Molecular mechanisms of genetic recombination in bacteriophage

J. Mol . Biol. (1964) 8,

51~540

Molecular Mechanisms of Genetic Recombination in Bacteriophage ll. Joining of Parental DNA Molecules of Phage T4 JUN-ICID TOMIZAWA AND NAOYO ANRAXU

Department of Ohemistry , The National Institute of Health of Japan Shinaga'lL'a-ku, Tokyo, Japan (Received 29 November 1963) DNA Was ext racted from E. coli infected with a mixture of s2P·labelled and bromouracil-labelled T4 phage particles after incubation for 60 min in the presence of 8 x 10-3 M-KCN. This DNA Wa8 mixed with DNA extracted from cells infected with a mixture of 3R·labelled and bromouracil.labelled T4 phage particles. The resultant mixture Wa8 cen t rifuged in a CsCI density gradient and fractionated. A considerable amount of 3Sp without accompanying 3R was found at a density intermediate between that of [32P]DNA and bromouracil-labelled DNA. The complex with [3SP]DNA found in the intermediate fractions has the following properties: (I) the com plex is composed of heavy and light components and is not a simple aggregate; (2) both components are sensitive to deoxyribonuclease and all the 32p in the complex can be made acid-soluble by t h is enzyme ; (3) the density of the complex does not change after treatmen t with ribonuclease; (4) the heavy and light components separate after heating at a te mperat ure at which DNA extracted from bromouracil-labelled phage separates into single strands, but do not separate below the melting temperature of T4 phage DNA; (5) the analysis of the fragments produced by shear degradation indicates linearity of the complex as a whole. The com plex is concluded to be a linear molecule com posed of on e s2P·labelled " light" DNA and one bromouracil-labelled "hea vy" DNA component joined end-to-end by hydrogen bonds. Structural models of the complex DNA and the possible genetic implication of formation of such molecules ar e discussed.

1. Introduction Isotopically-labelled DNA in parental particles of T-even phages was found to be dispersed among an appreciable number of progeny particles after one cycle of growth in a sensitive cell (see Hershey & Burgi, 1956; Dclbriick & Stent, 1957; Kozinski, 1961). When precautions are taken to prevent breakage by shearing, the DNA can be extracted as a single continuous molecule (Davison, Freifelder, Hede & Levinthal, 1961; Rubenstein, Thomas & Hershey, 1961; Cairns, 1961). From these two results it can be concluded that DNA molecules fragment during reproduction of the phage chromosome. Experiments with phage >. show a correlation between dispersion and genetic recombination and suggest the occurrence of chromosomal breakage and reunion which leads to recombination prior to replication (Meselson & Weigle , 1961 ; Kellenberger, Zichichi & Weigle, 1961). The analysis of these pro· cesses on a molecular basis is made difficult by the complication introduced by replication of the products form ed dire ctly after recombination. From the experiments presented in the preceding report (Tomizawa & Anraku, 1964) a possibility 516

JOINING OF PARENTAL DNA MOLECULES

517

emerged in which this difficulty could be eliminated. In this paper experiments are described to show joining of DNA molecules derived from two parents during treatment with KeN of cells infected with T4 phage particles.

2. Materials and Methods (a) Phage and bacterial strains Phage T4B (Benzer, 1955) and E. coli BB (McFall & Stent, 1958) were generally used in phage crosses. E. coli K12 T-, a thymine-requiring mutant (kindly supplied by Dr. T. Okada), was used for labelling phage particles with [3H]thymidine. (b) Media

Media used for bacterial growth and phage crosses are those described in the previous paper (Tomizawa & Anraku, 1964). 'I'ris-Casamino acids-glucose medium (Kozinski & Szybalski, 1959) contains 0·5 g Difco vitamin-free Casamino acids, 1 g glucose, 0'5 g NaCl, 1·6 ml, 0·1 M-Na sS0 4 , 3'2 mI. 0·1 M.KH sP0 4 , 1 ml. 1 M-MgS0 4 , 1·4 ml. 1 M.CaCIs, 0·3 ml. 0·01 M.FeC13 and 100 mI. 1 M-tris buffer, pH 7·3, per litre of water. The phosphorus content of the medium is 11 p.g/ml. DNA was dissolved in saline-citrate buffer: 0·15 M·NaCI-0·OI5 M-sodium citrate; pH 7·0. (c) Preparation of labelled phage

[32P]phage: E. coli BB was grown to a density of 5 x 10 B/m !. in tris-Casamino acidsglucose medium supplemented with 0·8p.c/p.g phosphorus of 3SP0 4 • 20JA-g/mI. of r.-tryptophan was added and the cells infected with phage T4 at a multiplicity of 0·1. The infected cells were incubated for 3 hr and lysed with chloroform. The specific activity of ssp was so chosen that the phage had an average of one 3Sp atom per particle. In addition, labelled phage preparations were made within 1 day before use. Thus artefacts caused by decay of up were minimized and are probably negligible. 3sP-Iabelled T2 phage was prepared in a similar way. [3SP]particles refer to sSP·labelled T4 phage particles. [3H]phage: E. coli KI2 T- was grown (4 x lOB/mI.) in broth supplemented with 4JA-gjmI. of [3H]thymidine specific activity of 14p.c/p.g of thymidine, infected with phage T4 at a multiplicity of I, incubated for 2 hr and lysed. BU-phaget: The method described by Schildkraut, Wierzchowski, Marmur, Green & Doty (1962) was generally followed. E. coli BB (5 x lOB/mI.) grown in t.ris-Casamino acids-glucose medium was incubated for 20 min in the same medium supplemented with 4p.g!ml. of 5-fluorodeoxyuridine (kindly supplied by Hoffman-LaRoche, Inc., Basle, Switzerland), and 200ftg!ml. ofBUDR. t-Tryptophan, 20ftgjmI. was added and the cells were infected with phage T4 at a multiplicity of 2 and incubated for 4 hr and lysed. BU-[3SP]phage was prepared by supplementing tris-Casamino acids-glucose medium with 0·8p.c!p.g phosphorus of 32P04 • Precautions were taken to prevent inactivation of the particles by visible light. (d) Phage crosses and extraction and handling of DNA Phage crosses and treatment with KCN were generally performed according to the procedure reported in the previous paper (Tomizawa & Anraku, 1964), except that infected cells were not immediately treated with the chemical but were first incubated for 5 min in broth, and then KCN was added. This procedure was adopted since infected cells treated immediately with KCN were rather fragile and a considerable portion of the infective centres was lost, especially during the centrifugation after the treatment. Incubation for a short period protected the infective centres. A total of 1·5 x 101 0 starved E. coli BB cells was centrifuged, suspended in 3·0 m!. of adsorption buffer supplemented with 20p.g!ml. of L-tryptophan and infected with 1 [3SP]· and 8 Bfl-particles per cell. After 5 min of adsorption, 27 ml. of warm broth were added

t Abbreviations used: -BDDR = 5.bromodeoxyuridine; BU-DNA = DNA synthesized in the presence of BUDR; B'U-phage = phage particles containing BU-DNA; TeA = trichloroacetic acid.

518

J. TOMIZAWA AND N. ANRAKU

and the culture incubated for 5 min with shaking. An equal volume of warm broth containing 1·6 x 10-2 M-KCN was then added and the culture was incubated for 60 min. After addition of KCN the culture was not shaken since otherwise mass lysis was induced. The culture was then cooled in ice water and an equal volume of ice-cold buffer was added. The treated cells were sedimented by centrifugation for 7 min at 5000 rev.(min, resuspended in 1·0 ml. cold saline-eitrate buffer, transferred to a small test-tube containing 0·1 ml. of solution of crystalline lysozyme (5 mg(ml.) and 0·1 ml. of 0·1 M-EDTA, and immediately frozen by immersion in a dry ice-acetone bath. As a control, E. coli BB cells were infected with I [3H]- and 8 BU-particles. After 5 min each of adsorption in buffer and incubation in broth the infected cells were centrifuged, resuspended and frozen in the same way as the test sample. A frozen sample was immersed in a water bath at room temperature and refrozen as soon as ice melted. Freezing and thawing were repeated 3 times. Then 0·06 mI. of 20% sodium dodecyl sulphate was added and the tube was kept for 10 min at 37°C and cooled in ice water. Cold water-saturated phenol (1'5 rnl.) was added to the lysate which was then shaken by hand with a rectilinear motion of 15 em amplitude for 3 min at about 120 oscillations/min. After centrifugation for 10 min at 3000 rev.jmin, the phenol layer was removed. The treatment with phenol was repeated and the viscous water layer was then dialysed against saline-citrate buffer at 4°C. About 85% of parental 32p was found in the cells after the treatment with KCN. From the cells, about 80% of 32p was recovered. The radioactive material was insoluble in 5% cold TCA after treatment with 1 N-NaOH for 18 hr at 37°C. The DNA of phage particles was extracted with water-saturated phenol by the slow rotation method described by Frankel (1963). The DNA thus extracted mainly contained intact molecules as shown by zone centrifugation. To minimize unnecessary shear degradation of DNA, transfers of DNA solutions were made with measuring pipettes with a screw delivery. In this report phage(T2, T4, T5 or A)-DNA refers to DNA extracted from phage particles. (e) Infection with DNA preparations of phage A

Phage A-DNA was prepared by a phenol method from a concentrated stock of the phage. The method of infection generally followed Kaiser (1962). E. coli C600 (Aimm434) (generously given by Dr. F. Jacob) was used as a host bacterium, Aimm434 as helper phage and E. coli C600 (Aimm434) as plating bacteria. (f) Density-gradient equilibrium centrifugl}tion of phage and DNA

Phage particles were suspended in about 3 ml. of a CsCI solution buffered with 0·01 M-tris buffer at pH 8,5, at a final density of 1'505 g em-a. The solution was warmed for 1 hr at 45°C and then centrifuged in the SW39 rotor in the Spinco model L ultracentrifuge for 20 hr at 23,000 rev.fmin at about 4°C. DNA was suspended in buffered escI solution (pH 8'5) at a final density of 1·725 g cm-3 , and centrifuged for 36 to 60 hr at 37,000 rev.fmin at about 15°C (Meselson & Stahl, 1958). After the run, 40 to 60 fractions were obtained by puncturing the bottom of the tube (Weigle, Meselson & Paigen, 1959) in a manner similar to that described by Martin & Ames (1961). When fractions had to be used for further experiments, the bottom of the tube was punctured by a sewing needle to collect drops in order to avoid shear degradation of DNA during passage of the fractions through a hypodermic needle. Carrier T4 DNA (2p.g) was added to each fraction to protect the DNA from degradation and from loss due to the sticking of DNA to the walls of test-tubes. Addition of carrier DNA was found essential to recover the radioactive material in a fraction with low DNA concentration. Collection of drops in tubes containing carrier DNA was preferred. Densities of the fractions were calculated from refractive index readings at 25°C measured by a refractometer. (g) Zone centrifugation

Zone centrifugation of DNA was carried out in a density gradient of sucrose, 5% to 20% (Britten & Roberts, 1960), dissolved in saline-citrate buffer. Less than 2p.g of DNA in 0·1 ml. of the same buffer was layered on 5 ml. of sucrose centrifuged 3 to 5 hr

JOINING OF PARENTAL DNA MOLECULES

519

at 30,000 rev.fmin at about IOcC in a Spinco SW39 rotor. The sample was fractionated by drop collection. Assuming structural homology, relative molecular length (L) of DNA species was estimated from the distances sedimented (D), applying the relationship proposed by Burgi & Hershey (1963), ( 1)

This formula is valid for linear molecules. (h) Heat denaturation of DNA

DNA samples, 1 to 4fLgfml., dissolved in saline-citrate buffer with or without 1% freshly neutralized formaldehyde or in CsCI centrifuging medium, were heated at different temperatures for various lengths of time and cooled rapidly in ice water. (i) Shear fragmentation of DNA

Labelled DNA was mixed with carrier T4 DNA to make 2 ml. solution in saline-citrate buffer containing 4fLg DNA. The mixture was placed in a small vial with a diameter of 22 mm and stirred at O°C by a metal rod with a plastic disk of diameter 13 mm attached to a homogenizer (Nihonseiki}. Significant fragmentation of whole molecules of T4 DNA was not observed at a stirring speed of 750 rev.fmin, and the majority of whole molecules fragmented roughly into half and quarter molecules by stirring for 60 min at 1500 and 2000 rev.fmin, respectively. A DNA mixture (4fLgfml.) in saline-citrate buffer was sonicated in a lO-kc sec sonic disintegrator (Kubota) for 30 min at about O°C. This produced fragments of a mean molecular length of about If40 of a whole molecule. ,\ DNA was stirred for 30 min at 3000 rev.fmin to obtain half molecules. (j) Treatments with nucleuses

Crystalline DNase (Worthington, once crystallized) was dissolved in 1 % serum albumin and stored. Since the action of DNase was completely inhibited by 0·05 M-CsCI, samples which had been dialysed against 0·02 M-tris buffer at pH 7·1 were used. Crystalline RNase (Worthington, crystallized from alcohol) was dissolved in water and heated at lOO°C for 10 min to inactivate any contaminating DNase. Samples with or without 0·05 M-CsCI were treated. The action of RNase is reduced to about 10% by CsC1. (k) Measurements of radioactivity

Equal portions of each fraction obtained after density-gradient centrifugations or zone centrifugations were placed on filter paper disks (Toyo filter paper no. 2, diameter 2 cm). Dried disks were treated with cold TCA and alcohol. Air-dried disks were then placed in glass vials filled with toluene-containing scintillators, 1,4-bis-2-(5-phenyloxazolyl)benzene (POPOP) and 2,5-diphenyloxazole (POP). Sometimes portions of each fraction obtained after zone centrifugation were taken directly in glass vials and semi-dried. To each vial 0·5 ml. Hyamine (Packard) was then added. After incubation for 60 min at 37°C with shaking, the vials were filled with toluene-containing soint.illat.ors. 32p and 3H were counted separately using a liquid scintillation spectrometer (Tri-Carb, Packard). The radioactivity of a single nuclear species was measured by a Geiger-Muller counter (Aloka, J.R.C.) or a gas-flow counter (Science Research Institute, Japan).

3. Properties of BU-Particles Light [3H]- and BU-[32P]particles were added to CsCl and fractionated after centrifugation. The radioactivity and infectivity of each fraction were assayed. The radioactivity of light and heavy particles formed separate bands with peaks at densities of about 1·49 and 1'54gcm-3 , respectively. These values are about 0·04 g cm-3 heavier than those reported by Kozinski & Kozinski (1963). The band of radioactivity of the heavy particles was slightly broader than that of light particles,

520

J. TOMIZAW A AND N. ANRAKU

suggesting slight heterogeneity of incorporation of BU . Infectivity assay showed th at t he heavi er particles had , on t he average, less infectivity per unit radioactivity, but even t he heaviest fra ctions contain ed infective particles. These results show that a certain fra ction of BD-particles la ck infectivity (Dunn & Smith, 1954) and that, on the average, the greater the content of BD the less infecti ve are the particles. Therefore, it is important for t he experiments reported here to characterize the damage du e to the incorporation of BU. Th e ability of BU-particles to ad sorb to E. coli BB cells was measured with purified BU-[32P]particles and it was found not to be different from light [32P]particles. Dete ctable numbers of particles lacking adsorbing a bility t o the cells (Kozinski & Kozinski, 1963) were not found in our preparations. Non-infective particles still retained ability to kill ba cteria. Assuming a Poisson distribution of lethal particles on bacteria, the number of lethal particles can be calculated from t he total number of bacteria mixed with t he particles and that of bacteria surviving after mixing. The number of lethal par ti cles thus assayed was 2 to 3 times greater than t he number of active particles, depending on preparations, and agrees well with the number evaluated on the ba sis of th e DNA content of a preparation. The number of BU-particles refers to that of the bacteria-killing particles. Functional activity and genetic potentiality of the rlI A eistron of a BUparticle were examined by the sam e method used to characterize the damage induced by u.v. irradiation (Kreig , 1959). E. coli KI2('\) cells were infected at a multiplicity of 0·1 of BD-T4r+ particles, 70% of which lack infectivity, and a mul tiplicity of 2 of normal T4rII A mutant particles. Infectiv e centres were plated on E . coli B(S) and E . coli KI2('\). Practi cally all th e cells infected with BD-T4r+ particles formed plaques on both st rains. Therefore t he rIl A cistron of most Bfl-particles appears to be intact both functionally and genetically. We therefore conclude that damage due to incorporati on of BD is not extensive and that intact parts of a genome could be rescued by genetic recombination. In other words, chromosomes of the noninfective particles can participate in genetic recombination. Thus t he use of BU as a densit y lab el in the experiments can be justified.

4. Properties of DNA extracted from Ordinary T4- and BU-T4 Particles (a) CsCl density gradient centrifugation

DNA ext ract ed from BU-particles cent rifuged for 60 hours in CsCIdensity gradient form ed a band with a peak at a densit y about 1·795 g cm-3. The band was broader than that of DNA extracted from light phage particles, indicating heterogeneity of incorporation of BU. Band profiles of light [3H]DNA and BU -[32P]DNA extracted from phage particles are presented in "F ig. 1. The band of BD-DNA is slight ly broader than those reported (Schildkraut et al., 1962; Kozinski, 1961). The separation of the bands is, however , good enough for the present purpose. It was not difficult to fra ctionate a phage preparation to obtain more homogeneously dense parti cles, but the use of su ch preparations did not significantly improve the outcome of the experiments. Becaus e of the heterogeneity , the term "density of BD·DNA" cannot be defined precisely. The term is used to indicate the range of densities at which the majorit y of BD-DNA molecules band in the density gradient under the experimental conditions used. The term may well be understood as the density at which the BUDNA form s a peak.

JOINING OF PARENTAL DNA MOLECULES

521

Density (9 cm-3}

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FIG. 1. CsCI density gradient analysis of [3H]DNA and BU·[3'P]DNA extracted from phage particles. (0) for 3H and (.) for 3·P. Total number of fractions is 44.

(b) Thermal denaturation

The melting temperature of T4 DNA in saline-citrate buffer measured by the optical method (Marmur & Doty, 1961) was 84·5°0 and that of BU-DNA was 3 to 4°0 higher (Kit & Hsu, 1961; Szybalski & Menningmann, 1962; Kozinski & Beer, 1962; Inman & Baldwin, 1962). A mixture of (3H]DNA and BU-[32P]DNA was heated at a given temperature for 10 minutes and then cooled in ice water. The concentration of DNA was about 1 f'gfml. The samples were added to OsOI and centrifuged for 36 hours. The separation of the bands of single- and double-stranded DNA molecules was not necessarily sharp, probably due to the presence of partially denatured or partially renatured products. The extent of strand-separation of ordinary or BU-DNA can, however, be estimated from the band profile of the heated DNA. Samples of [3H]DNA, heated at 84°0 and 86°0, gave bands with the same density as that of the unheated sample. When heated at 88°0, about 60% of the label formed a band at the density corresponding to single-stranded polynucleotides. Heating at 90°C was found necessary for complete separation of both strands of [3H]DNA. For BU.(32P]DNA, heating at 86°0 did not significantly change the band profile from that of an unheated sample. Heating at 88°0 caused about 25% of the label to band as the single.stranded form. Complete separation of both strands of BU· [32P]DNA required heating above 93°0. Since no interaction between [3H]DNA and BU-(32P]DNA was observed by these treatments, any renaturation during rapid cooling of the heat-denatured DNA molecules must take place between the originally paired strands; the strands may be prevented from separating by a few residual base pairs within a DNA molecule. This is in agreement with the results obtained by

J. TOMIZAWA AND N. ANRAKU

522

both optical measurements (Schildkraut, Marmur & Doty, 1961; Geidschek, 1962) and centrifugation analysis (Freifelder & Davison, 1962). In the experiments to be reported here, heating at 94°C in saline-citrate buffer, at 100°C in CsCI centrifuging medium or 93°C in saline-citrate buffer with 1% HCHO was used to separate the strands of T4 DNA with or without incorporated BU. Heating at 84°C was chosen as the condition not to separate them. By such heating, debromination, which causes measurable change in density of BU-DNA, was not observed. (c) Zone centrifugation of fragments formed by shear or enzymic degradation

Stability of BU-[32P]DNA against shearing force or enzymic digestion was compared with that of [3H]DNA. To estimate the size of fragments produced by these treatments the following samples were sedimented through sucrose gradients:

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FIG. 2. Zone centrifugation analysis of mixture of(3R]DNA and BU-[32P]DNA extracted from phage particles with or without sonication or enzymic digestion. (0) for 3R and (.) for SIP. (a) Untreated mixture centrifuged for 3 hr. Total number of fractions is 60. (b) Mixture sonicated for 30 min and centrifuged for 5 hr. Total number of fractions is 60. (c) Mixture treated with 2 x 10- 3 /Lg/ml. of DNaae for 10 min at 25°0 and centrifuged for 3 hr. Total number of fractions is 30.

(a) DNA extracted from [3H]_ and BU-[32P]particles and mixed with non-labelled carrier DNA to give a final concentration of 2lJ-gJml. in saline-citrate buffer (Fig: 2(a)); (b) a DNA mixture, at a concentration twice that of above sample, sonicated for 30 minutes (Fig. 2(b)); (c) a DNA mixture extracted from [3H]_ and BU_[32P]par_ ticles dialysed and treated with 2 x 1O-3IJ-gJml. of DNase for 10 minutes at 25°C in the following mixture (Fig. 2(c)): DNA, including [3H]DNA, BU-[32P]DNA and carrier DNA, 10IJ-gJml.; 4xlO-3M-MgCI 2; 2xlO-2M-tris buffer, pH 7'1; 5 x 10-3 M-EDTA (added at 10 minutes). Samples (a) and (c) were centrifuged for 3 hours and sample (b) for 5 hours. The results presented in Fig. 2 give the following information. (1) DNA extracted from phage particles is quite homogeneous. (2) BU-[32P]DNA sediments about 10% faster than [3H]DNA with the same molecular length. (3) [3H]DNA and BU-[32P]DNA are equally sensitive to sonication. With a low rate of shear, no significant difference in the sensitivities of light and heavy DNA molecules was observed. These findings are in disagreement with the statement by Szybalski (1962). (4) [3H]DNA and BU-[32P]DNA are equally sensitive to DNase.

JOINING OF PARENTAL DNA MOLECULES

523

T4 [3H]DNA, T2 [32P]DNA and both native and sheared ,\ DNA were mixed and centrifuged through a sucrose gradient for 3 hours. Radioactivity of T4 and T2 DNA's and infectivity of A DNA in each fraction were assayed. Bands of T4 and T2 DNA's overlapped almost perfectly and they sedimented 1·6 times faster than the fast-sedimenting component oftransforming'\ DNA. The fast-sedimenting component of transforming ,\ DNA sedimented 1·25 times faster than the slow-sedimenting component.r The fast- and slow-sedimenting components are supposed to be whole and half molecules of ,\ DNA, respectively (Kaiser, 1962). These results indicate the validity of equation (1) for estimation of molecular length by sedimentation and also show that the majority of DNA molecules extracted from T4 phage particles are intact whole molecules.

5. Properties of DNA extracted from Infected Cells (a) Sedimentation in CsCI density gradient

DNA extracted from cells infected with 1 [32P]_ and 8 BU-particles and treated with KCN from 5 to 65 minutes was mixed with DNA from cells infected with 1 [3R]_ and 8 BU-particles but without incubation with KCN and suspended in buffered CsCI. After centrifugation for 60 hours, 44 fractions were collected and the radioactivity of each fraction was assayed (Fig. 3). [3H]DNA showed a single band with a peak at a density characteristic of T4 DNA and its shape was almost symmetrical. On the other hand, the band of [32P]DNA was skewed towards heavier densities up to the density of BU-DNA. For further analysis of this result and also for testing the reproducibility of the experiment, fifty such experiments, with or without slight changes in various conditions, were performed. 32p unaccompanied by 3H was always found in fractions with densities intermediate between that of light DNA and BU-DNA. However, the proportion of 32p in the intermediate fractions varied from experiment to experiment. Even though apparently the same procedures were used, different extents of skewness were observed. This does not seem to be done to the difference in extraction and sedimentation procedures. At the moment we do not have a consistent interpretation of the differences. However, the material in the fractions with intermediate density invariably showed the same characteristic properties as those presented below. The experiment with the results presented in Fig. 3 gave the largest proportion of 32p in the fraction with intermediate densities. DNA extracted before treatment with KCN has never shown skewing. Preparations of [32PJ. and BU-DNA mixed together in broth with or without KCN were added with CsCI and centrifuged. 32p in DNA formed the same band as that of a single component of light [32PJDNA. A sample of the mixture of fractions 22, 23 and 24 in Fig. 3 was recentrifuged in a density gradient (Fig. 4(a)). 3H formed a single band at the density of the light DNA. On the other hand, 32p showed bimodal distribution with one peak at the light density and one at the density of the original fractions. The bimodal distribution of 32p is due to the presence of the light [32P]DNA in fraction 24 of the first run as expected from the presence oPH in the fraction. A band found after recentrifugation of a fraction at a density 1·755 g cm-3 obtained in another similar experiment is shown in Fig. 4(b). These results undoubtedly show that a part of [32P]DNA changes its density. t This part of the experiments was carried out in collaboration with Mrs. T. Ogawa.

524

J. TOMIZAWA AND N. ANRAKU Density (9 cm-3)

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FIG. 3. CsCI density gradient analysis of mixture of DNA extracted from cells infected with 1 [s'P} and 8 BU-particles and treated from 5 min to 65 min with 8 x 1O-s M-KCN and DNA extracted from cells infected with 1 [3R]_ and 8 BU-particles incubated for 5 min. Total number of fractions is 44. (0) for 3R and (.) for 3·P.

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525

JOINING OF PARENTAL DNA MOLECULES

(b) Sedimentation properties in zone centrifugation

To estimate the physical size of the labelled molecules, extracts from the infected cells were analysed by sedimentation in sucrose gradient. The result obtained from the extract from cells infected with [32P]_ and BD-particles and treated with KCN is presented in Fig. 5(a). Figure 5(b) represents the sedimentation profile of [32P]DNA 1

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FIG. 5. Zone centrifugation analysis. (a) DNA extracted from cells infected with 1 [3SP]_ and 8 BU-particles and treated with KCN from 5 min to 65 min. [3H]DNA was added before centrifugation. Arrows indicate the positions for whole, half and quarter molecules of T4 DNA. Total number of fractions is 30. (b) Fraction of density 1·73 g cm- 3 obtained after density-gradient centrifugation of the above sample and dialysed against saline-citrate buffer and [3H]DNA added as a reference. (0) for 3H and (.) for 32P.

in a fraction of density 1·73 g cm-3, taken from fraction 22 in Fig. 3, and run after dialysis against saline-citrate buffer. The sedimentation behaviour of [3H]DNA extracted from the cells without KCN treatment showed no significant difference from that of [32P]DNA extracted after the treatment. Since phage DNA molecules fragment during similar extraction procedures, the distributions do not necessarily reflect the size of molecules in the infected cells. They simply reflect distribution of label in molecules of different physical sizes in the extracts. The majority of the label was found in molecules with sizes corresponding to whole or half molecules of T4 DNA. It must be noted that even though the DNA in the fraction of intermediate density is a complex of [32P]DNA and BD-DNA, as will be discussed, its sedimentation behaviour did not show significant difference from the bulk of the [32P]DNA or [3H]DNA extracted from the cells. (c) Thermal denaturation

The mixture of fractions 22, 23 and 24, whose profile is presented in Fig. 4, was heated for 30 minutes in the CsCI centrifuging medium, and rapidly cooled and centrifuged for 60 hours. The profile of the heated sample is presented in Fig. 6(a). All the radioactivity is found in a single band at the density of heat-denatured light DNA. Since, under these conditions, breakage of nucleotide bonds was suspected, DNA was heated under milder conditions. A fraction of density 1·74 g cm-3 obtained in another experiment was dialysed against saline-citrate buffer and added

J. TOMIZAWA AND N. ANRAKU

526

to [3H]DNA. The mixture was then heated at 93°0 with 1 % HOHO for only 10 minutes and cooled rapidly. The correspondence between the profiles of [32P]DNA and [3HJDNA shows not only that the strands of labelled DNA separated, but also complete separation of [32PJDNA and BD·DNA (Fig. 6(b)). Dialysed preparations of DNA in a fraction with intermediate densities were heated in saline-citrate buffer without HOHO at 94°0 and 84°0 for 10 minutes and cooled. A sample heated at Density (9 cm-3)

1·7

I·,

I·

40 0

n

1\ JI

30 ~

I

\ I \ I I

.oj

:>

Q.

£ 15 .oJ

B

20

'-

I

20

I

0

.

I

30

>c, > u

0

10

~

0

15 Fraction no.

FIG. 6. CsCl density gradient analysis of labelled material in the fractions with intermediate densities after thermal denaturation. (a) Mixture of fractions 22, 23 and 24 in Fig. 3 was heated for 30 min at 100°C in CsCl centrifugation medium and cooled rapidly. Total number of fractions is 40. (b) A fraction at a density 1·74 g cm- s was heated for 10 min at 93°C in saline-eitrate buffer with 1 % HCHO. (SH]DNA was added before heating. Total number of fractions is 56. (0) for sH and (.) for "P.

94°0 gave a pattern similar to that presented in Fig. 6, but in the sample heated at 84°0 there was no change in the original density. The results indicate that treatment of DNA of intermediate density at the temperature at which double-stranded lightor BD-DNA separates breaks the association of light and heavy components of the DNA. Treatment at a temperature which does not cause the melting of DNA does not cause dissociation. (d) Shear fragmentation

A fraction of a density 1·735 g cm-3 dialysed against saline-citrate buffer was mixed with [3HJDNA and carrier DNA to make 2 ml. of solution containing 2 J.Lgjml. of DNA. The mixture was stirred for 60 minutes at 1500 rev.jmin. A portion (0-1 ml.) was centrifuged through a sucrose gradient. Since the amount of 32p was too small for accurate assay after zone centrifugation, the extent of fragmentation of molecules with 32p was estimated from the [3H]DNA. Instead of analysing the distribution of 32p of the sheared molecules, excess [32PJDNA was added as a reference just before zone centrifugation. Figure 7(b) shows that [3H]DNA was broken into fragments corresponding roughly to half molecules of T4 DNA. Since the stirring speed, 1500 rev.jmin, is close to the second "critical speed" (Burgi & Hershey, 1961) at which half molecules of T4 DNA are fragmented into quarter molecules, molecules with 32p

JOININ G OF P ARENT AL DNA M O L EC U L ES

527

which ar e longer than a half molecule might be fragmented. The remainder of the st irred sample (1,8 ml.) was added to CsCI and centrifuged for 60 hours (Fig. 7(a». The 32p in DNA formed small peaks at the light densit y and near the density of the original fra ction and tailed off near t he heavy density. Th e lat ter peak was apparently formed from DNA not broken by stirring. After further fragmentation , [32P]DNA formed a single band with a peak near the light density and skewed toward the heavy density side of the band. With in creasing fragmentation less skewness was observed. The results obtained after intensive fragmentation by sonication are presented below. D ensity (9 cm- 3 )

1·8

1·7

32p

~ ....,

15

::>

c,

.e::

:s

.9 .....o

10

c

'" i5u

a: '"

5

Fraction no.

FIG . 7. CsCI density gradient analy sis and zon e cen t rifuga tio n an a lysis of a fraction of an in te rmed iate density after shear fragmentat ion. A fraction of a density 1·73 5 g cm - 3 was mixed with [3HIDN A an d stirred for 60 mi n at 1500 rev.jm in a n d an alysed . T o t he sa m ple for zone cen t rifug ation, excess [32P]D N A was added befo re centrifugation (see text ). ( 0) for 3R and ( e) fo r " P . (a ) D ensi t y gradien t an al ysis. T ot al n umber of fracti ons is 42. (b ) Zone cen t rifugat ion a nalys is. Tot al number of fractions is 33. Ar rows indicate t he pos ition of half an d quarter molecules of T4 D NA .

A fra ction at a density 1·755 g cm-3 dialysed again st saline-citrate buffer was mix ed with [3HJDNA and carrier DNA to make a 2·5-ml. solution contain ing 4p.g/ml. of DNA and sonicated for 30 minutes. A portion (2 ml.) was added to CsCI and centrifuged for 60 hours and another portion (0·1 ml.) was centrifuged in a sucrose gra dient for 5 hours. The results of the density gradient ana lysis show th at most of 32p forms a band with a peak near t he light density (Fig . 8). The band, however , shows slight but significant skewness toward t he heavy densit y side. No concentration of 32p at a density corre sponding to " hybrid " D NA was observed . After zone centrifugation [3H]DNA formed a band similar to t hat presented in Fig. 2(b). If equation (1) is valid . for soni cated molecules, t he size of [3H]DNA fragmen ts is estima ted to be about 1/40 of the whole molecule of T4 DNA. A fraction wit h added [3HJDNA and carrier was stirred at 750rev./min for 60 minutes. [3H]DNA was practically intact after stirring and the band of 32p in the CsCl density gradient was not significantly different from that of an uns tirred sample. 35

J. TOMIZAWA AND N. ANRAKU

528

From these results it may be concluded that molecules of intermediate density have a stability to shear similar to that of whole molecules of T4 DNA and that fragmentation of the molecules produces some molecules of light density and some of intermediate density. Molecules of heavy density must be simultaneously produced although they could not be detected because of the lack of a suitable label. [32P] molecules of "hybrid" density do not accumulate. A quantitative analysis of these results will be made in the Discussion, Density (g cm-3)

1·8

1·7

10 /0"'0"0\

~

(,

8

....::>

0-

S

0....

6

.... 0

'0 4 to

'"

> 0 u

CIi

'"

2

20 Fraction no.

FIG. 8. CsCl density gradient analysis of the product of sonication of a fraction of an intermediate density. A fraction of a density 1·755 g cm- 3 was mixed with [3R]DNA and sonicated for 30 min. Total number of fractions is 55. (0) for 3R and (.) for 3·P.

(e)

Treatments with nucleases

Each of the fractions of densities 1,72, 1-73 and 1·75 g cm-3 was mixed with 2jLg of carrier DNA and dialysed against 2 x.1O-2M-tris buffer, pH 7·1. The dialysed samples were mixed with [3H]DNA, non-labelled T4 DNA and MgCl2 to make 1 ml. of solution with the following composition: DNA, 10 jLg; 2 X 10-2 M-tris buffer, pH 7,1, 5 x 10-3 M-MgCI 2. These samples were then treated with 2 jLg of DNase at 25°C for 60 min. Before and after the treatment, samples were taken in cold 5% TCA with 0·1 ml. of 1% serum albumin as carrier and the precipitated DNA was washed once with 5% TCA. After the treatment, neither 32p nor 3H was detected in the precipitates. Thus the [32P]DNA of intermediate density was completely hydrolysed to acid-soluble materials with DNase. To analyse the products of brief digestion with DNase, the fraction of density 1·76 g cm-3 was dialysed, and 2 ml. of a solution with the following composition was made: DNA, including [32P]DNA in the fraction, [3H]DNA extracted from phage and nonlabelled T4 DNA, 10 jLgjml.; 2 x 10-2 M-tris buffer of pH 7·1, 5 x 10-3 M-MgCI 2_ The solution was then treated with 5 x 10-4 jLgjml. of DNase at 25°C. After 10 minutes the reaction was terminated by the addition of 0·2 ml. of 0·1 M-EDTA. A portion (0-1 ml.) of the sample was used for zone centrifugation. Since the amount of 32p in the sample used for zone centrifugation was too small to measure accurately after fractionation, the extent of action of DNase was estimated from the band profile of [3H]DNA. [32P]DNA was added as a reference just before centrifugation (Fig. 9(b)).

JOINING OF PARENTAL DNA MOLECULES

529

Although accurate measurement of the extent of enzyme action from the result is difficult, it may be estimated that [3H)DNA was, on the average, subject to a little more than one random double-strand breakage per molecule. The rest of the sample (1,8 ml.) was added to CsCI and centrifuged. The results (Fig. 9(a» were analogous to those obtained after shear fragmentation (Fig. 7(a». A large peak at the density of the original fraction is due to non-fragmented complex molecules. The density of the fragmented molecules varied from the density of the light DNA to that of BU-DNA. The results will be discussed later. Density (9 cm- 3 )

re

I

~

... :J

a.

.s

"30

...

1·7

I

'l 10

'0

c '>0u"

5

'"

0::

Fraction no.

FIG. 9. CsCI density gradient analysis and zone centrifugation analysis of a fraction of an intermediate density after brief digestion with DNase. A fraction at a density 1·76 g cm- a was mixed with [3HJDNA and treated with 5 x 10-4 ",g/m!. of DNase at 25°C for 10 min. To the sample for zone centrifugation, excess [32PJDNA was added before centrifugation (see text). ( O) for aH and (.) for a2p. (a) Density gradient analysis. Total number of fractions is 42. (b) Zone centrifugation analysis. Total number of fractions is 55. Arrows indicate the position of half and quarter molecules of T4 DNA.

The fraction with a density 1·75 g cm-3, without dialysis, was mixed with [3HJDNA, carrier DNA and saline-citrate buffer to make a concentration of DNA of 5/-kg/ml. and treated with 50/-kg/ml. of RNase for 30 minutes at 25°C. The treated sample was added to CsCI and centrifuged for 60 hours. A band of 32p with a peak at the density of the original fraction was observed. Although CsCI in the reaction mixture inhibited the action of the enzyme to about 10%, the amount of enzyme was shown to be about 104 times as much as that required to digest an amount of bacterial [32PJRNA corresponding to that of the [32P)DNA in the fraction considered. It is therefore concluded that both the light and heavy components of the complex of intermediate density are resistant to RNase.

6. Synthesis of DNA during Treatment with KeN E. coli BB cells were grown in broth, infected with 1 [3H]- and 8 BD-particles per cell in buffer without phosphorus, centrifuged and resuspended in warm broth. Following incubation for 5 minutes, an equal volume of warm broth containing

530

J. TOMIZAWA AND N. ANRAKU

1·6 X 10-2 l\I-KCN and 32PO 4 was added. After further incubation for 60 minutes the cells were sedimented and the DNA extracted as usual, except that the extract was treated for 30 minutes at 37°C with 100 fLgfml. of RNase. The extract was centrifuged for 60 hours in a density gradient. After centrifugation and fractionation, the radioactivity in each fraction was assayed. Since we know the radioactivity of 3H per infected ceIl, we can calculate accurately the amount of incorporated 32p per infected cell from the relative amount of 3H and 32p in DNA fractions. Only 0·06 to 0·3 phage unit per infected ceIl of 32p was found in DNA fractions. No concentration of 32p in fractions of intermediate density was observed.

7. Discussion (a) Shift in density of [32P]DNA Density gradient analyses of DNA from KCN-treated celIs mixedly infected with [32P]_ and BD-particles showed the 32p at fractions with densities greater than that of the input [32P]DNA. Recentrifugation showed that the density of the radioactive material is intrinsic. Therefore, we come to the inevitable conclusion that light [32P]DNA must associate in some way with heavier component(s). Association with RNA as a cause of the shift in density of [32P]DNA was first suspected. RNA had not been removed from the extract. Most of the RNA is sedimented in the centrifugation, the remainder tailing off at the fractions containing BD-DNA. [32P]DNA was not found at densities heavier than that of the BD-DNA. In addition, [3H]DNA used as a control did not change its density, although there would have been the same possibility of its interaction with RNA during the extraction. Therefore, non-specific aggregation of [32P]DNA and RNA could be rejected as a cause of the shift in density of [32P]DNA. Natural association of DNA and RNA has been reported (see Marmur, Rownd & Schildkraut, 1963). Spiegelman, Hall & Storck (1961) found a hybrid which appeared to be composed of one single-strand of DNA and one strand of RNA, and Schulman & Bonner (1962) reported a complex probably made up of a double-stranded DNA and a single-strand of RNA. However, the complex DNA reported here has the following distinctive characteristics. (1) By treatment with deoxyribonuclease all the radioactive materials in the complex can be made acid-soluble. (2) The heavy component of the complex is also sensitive to deoxyribonuclease. (3) Treatment of the complex with ribonuclease does not change its density. (4) The complex dissociates at the temperature at which double-stranded T4 DNA dissociates. (5) Results of shear fragmentation of the complex strongly suggest linearity of the complex molecule with radioactive light and non-radioactive heavy components joined end to end. In view of these properties it is extremely unlikely that RNA is the component of the complex which makes it heavy. We will now turn to a discussion of the association between DNA molecules. Aggregation of DNA from phage seems to be specific; thus ,\ DNA shows no tendency to form a complex with T5 DNA (Hershey, Burgi & Ingraham. 1963). In the extract from the infected cells, the majority of phage DNA was BD-DNA whose main band in the density gradient appeared at the same position as BD-DNA

JOINING OF PARENTAL DNA MOLECULES

531

extracted from particles. Since no concentration of [32P]DNA at or near th e densi ty of BU-DNA was observed, it is unlik ely that many molecules of DNA aggregate to form a complex. [32P]DNA with an intermediate density did not sedim ent faster than a whole molecule of phage T4 . As a control, [3H]DNA was similarly ext racted from cells infected with fH]- and BD-phage particles but not treated with KCN. There was no shift in the density of [3H ]DNA, which eliminates t he possibility of aggregation during extraction and handling. The stability of t he complex against shearing forces strongly suggests that it is not a simple aggregate. Th erefore the shift in density of [32P]DNA must be due to the association in some way with BUDNA which took place during the treatment of infected cells with KeN . For the sake of convenience, we shall refer to a " molecule" formed by such an association as a "joint molecule". The relevance of this terminology may be judged from th e discussion below. (b) Non-essentiality of DNA synthesis for formation of joint molecules

In cells mixedly infected with [3R]_ and BU-particles and incubated in a medium with 32P0 4 and KCN, very little 32p was found in DNA. Although the possibility cannot be eliminated that newly-synthesized DNA has some special role in the association of heavy and light components, the fact that there is no concentration of [32P]DNA synthesized during treatment with KCN in fractions of intermediate densities allows the conclusion that synthesis of DNA is not essential for formation of joint molecules. This inference is further supported by the finding th at the DNA extracted from KCN-treated cells infected with [32P]_ and BU-particles contains some 32p with a density corresponding to that of BU-DNA (Fig. 3). If substantial syn thesis were essential for joining, very den se label would not be found . The incubation of the infected cells for 5 minutes before addition of K CN may add some complications, but this procedure was necessary for t echni cal rea sons. The elevated frequencies of genetic recombination after treatment with K CN previously observed in cells which were not pre-incubated (Tomizawa & Anraku, 1964) were found to be little affected by th e pr e-incubation. In addition, no synthesis of DNA was detected during this period. (c) Structural models of joint molecules

The results discussed above suggest that two parental DNA molecules associate to form a joint molecule. To facilitate understanding of the subsequent discussion we will first describe the structural models for a joint molecule a nd then discuss the supporting evidence. The models proposed are presented in Fig. 10. They have the (a)

(b)

:>...,-,-,-~,-,-",-cc",-,-......~~,-,-,-,-,-,-,--o...,-,-,-,--c F IG. 10. Models of a joint mol ecul e. Double-stranded DNA mol ecul es join t ogether at a homo logous ov erlapping region by h ydrogen b onds. Mod els (a) and (b ) differ in the number of paired ov erlapping regions.

532

J. TOMIZAWA AND N. ANRAKU

following charact erist ics. First, t he molecule is composed of two components and each component has the dou ble-stranded struct ure of the parental molecules. Secondly, even t hough each component ha s two ends of its own, t he' two component s form a single linear structure as a whole. Thirdly , t he compo nent s are joined end to end at overla pping homolo gous regions by hydrogen bonds bet ween complementary base-pairs. Fourthly, the overl apping r egion is short compared with the t otal length. From the r esults of purely genetic experiments, Doermann & Boehener (personal communication) proposed the same mod els for the structure of a heterozygote. (d ) Properties of the bonds connecting the heavy and the light components of a j oint molecule

Aft er various heat treatmen ts which separa te double-stranded BU -DNA into two single strands, all the [32P] component in a joint mol ecule appeared at fractions at which single-stranded light DNA form ed a band. In other words, the light and heavy components separate d completely by the same treatment which splits hydrogen bonds between complementary base pairs of T4 DNA. On th e other hand, after heating at a temperature slightly lower than the melting t emperature of T4 DNA, joint molecules retained their original density. This treatment breaks nucleotide bonds rarely if at all. Therefore, the bonds connecting the compon ents must be hydrogen bonds, probably formed betwe en complementary base pairs. In these chara ct eristics, a joint molecule is not different from the " hy brid" molecule formed aft er replication of heavy DNA in a light medium (l\tIeselson & Stahl , 1958) or formed after den aturation and subsequ ent renaturation of heavy and ligh t DNA (Doty, Marmur, E igner & Schildkra ut, 1960). (e) S tructu re of a joint molecule as a whole

The sed imentation properti es of joint molecules in CsCl or sucrose density gradients are quite similar to those of T4 DNA molecules. Stirring a t 750 r ev.jmin, which produ ces a shearing force lower than that required to fragment whole molecules of T4 DNA, docs not significantly break joint molecules. On the ot her hand, stirring at 1500 rev.fmin leads to considerable br eakage of joint molecul es. These resul ts suggest a linear structure for the joint molecule. To confirm the lin earity of a join t mole cule the product s of shear fragmentation were analysed. Before presenting t he quan titative analysis some pertinent properties of joint molecules may be discussed. Afte r fragmentation of lab elled DNA in fra ctions with intermediate densities, a considerable portion of the label still appeared at the intermediate fractions (Figs. 7(a) and Sia l). This shows that the bonds connecting the two components, light and heavy, are not selectively broken by shearing. Analysis by zone centrifugation (Fi g. 2(b)) showed that the stability of BU·DNA to shearing is similar to that of the light DNA. Therefore, if a joint molecule has a linear structure, the mode of a cti on of shearing force on a joint molecule must be similar to that on an ordinary linear m olecule. Analysis of the distribution of radioactivity of th e sheared fragments of joint molecules in a CsCI den sity gra dient supports linear ity of a joint molecul e. Th is will be seen in greater detail in th e following sections. (f ) D istribution of

32 P

in joint molecules in CsCl density gradient

In the pr ecedin g discussion it was suggest ed that joint molecules are composed of light [32P]DNA_ and BU-DNA-components in various ratio s. With t he following assum pt ions, the distribution of 32p in joint molecules in t he CsCI density gra dient

JOINING OF PARENTAL DNA MOLECULES

533

can be theoretically derived: (1) all BD-DNA molecules have the same density; (2) all molecules have a constant length and molecules with any given density form the same Gaussian distribution with its maximum at that density; (3) ratios of [32P]DNA_ to BD-DNA-components in the molecules range from infinity to zero and all values occur with equal probability. If we denote the number of 32p atoms in a light molecule without ED by a, the number of 32p atoms per molecule with a density x is given by

a(Xb-X) xb-xp

where x b and xp are the densities of BD·DNA and the light DNA, respectively. The fraction of 32p which contributes to a density xh of molecules of a density x is

((X-Xh)2)

1 ~(27T)a exp -

2a2

where a is the standard deviation of distribution of molecules of a given density. Therefore the number of 32p atoms per molecule at a density xh is cIXh)

=

f:: ~(2:)a (~b-=-~)

= ~(27T)a~Xb-Xp)

[a

exp ( -

2{ex

p(

(X;a~h)2) dx

_(Xb~:h)2)_exp( _(XP2~:h)2)}

-(xb-xh)

l

(2)

( y2 ) ] exp --2 dy 2a

Xb- x b

xp-xh

where y = X-xh' The integral of the last term in the equation can be evaluated with the help of numerical tables of the probability integral. Curves corresponding to two cases of interest are drawn in Fig. Ll.. The smaller value of o corresponds to the standard deviation observed for the DNA extracted from infected cells, and the other value is that of DNA sonicated for 30 minutes. With the same assumptions we can theoretically derive the curves representing the density profiles of 32p in the mixtures of the light molecules and joint molecules in any given proportions. Obviously, the properties of DNA extracted from infected cells do not strictly fulfil any of the assumptions stated above and the fractionation of the sample introduces inevitable artefacts. Nevertheless, by comparing the curves obtained experimentally with those derived theoretically, some significance of the experimental curve may be ascertained. For this purpose strict satisfaction of the assumptions may not be necessary. In apparent contradiction to one assumption, for example, BD-DNA is not a homogeneous molecular species, and yet consideration of the density heterogeneity of BD-DNA does not change the shape of the theoretical curves very much. The length of the molecules is actually not constant. Nevertheless, the observed distribution of 3H in DNA extracted before treatment with KCN can be approximated by a Gaussian distribution with a = 0·11 g cm-3 (this value is greater than that theoretically expected mainly because of artefacts caused by fractionation procedures; similar broadening effects are expected for molecules of any given density). Similarity in the deviation of the distribution of radioactivity in joint molecules of a given density in the density gradient to that of the light

534

J. TOMIZAWA AND N. ANRAKU

G(x)

x FIG. 11. Distribution of a.p of joint molecules in CsCI density gradient as derived theoretically. Curves A and B correspond to o = and 0·20 g em-a, respectively.

o-n

Density (g em-3)

\·8

·7

1'7

30

20

~ ...., ::>

a. .5

0....,

.B

....0

10

.,C> .,0u ex:: 15 Fraction no.

FIG. 12. Comparison of distribution of radioactivity in CsCI density gradient experimentally obtained for DNA extracted from infected cells and that calculated for a mixture of the light molecules and joint molecules. Results presented in Fig. 3 are analysed. Dotted lines are experimental curves and continuous lines are calculated ones. Curves in (a) are for distribution of a.p. A thick continuous line shows distribution of up as calculated for a mixture composed of 1/5·5 of total s'p in joint molecules. Thin continuous lines are distributions of up as calculated for the light molecules and for joint molecules. Curves in (b) are for distribution of [SHJDNA. The continuous curve shows a Gaussian distribution of a = 0·11 g cm- s.

JOINING OF PARENTAL DNA MOLECULES

535

molecules is suggested from the results of density gradient and zone centrifugations (Figs. 4 and 5). Many things remain to be discussed, but, to a first approximation, the first two assumptions are probably adequate. Consequently, if the last assumption is acceptable, we may be justified in comparing the distribution of 32p in the density gradient obtained experimentally with that derived theoretically. A theoretical curve which corresponds to having 1/5·5 of the total 32p in the joint molecules agrees fairly well with the experimental curve (Fig. 12). The agreement supports, in turn, the validity of the assumption in question, namely, a flat distribution of molecules between the density of the light DNA and that of EU-DNA. If a joint molecule has a linear structure with two components, light and heavy, joined end to end, the agreement would indicate that the two components join anywhere in a joint molecule with equal probability. (g) Linearity of a joint molecule and some properties of the heavy components

If the products of sonication of joint molecules have a constant molecular length and the effect of contribution of an overlapping region on density of the product containing such a region is not significant, the distribution of 32p in fragments Density (9 cm-3)

10

Z .0> :;J

8

a. .S

-0 .0>

6

.8

....0

4

?:-

'"

> 0 u

~

2 20 Fraction no.

FIG. 13. Comparison of distribution of radioactivity in CsCl density gradient experimentally obtained by sonicated products of joint molecules and that calculated for a mixture of the light molecules and joint molecules. Results presented in Fig. 8 are analysed. Dotted lines are experimental curves and continuous lines are calculated ones. Curves in (a) are for distribution of 32P. A thick continuous line shows distribution of 32p as calculated for a mixture composed of 1/15 of total 82p in joint molecules. Thin continuous lines are distribution of 32p as calculated for the light molecules and joint molecules. Curves in (b) are for distribution of 3H. A continuous curve shows a Gaussian distribution of a = 0·20 g cm- 8 •

in the density gradient can also be calculated from the equation. Assuming that the product of 30 minutes' sonication fulfils such requirements, the results presented in Fig. 8 will be analysed. The distribution of fragmented [3H]DNA shows perfect agreement with a Gaussian distribution of a = 0·20 g cm-3 (Fig. 13(b)). The same deviation of the distribution for molecules of any given density is assumed. Then if the ratio of the amount of 32p in joint molecules to that in the light molecules is known, the distribution of 32pof fragments in the density gradient could be calculated. However, the heterogeneity of the original joint molecules both in molecular length and in density makes it difficult to estimate the ratio of the amount of 32p in joint molecules to that in the light molecules of sonicated products. Therefore, we will

536

J. TOl\IIZAWA AND N. ANRAKU

take a species of joint molecules which has a length of half a molecule of T4 DNA and has a density, 1,755, composed of 40% of [32P]DNA- and 60% of BU-DNAcomponents, as a representative of the original joint molecules and calculate the ratio as for the products of sonication of such molecular species. If both components of a joint molecule are equally sensitive to shearing force, and sonication produces molecules of an average length of 1/40 of a whole molecule of T4 DNA, the [32PJDNA components must be fragmented into an average of eight pieces and only one of them has the properties of a joint molecule with, on the average, half of the label of a light [32PJfragment. Therefore about 14/15 of total 32p would shift to the light density and remaining radioactivity would be distributed among joint molecules with various densities. The distribution of 32p calculated for a mixture composed of 14/15 of 32p in the light molecules and 1/15 in joint molecules agrees quite well with that experimentally obtained (Fig. 13(a)). Considering the roughness of the estimation and the uncertainty inherent in the experiment the agreement may not be taken too seriously. The results, however, suggest that sonication of a joint molecule produces a fragment of an intermediate density, and the molecules thus produced are seen to be distributed flatly between the density of the light DNA and that of BU-DNA. Therefore the light and heavy components may have equal sensitivity as a function of shearing force. As shown in Fig. 2(b), BU·DNA and ordinary DNA are equally sensitive to shearing force. Thus the results provide evidence that a joint molecule is composed of [32PJDNA and BU-DNA. The results presented above also show that the joint between two components does not dissolve by sonication. They show further that the 32p is not concentrated at the middle density, indicating that the length of an overlapping region is small -in fact less than 1/40 of the whole molecule of T4 DNA. If joint molecules were fragmented into pieces of the size of an overlapping region, "hybrid" molecules would be produced, and from the size of the "hybrid" molecules thus produced, the length of the overlapping region could be estimated. However, after progressive fragmentation the band of 32p in the fragments becomes broader, and the density at which the amount of 32p in joint molecules is maximum approaches the middle density as calculated by equation (2). Therefore, it would be quite difficult to measure the length of an overlapping region by analysing the products of fragmentation. The results of stirring at 1500 rev.jmin are difficult to analyse quantitatively because of heterogeneity in density of the original sample and in the molecular length of the sample and the products. Before stirring, 32p formed a peak at a density 1·735 g cm-3 with similar deviation of distribution in the density gradient to the sample presented in Fig. 4. Let us consider a molecule with a density corresponding to a molecule composed of 60%" of [32PJDNA- and 40% of BU-DNA-components. If each component consists of a single unit and both components join end to end to make a linear molecule, breakage at the middle of the molecule produces one [32PJ_ light fragment and a fragment of 20% of [32PJDNA. and 80% of BU-DNA-components, which corresponds to a molecule of a density 1·775. The lighter fragment has five times as much 32p as the heavier fragment. The results presented in Fig. 7 show that considerable label appeared at the light-density fractions and the fractions of density around 1·775. Stirring may break linear joint molecules longer than half molecules of T4 DNA near the middle but may not break the molecules shorter than half molecules, thus giving the results presented in Fig. 7. The agreement of

JOINING OF PARENTAL DNA MOLECULES

537

these results with the expectation shows that the light and heavy components of a joint molecule must be represented as long units, most likely joined end to end, and the structure cannot be a mosaic of many small units. The results of brief treatment of joint molecules by deoxyribonuclease may be discussed here. Zone centrifugation analysis (Fig. 7(b)) shows that whole molecules of T4 DNA were estimated, on the average, to have been subjected to a little more than one random break per molecule. Joint molecules in the original fraction have various lengths, mostly a little shorter than whole molecules of T4 DNA (Fig. 5(b)). As already proved, the 32P-Iabelled light component is sensitive to deoxyribonuclease, and if the heavy component is equally sensitive to the enzyme, joint molecules would be subjected to, on the average, about one break. On the other hand, if only the light component is sensitive and the heavy component is resistant, the average number of breaks per molecule must be less than one. The results presented in Fig. 7(a) show that a considerable fraction of the joint molecules were broken, and the density of the fragmented molecules varies from the density of the light DNA to BU-DNA. Substantial label also appeared at the light fractions. Such a change in density must be produced by only a single breakage of a joint molecule. These results lead to the same conclusion as that from shearing experiments. If the molecule is composed of one each of the light and heavy components the results indicate that the heavy component of the complex is also sensitive to deoxyribonuclease. If the heavy components were resistant, 32p would appear only at the fractions with the light density and those with a density heavier than that of the original material, but none would be found in the fractions with density between the light DNA and the original material. This is in contradiction to the results. As shown in Fig. 2(c) the sensitivity of BU-DNA to deoxyribonuclease is indistinguishable from that of the light DNA. Thus the results of the enzyme digestion further support the conclusion that a joint molecule is composed of one each of the light DNA- and BU-DNA-components joined end to end. The results so far discussed clearly indicate that structure of a joint molecule can be represented by the models presented in Fig. 10. We will now choose the more likely one. Levinthal & Davison (1961) found that T2 DNA, which had suffered so substantial a decay of 32p that the polynucleotide chains were interrupted, degraded at a lower rate of shear than was required to break undamaged molecules. According to this, the second model might involve weak points at free rotating bonds located at both ends of an overlapping region. Since there is no indication that such breaks are produced by shear fragmentation, we prefer to choose the first model. More direct experiments could be designed since the nature of hydrogen bonds in light DNA, BU-DNA and "hybrid" DNA is different (Inman & Baldwin, 1962). (h) The possible genetic meaning of the joint molecule

Since their discovery (Hershey & Chase, 1951), heterozygotes have been thought to be the intermediate products that yield recombinants. They are recognized because they contain both alleles of one or more of the markers employed in the cross. In other words, they have genetic overlaps. All phage heterozygotes are partial in the sense that the average length of an overlapping region is probably less than 1 % of the total genome (calculated by Doermann & Boehener, personal communication). The frequency of overlaps is sufficient to account for all observed recombination

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among loosely-linked markers (Levinthal, 1954; Trautner, 1958). For closely-linked markers, recombinants arise as segregation products of heterozygotes (Edgar, 1961). During KCN treatment of infected cells, joint molecules accumulate and, at the same time, genetic recombination seems to proceed (Tomizawa & Anraku, 1964). With several assumptions, the extent of the correlation between the observed amount of joint molecules and the frequency of genetic recombination during treatment with KCN might be obtained. Unfortunately we cannot make a valid calculation here for two reasons. First, there is the uncertainty of the length of the intracellular joint molecules, and secondly, it is difficult to measure the frequency of genetic recombination in the experimental system reported here in which more than half of one of the parental particles lack viability. Since, however, the joint molecules we studied have probably suffered mechanical fragmentation, the observed amount of 32p appearing in these molecules would imply participation of a considerable number of parental [32P]DNA molecules in the formation of joint molecules; a conclusion not incompatible with the expectations based on the genetic results presented in the preceding report (Tomizawa & Anraku, 1964). Furthermore, there is no doubt that the joining of two DNA molecules derived from two parents must be accompanied by combination of some genetic markers of both parents. It may, therefore, be concluded that joint molecules are produced during the process of genetic recombination. A joint molecule having the structure proposed here also satisfies the general properties of a heterozygote. In fact, as we have said, the same models were also proposed on purely genetic experiments. Recent findings of the presence of two kinds of heterozygotes, internal and terminally redundant (Sechaud, Streisinger, Lanford, Reinhold & Stahl, 1964), however, require reconsideration of the genetic results so far obtained which have not differentiated between the two kinds. The kind of heterozygote to which the joint molecule might correspond is the internal one, with or without a terminal redundancy. This does not necessarily mean that a joint molecule is the only kind of internal heterozygote. Heterozygotes with different molecular structure, such as a duplex DNA molecule with a region lacking base-pair complementarity (Levinthal, 1954), may possibly originate from a joint molecule. It is also possible that formation of a structure as a joint molecule is a preliminary stage of recombination which might be followed by joining of parental polynucleotides by nucleotide bonds. We have isolated phage particles which contain, in a single particle, DNA derived from two parents, and the results of the analysis of DNA in phage particles will be reported separately. (i) Formation of joint molecules

The experimental results strongly indicate joining of two molecules of DNA at homologous regions to form a single joint molecule. If a linear phage chromosome terminates at a point randomly distributed over a formally circular chromosome (Streisinger, Edgar & Denhardt, 1964), molecules with preformed homologous ends must be extremely rare. Since we found quite a large fraction of the total [32P]DNA in the joint molecules, joining presumably occurs either at a terminal region of one molecule and an internal region with homologous structure of another molecule, or at homologous internal regions of two molecules. Joining of two molecules to form a single linear molecule in this way must be preceded or followed by breakage of at least one molecule. Thus breakage and joining of two linear molecules must produce one joint molecule and one or two odd molecules. Each joint molecule is different

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as regards position of the joint and molecular length. Each odd molecule is different in length but generally still long enough to enable subsequent joining with another molecule. When bacteria co-infected with 32P-Iabelled phage and unlabelled phage were frozen to allow decay of 32p atoms and then thawed to allow phage growth, very effective rescue of genetic markers in the fragments op2P-labelled chromosomes was observed (Stent, 1953; Anraku & Tomizawa, to be published). This indicates that the fragments of phage chromosomes are able to recombine. If phage chromosomes in infected cells can be physically circular, breakage and joining of two circular molecules at a homologous overlap produce a linear joint molecule about twice as long as a single chromosome without producing any odd molecules. After that, the behaviour would be indistinguishable from that of linear molecules as described above. In either case linear molecules longer than a single phage genome might be produced by recombination. Long molecules of phage DNA as observed by Frankel (1963) might be joint molecules, or might originate from them. At some stage of maturation a longer molecule might give rise to a final product of uniform genetic and molecular length. A terminally redundant chromosome might originally form through these breaking- and joining-processes. Fragmentation of phage chromosomes might be independent of recombinational acts, and the fragments thus produced might reassemble later. According to this, fragmentation would not be specific, but only reassembly characterizes recombination. However, since a cell infected with a single phage particle survives to form a plaque after treatment with KeN, the phage chromosome could not be fragmented during the treatment. If the chromosome were fragmented, fragments so produced could not re-assort to form a complete chromosome because of the lack of homologous regions. Therefore, unless there is some mechanism which discriminates between single- and multiple-infections, non-specific fragmentation of chromosomes prior to recombination is unlikely to occur. Since bacterial DNA in cells infected with phage at a multiplicity of 5 retained high molecular weight after treatment with KeN, this would suggest the absence of at least extensive activation of hydrolytic enzymes for DNA during the treatment. Therefore we prefer the interpretation that breakage itself is involved in the specific processes of recombination, probably following the pairing of homologous regions of two chromosomes. We wish to thank Dr. H. Ozeki for his criticisms and Mr. H. Ogawa for his help in solving mathematical problems. We also appreciate the kindness of Drs. I. Tagaya, T. Komai, C. Nishimura, H. Uchida and Mr. M. Honda who allowed us to use analytical instruments. This work was aided in part by Research Grant GM8384 from the National Institutes of Health, United States Public Health Service. REFERENCES Benzer, S. (1955). Proc, Nat. Acad. Sci., Wash. 46, 344. Britten, R. J. & Roberts, R. B. (1960). Science, 131, 32. Burgi, E. & Hershey, A. D. (1961). J. Mol. Biol. 3, 458. Burgi, E. & Hershey, A. D. (1963). Biophys. J. 3, 309. Cairns, J. (1961). J. Mol. Biol. 3, 756. Davison, P. F., Freifelder, D., Hede, R. & Levinthal, C. (1961). Proc. Nat. Acad. Sci., Wash. 47, 1123. Delbruck, M. & Stent, G. S. (1957). In The Ohemical Basis of Heredity, p. 119. Baltimore: Johns Hopkins Press.

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