose gradient in the presence of sodium dodecyl sulfate, of the DNA synthesized in phage 429-infected B. subtilis at 50 min postinfection, labeled with [3H]thymidine during a 1.5-min pulse. Two peaks of radioactivity appear, one of them (peak A) with a sedimentation coefficient similar to that of a marker of native 429 DNA. The peak with a lower sedimentation rate (peak B) disappeared after a chase with an excess of cold thymidine (Salas et al., 1981). Peak B was also present when the pulse-labeled DNA was digested with proteinase K prior to neutral sucrose gradient centrifugation (results not shown). Uninfected bacteria, pulse labeled under the same conditions as the infected cells, showed no radioactive material at any of the positions corresponding to the two major peaks seen in infected cells (Fig. 1A ). (6) Electron Microscopy of 429 Replicative Intermediates Present in Peak A The fractions corresponding to peak A from a preparative gradient were sedimented through a neutral sucrose gradient in the absence of sodium dodecyl sulfate. The radioactive fractions were pooled, dialyzed, maintained at 4” from 1 to 6 weeks to allow protein-protein interaction in vitro, and examined with the electron microscope as described under Materials and Methods. Figure 2 shows the different kinds of protein-protein interactions in type I molecules (double-stranded DNA with single-stranded tails located at a random position) and type II molecules (DNA partially double stranded and partially

PROTEIN-CONTAINING

single stranded, the transition point being random) (Inciarte et al., 1980) which would give rise to circular DNA molecules assuming the presence of 5’ linked protein at the ends of both the parental and daughter DNA strands. Figures 3 and 4 show examples of such circular type I, type II, and type I/II molecules (a combination of type I and type II molecules). Figure 3A shows a type I molecule in which the protein at the end of the displaced parental single strand has interacted with the protein at the end of the daughter double strand. Figure 3B shows a type I molecule in which the protein at the end of the parental displaced single strand has interacted with the protein at the end of the parental double strand; the single-stranded region has the same length as the daughter double-stranded region. Figure 3C shows a molecule in which the protein at the end of the daughter double strand interacts with the protein at the end of the parental double strand. Figure 3D shows a molecule in which the two proteins at the ends of the two parental regions (parental displaced single-stranded region and parental double-stranded region) and the protein at the end of the daughter double-stranded region interact. Figures 4A and B show two circular type II molecules in which replication has proceeded to a different extent. In these molecules the protein at the end of the parental single strand interacts with the protein at the end of the daughter double strand. Circular type I/II molecules were also seen (Fig. 4C and D). Figure 5 shows three dimers, two of them formed by a circular double-stranded DNA molecule and a type I (Fig. 5A) or a type II (Fig. 5B) molecule interacting either through the protein at the end of the parental double strand or through the protein at the end of the daughter double strand. In the dimer shown in Fig. 5C, the proteins from one circular type II molecule interact with the protein from the daughter double strand of a linear type II molecule. Other dimers were also observed and they consisted of two circular molecules or of two linear molecules, at least one of them being a replicating type I or type II molecule. Fig-

REPLICATIVE

429 DNA

Type

I molecules

t 0

.

t

*o-?-t Type

II molecules

0

.

6 FIG. 2. Diagrams of the different types of circular DNA resulting from protein-protein interactions in type I and type II molecules. The solid circles (0) represent the parental protein at the ends of the parental DNA. The open circles (0) represent newly synthesized protein at the end of the daughter DNA strand. In the diagrams of the different types of molecules in this and the following figures, the heavy line represents double-stranded DNA and the light line represents single-stranded regions.

ure 6A shows a circular dimer formed by two type II molecules in which the protein at the end of the daughter double strand of each molecule interacts with the protein at the end of the parental single strand of the other molecule. Figure 6B shows two linear type II molecules interacting through the proteins at the end of the daughter double strand. Figure 6C shows a circular type I molecule in which the proteins at the three ends interact with the protein at the end of mature doublestranded DNA. Aggregates higher than dimers were also seen but, taking into account the difficulty of their analysis, they were not considered for the statistical studies. Table 1 shows that the percentage of circular relative to total (circular plus linear) type I (and type I/II) and type II molecules, 60 and 48%, respectively, is

6

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ET AL.

PROTEIN-CONTAINING

similar or even higher than that of circular double-stranded mature DNA (34% of the total), indicating that protein-protein interaction occurs in replicating molecules. Moreover, the different kinds of protein-protein interactions shown in Fig. 2 for type I molecules occur with a similar frequency. Type III molecules (DNA partially double stranded with single-stranded regions at both ends) and type IV molecules (DNA with double-stranded regions at the two ends and an internal singlestranded region) (Inciarte et al., 1980) can be distinguished from type II molecules only in their linear form (see Table 1). Therefore, possible circular type III and type IV molecules would be included in the number of circular type II molecules. Nevertheless, the percentage of circular type II molecules would not be decreased to a significant extent since the amount of type III and type IV molecules is low relative to that of type II molecules. It should be pointed out that the amount of type I (and I/II) molecules (13% of the total amount of replicating molecules) is lower than that of type II molecules (73%). This is probably due to the fact that (1) type I molecules have more ends with protein for interaction than type II molecules and more nonanalyzable aggregates are probably formed from type I than from type II molecules and (2) the displaced parental strand of type I molecules seems to be more susceptible to degradation than the single-stranded DNA from type II molecules (see later). Similar results were obtained when the replicating molecules isolated by CsCl gradient sedimentation after treatment with guanidinium chloride were analyzed by electron microscopy (results not shown). These DNA molecules contain only protein p3 (see later).

REPLICATIVE

7

&29 DNA

(c) Nature of the DNA in Peak B

Molecules

Present

Analysis by alkaline sucrose gradient centrifugation of the fractions corresponding to peak B (see Fig. 1A) showed that the material from this peak had a sedimentation rate slower than that of denatured mature $29 DNA. An average molecular weight of 1.9 X lo6 was calculated for peak B from its sedimentation rate. From CsCl centrifugation to equilibrium, a density of 1.714 g crne3 was obtained compared with a density of 1.705 g cm-3 for peak A. About 90% of the radioactivity of peak B was degraded by nuclease Sl, specific for single-stranded DNA. These results suggest that the material from peak B is mainly singlestranded DNA. Electron microscopy of the material from peak B confirmed its single-stranded nature. Measurement of 246 filaments gave an heterogeneous length distribution, ranging from 0.4 to 5.4 pm. The average length obtained was 2.4 Frn, which corresponds to a molecular weight of 1.8 X lo6 in good agreement with the value obtained by sedimentation. When glyoxal was added during the incubation of the infected lysates with sodium dodecyl sulfate (see Methods) the amount of radioactivity present in the position corresponding to peak B greatly decreased (see Fig. 1B). Moreover, when replicating molecules corresponding to peak A, isolated after treatment of the lysates with glyoxal, were analyzed by electron microscopy, an increase in the number of type I and type I/II molecules was observed (see Table 2). If we consider only the replicating population, the number of type I plus type I/II molecules increased from 13% (replicating intermediates iso-

FIG. 3. Electron micrographs of circular type I replicating 429 DNA molecules. (A) The protein at the end of the parental displaced single-strand interacts with the protein at the end of the daughter double strand. (B) The protein at the end of the parental displaced single strand interacts with the protein at the end of the parental double strand. (C) The protein at the end of the daughter double-stranded region interacts with the protein at the end of the parental double-stranded region. (D) The proteins at the ends of the two parental regions (parental displaced single-stranded region and parental double-stranded region) and the protein at the end of the daughter double-stranded region interact. The insets show representations of the molecules. Bar = 0.5 pm.

8

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ET AL.

PROTEIN-CONTAINING

lated in the absence of glyoxal, Table 1) to 38% (replicating molecules isolated in the presence of glyoxal, Table 2). The contrary occurs with type II molecules which decreased from 73% in the absence of glyoxal (Table 1) to 56.5% in its presence (Table 2). The percentage of circular molecules (considering the whole population) decreased from 34 to 60% in the absence of glyoxal (Table 1) to 7.‘7-14.7% in the presence of glyoxal (Table 2). It seems, therefore, that the singlestranded DNA present in peak B is derived from type I molecules by cleavage of the single-stranded DNA tails. This effect is reduced by glyoxal treatment, which forms an adduct with guanylic acid residues in single-stranded nucleic acids (Broude and Budowski, 1971). (d) Length Determination ing Molecules

of $29 Replicat-

When length measurements were made on circular type I (type I plus type I/II) and type II molecules, conclusions as to the origin and mechanism of $29 DNA replication drawn were similar to those obtained from the analysis of deproteinized type I and type II replicating molecules (Inciarte et al., 1980). In this case, however, molecules in which the protein from the parental duplex strand and the protein from the daughter duplex strand interact to produce a circle (Fig. 3C) were not included since, as the protein is not detectable, it is not possible to determine the point of the interaction. Figure 7A shows a histogram of the length of the double-stranded region of the circular type I molecules (excluding those indicated above), with an average value of 7.35 f 0.40 pm. These length measurements include 77 type I molecules obtained and prepared for electron microscopy in three different experiments. When the lengths

REPLICATIVE

429 DNA

9

of double-stranded circular DNA molecules were determined, average values of 6.86 f 0.11 pm (53 molecules), 7.28 f 0.19 pm (52 molecules), and 7.56 -+ 0.15 pm (22 molecules) were obtained, with a mean value of 7.15 rt 0.30 pm, very close to that found for the double-stranded region of circular type I molecules. These small differences in length in different experiments seems to be a characteristic of the BAC spreading technique. Figure 7B shows the ratio between the length of the doublestranded region with the value closer to the single-stranded one and the length of the total double-stranded DNA in circular type I molecules. The random distribution obtained indicates the existence of molecules having replicated to a different extent, with those replicated less being more abundant. The ratio between the length of the single-stranded region in circular type I molecules and the corresponding doublestranded region shows a maximum at a value of 1 (Fig. 7C), after correction for the higher length obtained with the BAC spreading technique for single- than for double-stranded DNA (see below). A similar distribution was obtained when deproteinized linear type I molecules were analyzed (Inciarte et al., 1980). The length distribution of 123 circular type II molecules from three different experiments with an average length value of 7.50 +- 0.50 pm is shown in Fig. 8A. The values obtained for type II molecules in two experiments, with average lengths of 7.23 * 0.34 and 7.63 & 0.36 km, respectively, should be compared with those of 6.86 f 0.11 and 7.28 + 0.19 pm obtained for double-stranded DNA in the same experiments. The greater length obtained for type II molecules than for doublestranded DNA is due to the single-stranded DNA present in type II molecules and, since the proportion of single- relative to double-stranded DNA in those molecules

FIG. 4. Electron micrographs of circular type II and type I/II replicating 429 DNA molecules. (A, B), type II molecules in which the protein at the end of the daughter double-stranded region interacts with the protein at the end of the parental single-stranded region. (C, D), type I/II molecules. The protein-protein interactions are similar to those described in Figs. 3C and D. The insets show representations of the molecules. Bar = 0.5 pm.

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ET AL.

PROTEIN-CONTAINING

REPLICATIVE

is known, the increase in length of singlestranded DNA with respect to doublestranded DNA can be calculated to be 16.5%. Figure 8B shows the ratio between the length of the double-stranded region and the total length of the DNA (double and single stranded) in type II molecules. A random distribution can be seen, indicating the existence of molecules having replicated to a different extent, although those having replicated further are more abundant. (e) Binding

of Replicating 429 DNA ecules to Cellulose Nitrate Filters

Mol-

To confirm the presence of protein in replicating $29 DNA molecules the fractions corresponding to peak A were dialyzed against 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, and passed through cellulose nitrate filters in the presence of 0.2 M NaCl. Under these conditions protein-free DNA is not fixed by the filters while protein-containing DNA is retained. Table 3 shows that 82% of the radioactivity present in peak A was retained by the filters while only 0.3% of proteinase K-treated $29 DNA was fixed to the filters. After heat denaturation, 62% of the radioactivity present in peak A was retained by the filters while only 0.2% of protein-free DNA, used as a control, was bound. The smaller percentage of radioactivity retained by the filters after heat denaturation may be due to single-strand nicks in the replicating DNA molecules. of the Protein Present in the DNA Molecules from Peak A

(f) Characterization

Figure 9 shows that when DNA molecules corresponding to peak A, labeled with [35S]sulfate and rH]thymidine and purified by CsCl gradient centrifugation,

629 DNA

11

were subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, no protein migrated into the gel (slot a) unless the DNA was previously digested with nuclease Sl after denaturation (slot b) (Salas et al., 1978). In the latter case, a single protein band with a mobility very similar to that of protein p3 (slot d) could be seen. DISCUSSION

$29 DNA molecules isolated from viral particles without treatment with proteolytic enzymes can form a variety of circular structures and concatemers due to protein-protein interaction (Ortin et al., 1971; Salas et al., 1978). When $29 replicative intermediates were isolated from phage-infected cells omitting a treatment with Pronase, different circular structures, dimers, and higher aggregates were obtained from type I, type II, and type I/ II molecules. In the case of type I (and type I/II) molecules, the circles produced are consistent with the interaction of the protein at the ends of the two parental strands, the interaction of the protein at the end of the daughter double strand with the protein at the end of either of the two parental strands, or the interaction of the three proteins. Circular type II molecules can be formed by interaction of the protein at the end of the parental single strand and that at the end of the daughter double strand. The above results suggest the presence of protein at the ends of all DNA strands, parental and daughter, in replicating 429 DNA molecules. Polyacrylamide gel electrophoresis of the 35S-labeled DNA molecules showed that the only protein present was p3. This result strongly suggests that the protein present at the ends of the two parental strands and daughter strand in type I molecules and

FIG. 5. Electron micrographs of dimers. (A, B) The proteins of a circular double-stranded genome interact with the protein at the end of the parental double-stranded region of a type I molecule and with the protein at the end of the daughter duplex region of a type II molecule, respectively. (C) Two type II molecules in which the protein at the end of the duplex strand from a linear molecule interacts with the proteins from a circular molecule. The insets show representations of the molecules. Bar = 0.5 pm.

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ET AL.

PROTEIN-CONTAINING

REPLICATIVE

TABLE FREQUENCIES

DNA

Type I + Type Circular Linear

SEEN IN THE PREPARATION OF REPLICATING IN THE ABSENCE OF GLYOXAL Circular moleculesa (%o)

Number

Double-stranded Circular Linear

Tpye II Circular Linear

1

OF THE DIFFERENT TYPES OF MOLECULES INTERMEDIATES OF $29 DNA ISOLATED

molecule

13

629 DNA

Replicating molecules (%)

34

1’76 338 I/II 27 18

60

13

125 136

48

73

Type III Linear

43

12

Type IV Linear Dimersb

6 130

2

Note. The results are the average of two different experiments. 25 and 43 days, respectively, before analysis with the electron a Calculated for each different type of DNA molecule. b The dimers have been considered as units.

at the end of the parental and daughter strand in type II molecules is p3, the protein covalently linked to the 5’ ends of $29 DNA (Salas et al., 1978). Length determinations of the circular type I (and I/II) and type II molecules are in agreement with previous results obtained on linear replicating $29 DNA molecules obtained by treatment with Pronase (Inciarte et al., 1980) and they support the conclusion that $29 replication starts at the ends of the DNA and proceeds by a mechanism of strand displacement. Adenovirus DNA, which also has a protein covalently linked to the 5’ ends (Rekosh et al., 19’77; Carusi, 1977) initiates

The DNA microscope,

preparations

were

left at 4’ for

replication at the ends and replicates by a similar strand displacement mechanism (Ariga and Shimojo, 1977; Lechner and Kelly, 1977; Sussenbach and Kuijk, 1977; Sussenbach and Kuijk, 1978). Circular replicating type I and type II molecules were also found, consistent with the presence of the terminal protein at the ends of the parental and daughter DNA strands (Girard et al., 1977; Kelly and Lechner, 1979). Also, by using other techniques, evidence for the presence of protein at the ends of parental and daughter adenovirus DNA strands has been obtained (Robinson et al., 1979; Stillman and Bellett, 1979; Van Wielink et al., 1979).

FIG. 6. Electron micrographs of dimers. (A) Two type II molecules in which the parental protein at the end of one molecule interacts with the daughter protein at the end of the other molecule, and vice versa, giving rise to a circular molecule of double length. (B) Two type II molecules in which the protein at the end of each daughter double-strand interact giving rise to a linear molecule of double length. (C) One circular type I molecule in which the three proteins at each end have interacted and they also interact with the protein at the end of a linear double-stranded molecule. The insets show representations of the molecules. Bar = 0.5 Km.

14

SOGO

ET AL.

TABLE FREQUENCIES

DNA

2

OF THE DIFFERENT TYPES OF MOLECULES SEEN IN THE PREPARATION OF REPLICATING INTERMEDIATES OF 629 DNA ISOLATED IN THE PRESENCE OF GLYOXAL

molecule

Circular moleculesa (%)

Number

Double-stranded Circular Linear

Replicating molecules (%I

156 2014

7.7

62 421

14.7

38

Type II Circular Linear

61 656

9.3

56.5

Type III Linear

65

5.1

Type IV Linear

5

0.4

Type I + Type Circular Linear

Dimersb

I/II

92

Note. The results are the average of four different experiments analysis with the electron microscope, with similar results, ’ Calculated for each different type of DNA molecule. b The dimers have been considered as units.

The results obtained both in the case of phage 429 and adenovirus replication, which suggest the presence of the terminal protein at the ends of the parental and daughter DNA strands, support the model proposed for the initiation of replication of $29 (Inciarte et al., 1980; Harding and Ito, 1980; Mellado et al., 1980) and adenovirus (Rekosh et al., 1977) DNAs. In the model, a newly synthesized molecule of the terminal protein acts as a primer for the initiation of replication by interaction with the parental protein, reaction with the dNTP corresponding to the 5’ end, and formation of a protein-dNMP covalent linkage, thus providing the 3’OH group needed for elongation by the DNA polymerase (see Fig. 10). Evidence that the newly synthesized molecule of the terminal protein recognizes the parental protein in the DNA has been obtained, both in 429 and adenovirus replication. In the case

left

at 4” between

1 and 8 weeks

before

of phage $29, mixed infection at 42” with a ts3 and a ts2 mutant produced only progeny with the ts2 genotype suggesting that a functional parental protein p3 is required for replication (Salas et al., 1978). In the case of adenovirus, in vitro replication systems have shown the requirement of protein-containing DNA as a template for replicative synthesis (Challberg and Kelly, 1979a, b; Reiter et al., 1980; Ikeda et al., 1981; Stillman, 1981). There are several possible mechanisms to explain the formation of type II molecules. In adenovirus DNA, the inverted terminal repetition of about 100 nucleotides (Steenbergh et al., 1977; Arrand and Roberts, 1979; Shinaga,wa and Padmanaban, 1979; Tolun et al., 1979) has been proposed to play a role in circularizing the displaced parental strand to initiate replication and produce type II molecules, although no circular single-stranded DNA

PROTEIN-CONTAINING

REBLICATIVE

TABLE BINDING

2

4 (a+b),

6 8 pm

02

04

o/to+bi

OS 0.8

05

1.0 C/O

‘7.Length distribution, position of the growing points, and length of single-stranded branches in circular type I replicating $29 DNA molecules. Replicating 429 DNA molecules were isolated and prepared for electron microscopy as described in the text. (A) Length of the double-stranded DNA in circular type I molecules. (B) Position of the growing points in type I molecules. The histogram represents the ratio between the length of the double-stranded region with a length similar to that of the singlestranded tail, a, and that of the total length of duplex DNA, a + b. (C) Length of single-stranded branches in type I molecules. The histogram represents the ratio between the length of the single-stranded tail, c, and that of the double-stranded region with a similar length, a. FIG.

has been reported (Lechner and Kelly, 1977). In 429 DNA, which has a short inverted terminal repetition of six nucleo-

FIG. 8. Length distribution and position of the growing points in circular type II replicating $29 DNA molecules. Replicating 429 DNA molecules were isolated and prepared for electron microscopy as described in the text. (A) Length of the doubleand single-stranded regions in circular type II molecules. (B) Position of the transition between doubleand single-stranded regions in type II molecules. The histogram shows the ratio between the doublestranded region, d, and that of the total DNA length, d + e.

15

629 DNA 3

OF REPLICATING $29 DNA MOLECULES CELLULOSE NITRATE FILTERS

TO

DNA Molecule

Radioactivity bound (% )

Peak A Peak A, heat denatured

82 62

Protein-free $29 DNA Protein-free 629 DNA, heat denatured

0.3 0.2

Note. Samples containing 3H-labeled peak A (1665 cpm) isolated in the absence of glyoxal or 14C-labeled protein-free 429 DNA (3864 cpm) in TES buffer were filtered through cellulose nitrate filters as described under Materials and Methods, section (f). The heatdenatured samples were obtained by boiling the DNA for 7 min and cooling quickly in ice. To obtain protein-free DNA, phage 429, labeled with i4C-uracil, was treated with proteinase K and phenol extracted as described (Mellado et al., 1980).

tides (Escarmis and Salas, 1981; Yoshikawa et al., 1981), we have not found single-stranded circles although some single-stranded linear molecules with a length corresponding to the whole genome were present. Type II molecules could be formed by the following mechanism (see Fig. 10): before one of the parental strands is completely displaced, initiation of replication occurs at the other end by interaction of a newly synthesized molecule of protein p3 with the parental protein at the 5’ end of the opposite strand; when replication forks get close, two type II molecules originate. An alternative possibility is that the parental strand is completely displaced releasing a single-stranded DNA molecule. Interaction of a newly synthesized molecule of protein p3 with the 3’ end of the single-stranded linear DNA could initiate replication of the displaced strand and produce a type II molecule. The possibility of circularization of the displaced parental single-strand to initiate replication by interaction of a new molecule of protein p3 and the parental protein is less likely since no single-stranded circles have been found. Another open question is whether phage

SOGO b

ET AL. 0

d

> -0

< l+o

0 o-

> 1 + dATP >

l

1 +4dNTP

Type I

A

-pl2

-p12*

t &,A-------+ <

-~8

-pII =

p,ig

-p8.5 -P3 -pl5

-PI7

-PG

FIG. 9. Electrophoresis on polyacrylamide gels of the protein present in DNA molecules from peak A. Phage #29-infected B. subtilis was labeled with

Type II 0

FIG. 10. Model for 429 DNA replication and for the role of protein p3 in the initiation of replication. Continuous lines represent parental DNA and discontinuous lines represent newly synthesized DNA. The black dots at the 5’ ends of the DNA represent the parental protein p3 and the white dots represent newly synthesized molecules of protein p3. Only the initiation of replication of one of the DNA strands, at the left end, has been drawn for simplicity. Initiation of replication at the right end occurs with the same frequency (Inciarte et al., 1980). When initiation of replication takes place at the two ends on the same molecule two type II molecules are produced when the replication forks meet.

[%]sulfate and aH]thymidine as described under Materials and Methods and the DNA molecules corresponding to peak A isolated in a sucrose gradient were further purified by CsCl gradient centrifugation after treatment with guanidinium chloride and subjected to electrophoresis in slab gels containing a 10 to 20% acrylamide gradient as described under Materials and Methods. (a) The DNA molecules, labeled with 3H in the DNA (126,800 cpm) and with [35S]sulfate in the protein (3300 cpm) were heated for 5 min in a bath of boiling water in the dissociation buffer described under Materials and Methods. (b) The DNA molecules (126,800 cpm of 3H and 3300 cpm of a%) were treated with nuclease Sl as described (Salas et al., 1978) and the mixture was then dissociated for electrophoresis as described above. Over 98% of the 'H radioactivity became acid soluble. (c) Phage 429 labeled with [%]sulfate. (d) B. subtilis minicells infected with phage $29 and labeled with a %-protein hydrolysate as described (Garcia and Salas, 1980).

PROTEIN-CONTAINING

$29 and adenovirus replication in vivo occur on linear DNA or on DNA circularized by protein-protein interaction. To answer this question $29-infected cells were treated immediately after lysis with 0.1% glutaraldehyde under conditions shown to maintain #29 DNA in a circular form by interaction of the proteins at the ends of the DNA. The amount of circular type I (and type I/II) molecules relative to total was 9.2% and that of circular type II molecules was 5%. In a parallel control, not treated with glutaraldehyde, the amount of circular type I (and type I/II) molecules was 10% and that of circular type II molecules was 3%. On the other hand, in the sample treated with glutaraldehyde, linear concatemers were found, probably due to fixation of the proteins at the ends of linear DNA. These results suggest that 429 replication occurs on linear DNA. However, a final answer to this question will require further experiments. The availability of an in vitro 429 replication system dependent on exogenously added protein p3-DNA complex might help to solve this problem. ACKNOWLEDGMENTS The help of M. Lozano, M. Caballero, and E. Garcia in the processing of the data from the electron microscope and that of J. de la Torre in the preparation of the photographs from the electron microscope is gratefully acknowledged. This investigation was aided by Research Grant 1 ROl GM27242-02 from the National Institutes of Health and by Grants from Comision Asesora para el Desarrollo de la Investigation Cientifiea y T&mica and Comision Administradora de1 Descuento Complementario (INP). J.A.G. and M.A.P. were fellows, respectively, from Caja de Ahorros y Monte de Piedad de Madrid, and Instituto National de Ayuda y Promo&n al Estudiante. REFERENCES ARIGA, H., and SHIMOJO, H. (1977). Initiation and termination sites of adenovirus 12 DNA replication. virology 78, 415-424. ARRAND, J. R., and ROBERTS, R. J. (1979). The nucleotide sequences at the termini of adenovirus 2 DNA. J. Mel Biol. 128, 577-594. BROUDE, N. E., and BUDOWSKY, E. I. (1971). The reaction of glyoxal with nucleic acid components. III. Kinetics of the reaction with monomers. Biochim Biophys.

A&

254,380-388.

REPLICATIVE

429 DNA

17

BROWN, N. C. (1970). 6-(Hydroxyphenylazo)-uracil: A selective inhibitor of host DNA replication in phage-infected Bacillus subtilis. Proc. Nat. Acad. Sti USA 67,1454-1461. CARRASCOSA, J. L., VI%JELA, E., and SALAS, M. (1973). Proteins induced in Bacillus subtilis infected with bacteriophage $29. virology 56,291-299. CARRASCOSA, J. L., CAMACHO, A., MORENO, F., JIM~NEZ, F., MELLADO, R. P., V&UELA, E., and SALAS, M. (1976). Bacillus subtilis phage +29: Characterization of gene products and functions. Eur. J. Biochem.

66,229-241.

CARUSI, E. A. (1977). Evidence for blocked 5’ termini in human adenovirus DNA. virology 76,390-394. CHALLBERG, M. D., and KELLY, T. J., JR. (1979a). Adenovirus DNA replication in vitro. Proc. Nat. Acod

Sci

USA 76, 655-659.

CHALLBERG, M. D., and KELLY, T. J., JR. (1979b). Adenovirus DNA replication in vitro: Origin and direction of daughter strand synthesis. J. Mol. Biol. 135,999-1012. ESCARMfS, C., and SALAS, M. (1981). Nucleotide sequence at the termini of the DNA of Bacillus sub tilis phage 429. Proc. Nat. Acad. Sci. USA 78,14461450. GARCfA, J. A., and SALAS, M. (1980). Bacteriophage 429 infection of Bacillus subtilis minicells. Mol. Gen. Genet. 180,539-545. GIRARD, M., BOUCH& J., MARTY, L., REVET, B., and BERTHELOT, N. (1977). Circular adenovirus DNAprotein complexes from infected HeLa cell nuclei. Virology

83, 34-55.

.

HARDING, N., ITO, J., and DAVID, G. S. (1978). Identification of the protein firmly bound to the ends of bacteriophage $29 DNA. Virology 84,279-292. HARDING, N., and ITO, J. (1980). DNA replication of bacteriophage 629: Characterization of the intermediates and location of the termini of replication. virology 104, 323-338. HERMOSO, J. M., and SALAS, M. (1980). Protein p3 is linked to the DNA of phage 429 through a phosphoester bond between serine and 5’dAMP. Proc. Nat.

Acad

Sci. USA 77, 6425-6428.

IKEDA, J. E., ENOMOTO, T., and HURWITZ, J. (1981). Replication of adenovirus DNA-protein complex with purified proteins. Proc. Nat. Acad. Sci. USA 78,884-888. INCIARTE, M. R., SALAS, M., and SOGO, J. M. (1980). Structure of replicating DNA molecules of Badus subtilis bacteriophage 429. J. firol. 34, 187-199. ITO, J. (1978). Bacteriophage 429 terminal protein: Its association with the 5’ termini of the 429 genome. J. ViroL 28, 895-904. KELLY, T. J., JR., and LECHNER, R. L. (1979). The structure of replicating adenovirus DNA molecules: Characterization of DNA-protein complexes from infected cells. Cold Spring Harbor Symp. Quad.

Biol.

43,721-728.

LECHNER, R. L., and KELLY, T. J., JR. (1977). The

18

SOGO ET AL.

structure of replicating adenovirus 2 DNA molecules. Cell 12, 1007-1020. MELLADO, R. P., PE~ALVA, M. A., INCIARTE, M. R., and SALAS, M. (1980). The protein covalently linked to the 5’ termini of the DNA of Bacillus subtilis phage 429 is involved in the initiation of DNA replication. firoZog2/ 104, 84-96. MORENO, F., CAMACHO, A., VI~~UELA, E., and SALAS, M. (1974). Suppressor-sensitive mutants and genetic map of Bacillus subtilis bacteriophage $29. Virology 62, 1-16. ORTfN, J., VIGUELA, E., SALAS, M., and V~SQUEZ, C. (1971). DNA-protein complex in circular DNA from phage $29. Nature New Biol. 234,275-277. REITER, T., FRITTERER, J., WEING~RTNER, B., and WINNACKER, E. L. (1980). Initiation of adenovirus DNA replication. J. Viral. 35, 662-671. REKOSH, D. M. K., RUSSELL, W. C., and BELLET, A. J. D. (1977). Identification of a protein linked to the ends of adenovirus DNA. Cell 11,283-295. ROBINSON, A. J., BODNAR, J. W., COOMBS, D. H., and PEARSON, G. D. (1979). Replicating adenovirus 2 DNA molecules contain terminal protein. virology 96, 143-158. SALAS, M., MELLADO, R. P., VI~UELA, E., and SOGO, J. M. (1978). Characterization of a protein covalently linked to the 5’ termini of the DNA of Bacillus subtilis phage 429. J. Mol. Biol. 119, 269-291. SALAS, M., PE~~ALVA, M. A., GARCfA, J. A., HERMOSO, J. M., and SOGO, J. M. (1981). Priming of phage 429 replication by protein p3, covalently linked to the 5’ends of the DNA. In “ICN-UCLA Symposium on Molecular and Cellular Biology and Structure and DNA-Protein Interactions of Replication Origins,” (D. S. Ray and C. F. Fose, eds.), Vol. XXI. Academic Press, New York. SHINAGAWA, M., and PADMANABHAN, R. (1979). Nucleotide sequence at the inverted terminal repetition of adenovirus type 2 DNA. B&hem. Biophys. Res. Commun. 87, 671-678. SOGO, J. M., INCIARTE, M. R., CORRAL, J., VI~UELA, E., and SALAS, M. (1979). RNA polymerase binding sites and transcription map of the DNA of B. sub tilis phage 429. J. Mol. Biol. 127, 411-436. SOGO, J. M., RODENO, P., KOLLER, Th., VI~?UELA, E., and SALAS, M. (1979). Comparison of the A-T rich regions and the Bacillus subtilis RNA polymerase

binding sites in phage $29 DNA. Nucleic Acids Res. 7, 107-120. STEENBERGH, P. H., MAAT, J., VAN ORMONDT, H., and SUSSENBACH,J. S. (1977). The nucleotide sequence at the termini of adenovirus type 5 DNA. Nucleic Acids

Res. 4,4371-4389.

STILLMAN, B. W., and BELLETT, A. J. D. (1979). An adenovirus protein associated with the ends of replicating DNA molecules. Virology 93, 69-79. STILLMAN, B. W. (1981). Adenovirus DNA replication in vitro: A protein linked to the 5’ end of nascent DNA strands. J. firol. 37, 139-147. SUSSENBACH, J. S., and KUIJK, M. G. (1977). Studies on the mechanism of replication of adenovirus DNA. V. The location of termini of replication. Virology 77, 140-157. SUSSENBACH, J. S., and KUIJK, M. G. (1978). The mechanism of replication of adenovirus DNA. VI. Localization of the origin of the displacement synthesis. mrology 84, 509-517. TOLUN, A., ALESTR~?M, P., and PETTERSSON, U. (1979). Sequence of inverted terminal repetitions from different adenoviruses: Demonstration of conserved sequences and homology between SA7 termini and SV40 DNA. Cell 17, 705-713. TSAI, R. L., and GREEN, H. (1973). Studies on mammalian cell protein (~8) with affinity for DNA in vitro. J. MoL Biol. 73, 30’7-316. VAN WIELINK, P. S., NAAKTGEBOREN, N., and SusSENBACH, J. S. (1979). Presence of protein at the termini of intracellular adenovirus type 5 DNA. Biochim. Biophys. Acta 563, 89-99. VOLLENWEIDER, H. J., SOGO, J. M., and KOLLER, Th.. (1975). A routine method for protein-free spreading of double- and single-stranded nucleic acid molecules. Proc. Nat. Accd Sci. USA 72, 83-87. WINNACKER, E. L. (1978). Adenovirus DNA: Structure and function of a novel replicon. Cell 14, 761773. YEHLE, C. D. (1978). Genome-linked protein associated with the 5’ termini of bacteriophage 429 DNA. J. firol. 27, 776-783. YOSHIKAWA, H., FRIEDMANN, T., and ITO, J. (1981). Nucleotide sequences at the termini of $29 DNA. Proc. Nat. Acad. Sci. USA 78, 1336-1340.