ENZYMATIC SYNTHESIS OF DNA, XXIV. SYNTHESIS OF INFECTIOUS PHAGE 4x174 DNA* BY MEHRAN GOULIAN,t ARTHUR KORNBERG, AND ROBERT L. ~INSHEIMER DEPARTMENT OF BIOCHEMISTRY, STANFORD UNIVERSITY SCHOOL OF MEDICINE, PAW ALTO, AND DIVISION OF BIOWQY, CALIFORNIA INSTITUTE OF TECHNOWGY, PASADENA Communiealed September 96, 1967 Past attempts at in vitro replication of transforming factor present in DNA have given negative or inconclusive Rigid proof was lacking that temp1at.e material had been excluded from the synthetic product. Even if a rigorous demonstration of net synthesis of transforming factor for a given genetic marker were forthcoming, it would still prove only that some relatively short sequence of nucleotides, sufficient for replacement of the mutant locus, had been synthesized. If enzymatic synthesis of infectious bacteriophage DNA were achieved, it would be made clear at once that relatively few, if any, mistakes had been made in replicating a DNA sequence of several thousand nucleotides. Escherichia coli DNA polymerase can replicate single-stranded circular DNA from phage M13 or 4x174' and in conjunction with a polynucleotide-joining enzyme produces a fully covalent duplex cir~le.~ Analyses of this product by equilibrium and velocity sedimentation and by electron microscopy have shown it to be indistinguishable, except for supercoiling, from replicative forms (RF)' of the viral DNA.6 By substitution of bromouracil for thymine in the comple- mentary strand ((-) circle), it should be possible on the basis of density difference to isolate this strand from the duplex circle and determine whether it has the infectivity known to reside in (-) circles.7.8 This report will describe: (1) the isolation of infective, synthetic (-) circles from the partially synthetic replicative form, (2) the ability of the isolated (-) circles to serve as templates for the production of infective, completely synthetic duplex circles, and (3) the isolation of infective, synthetic (+) circles from the latter. Thus, DNA polymerase carries out the relatively error-free synthesis of the 4x174 genome from the four deoxyribonucleoside triphosphates on direction from phage DNA templates. ResuEts.O-Isolatim of synthetic (-) circle and test of infectivity: A duplex circle was synthesized by replicating H3-#X174 DNA with DNA polymerase in the presence of a polynucleotide-joining enz.yme. Details for the production and isolation of this partially synthetic RF, containing BU and Pa in the (-) circle, were described in an earlier rep01-t.~ Separation of the synthetic (-) circle from the duplex form followed the plan outlined in Figure 1. The duplex circles were exposed to pancreatic DNase to an extent sufficient to produce a single scission in one of the strands in about half of the molecules. The resulting mixture of intact and nicked molecules was denatured by heating. The mixture, which now contained circular and linear HS-T (+) strands, and P=-W (- ) strands, in addi- tion to intact RF, was fractionated by equilibrium density-gradient sedimentation in CsCl (Fig. 2).1° Three peaks of radioactivity were evident, corresponding, in order of decreasing density, to single-stranded DNA containing W, a duplex hybrid 2321 2322 BIOCHEMISTRY: GOULIAN ET AL. PROC. N. A. S. partial I y synthatic RF I1 Isolation oC synthetic "P-BU t-) circles Fro. 1.Schematic representation of the preparation of synthetic (- ) circles and RF. For details sae text and Figs. 2 and 6. of and T, and single strands containing T, with mean densities of 1.809, 1.747, and 1.722 gm/ml, respectively. These values may be compared to the values of 1.732 and 1.725 previously determinedI3 for the native hybrid and T (+) single strands prepared in vivo or to the calculated values14 of 1.815 and 1.753 for the BU (-) strands, and the hybrid, respectively. In addition to the three peaks, there was an area on the heavy side of the hybrid zone, which in other experimental trials appeared as a more distinct shoulder and is attributable to some duplex circles that had failed to renature after the heat treatment (Fig. 2). Inasmuch as the (-) circle is infectious in the spheroplast assayJ78 * it was possible to test the enzymatically synthesized material directly for biologic activity. Four peaks of infectivity were found (Fig. 2). One corresponded to the position of heavy, P32-m, synthetic (-) single strands and another to that of light, H3-T (+) single strands. Specific infectivity values for the single-stranded regions could not be determined from these data because there was an unknown quantity of linear strands. The P32-gU and H3-T peaks were therefore each subjected to velocity sedimentation in a neutral , low-salt sucrose gradient to give a partial separation of the circles from linear forms. As seen in Figure 3 for the Pa2-= (-) strands, and in Figure 4 for the H3-T (+) strands, the infective material was found, in each case, in the leading shoulder of the peak which contains the circles - VOL. 58, 1967 BIOCHEMISTRY: GOVLIAA ET AL. 2328 FIQ. 2.-Equilibrium density-gradient sedi- - v-1 mentation anal sis of partially synthetic RF after limited Dkase action and denaturation. Partially synthetic RF, with Pa* and BU in the synthetic (-) strand,' was incubated for 20 min at 20" at a concentration of 0.1 mM, in 0.2 ml of 10% gl cerol-10 mM Tris HCl (pH 7.6)-2 mM Mg614.25 mpg/ml ancre- atic DNase. (The DNase (Wortiin on was stored at 0'11 and diluted immediately reaction was stopped by addition of EDTA before use in 10 mM Tris acetate (pH 5.5)-5 mM MgClz-0.2 M KC1-50% glycerol.) The I? to 8 mM. The mixture was heated at 90" 2 for 2 min and adjusted to a volume of 9.8 ml x with 0.01 M Tris HC1 ( H 7.6); EDTA was g A added to 1 mM, as wefi as 1 mg of bovine - plasma albumin and 9.961 gm of CsCI. 2 Centrifuption of this mixture (p = 1.750) was carried out in the S inco no. 50 angle rotor at 45,000 rpm at 25" !or 50 hr. Aliquots from each fraction were assayed for radio- activity on filter aper disks,' and for in- and Sinsheimer.1f Inhibition by CsCl in the 10 : 0 J 1' spheroplast assay was avoided by dilution. because of their more rapid sedimentation. Because of their content of TU, the (-) circles had a distinctly higher sedimentation rate than their T (+) comple- ments (compare sedimentation values relative to the DNA marker in Figs. 3 and 4). The specific infectivities estimated for the synthetic (-) circles and template (+) circles were 0.074 and 0.80, respectively (Table 1). The other two peaks of infectivity in Figure 2 corresponded to the position of denatured and native forms of duplex hybrid molecules. Their respective specific infectivities were 0.066 and 0.012 (Table 1). Proof that the infectivity of the P32-FU peak resides in the enzymatically synthesized DNA: (1) A peak of infectivity coincides with the Pa-m peak" in the density gradient (Fig. 2) and is separated from neighboring peaks. (2) Phage (+) circles are absent from the single-stranded, Pa-m peak as judged by the absence of detectable H3-labeled material. In view of the sensitivity of the radioactivity measurements, the upper limit for the amount of template material in the synthetic peak is 8 ppmoles/ml; this concentration is one-tenth of that necessary to account for the infectivity of the peak. (3) In velocity sedimentation in sucrose gradients, the peak of infectivity corresponds to the position of intact Pa-m (-) circles, and sediments more rapidly because of the presence of m than the analogous peak of intact H3-T (+) circles. (4) The photoinactivation of Pa-m (-) DNA as compared with H3-T (+) DNA (Fig. 5) demonstrates the more rapid inactiva- tion of most of the infectious particles in the CsCl gradient peak corresponding to - Pa-m (-) strands and is consistent with the known greater photosensitivity of BU-containing DNA.'* The presence of approqimately 5 per cent of the infectious material displaying an inactivation rate similar to that of T DNAlS (Fig. 5) indicates the extent of contamination by T (+) circles. Inasmuch as the (+) circles of Figure 2 have about ten times the specific infectivity of these (-) circles, the residual content of phage DNA in the fraction is estimated to be closer to 0.5 per cent than 5 per cent. 16- 1X recrystallized), 5 mg/ml in 0.01 N lP C1, -0 - 4 fectivity by the spieroplast assay of Giithrie n Fraction numbct- 2324 BIOCHEMISTRY: GOULIAN ET AL. PROC. N. A. S. I I I I I 0 t ^^ 25 30 I I I Fromion number Fraction number FIQ. 3.-Velocity sedimentation of PaLBT !-) synthetic DNA derived from partially synthetic RF. The Pa*-m peak fractions were pooled, dialyzed against 2 mM Tris HCl (pH 7.6w.2 mM EDTA and then concen- trated five fold to a volume of 0.1 ml by rotary evaporation under reduced pressure. -An ali- quot of 20 pl was centrifuged in a 5-20% sucrose gradient in 5 mM NaCI-5 mM Tris HC1 (pH 7.6)-1 mM EDTA, at 60,000 rpm and 10" for 360 min. Ha was not detectable (<0.3 plmolelfraction). The position of 4x174 DNA was obtained from a separate tube con- taining this DNA ns marker. FIG. 4.-Velocity sedimentation of H*-T, (+) phage DNA derived from partially syn- thetic RF. The H*-T peak (Fig. 2) was treated as described in Fi . 3 for P"-W except that half as much H3-I! was placed on the sucrose gradient). Replication of synthetic (-) circle, isolation of fully synthetic replicative fms, and a test of infectivity: The synthetic, Px-W (-) circles, separated from phage (+) circles, could now be used as templates for the production of fully synthetic RF (Fig. 1) which proved to be infective. Incubation conditions for synthesis of the RF were as previously employedJ6 except that H3-dCTP was the labeled sub- TABLE 1 INFEmIVITIES OF NATURAL AND SYNTHETIC 4x174 DNA Specifi! infectivity Plaques DNA (plaques/ Relative (ml-1 x 10-8) (rrmole mi-') particle) infectivity Ref. ( +) Circle, natural 37 64 0.80* 1 .o Fig. 4 (;) Ciyle, natural 4.2 8 R,F (native), natural 0.05 15 ;F (denat.), naturar 1 .o 16 , synthetic 6.2 150 0.074. 0.09 Fig. 3 , part. s nthetic 61 9,100 0.012 0.01 Fig. 2 U , part. synthetic 9.5 260 0.066 0.06 Fig. 2 , fully synthetic 24 120 0.36 0.3 Fig. 6 Specific infectioifu WM calculated on the bMis of 1 1 X 101 particles/prmole of nucleotide residues for single- stranded molecules and half that value for the duplexes. Relolire injectioity of the natural (+) circle was arbitrarily taken M 1.0 and the other %urea adjuated. with incluaion of a correction. for variations between different a~aya. from phage DNA standards that were run in each aasay. Technical difficulties resulting from the low concentrations of DNA have thus far prevented reliable estimates of the specific infectivities of native, fully synthetic RF and synthetic (+) circles. , part. synthetic 6,000 200,000 0.058 0.07 t o Includes a correction for estimated contamination with linear forma. t Sample assayed prior to exposure to DNase as in Fig. 2. VOL. 58, 1967 BIOCHEMISTRY: GOULIAN ET AL. 2325 hybrid peak contained H3 and P2 in approximately equimolar amounts and I I 2326 BIOCHEMISTRY: COllIJAh' ET AL. PROC. N. A. 5. r-----i I I 5 IO 15 20 Fraction number Fro. 6.-Alkaline sucrose-gradient dimen- tation of fully synthetic RF. Pal-BU (-) strands (40 pl of peak sample from hg. 2, dialyzed and concentrated as described in Fig. 3) were replicated in a volume of 0.1 ml as de- scribed reviously ;' the labeled nucleotide was HS-dCT!' (Sehwarz BioResearch, 1000 cpm/ ppmole), and dTTP rather than dBvTP ww used. After 180 min, the mixture was made 20 mM in EDTA, 0.1 M in NaOH, and centri- fuged in a sucrose gradient in 0.2 M NaOH- 0.8 M NaCI-1 mM EDTA, at 60,000 rpm and 1" for 100 min. The fractions were neutralized with 1 M Tris citrate (pH 5) before being assayed for radioactivity and infectivity. 0 105; C (D OI 12 2 0 xe v) - 2! 4 0 IO 20 30 LO Fraction number FIG. 'I.-Density-gradient sedimentation of synthetic RF in the presence of ethidium bromide. The synthetic.RF, prepared as de- scribed in Fig. 6, was purified by a preliminary density-gradient centrifugation in CsClethid- ium bromide, as described previouslyP The covalent duplex zone, identified by alkaline sucrose-gradient sedimentation of aliquots from the fractions, was collected and refractionated in the same type of CsCl-ethidium bromide gedient with results shown above. Fractions were diluted 2Wfold for the spheroplast assay but were not otherwise treated tozmove CsCl or ethidium bromide. Pa* in the BU, (- ) tem- plate was not measurable due to radioactive decay and low recoveries. Numerous $X174 mutants are knownz1 in which the change of a single nucleotide results in loss of infectivity under the assay conditions employed. The fact that isolated synthetic circles and fully synthetic RF forms made with these circles as templates had specific infectivity values in the range measured for natural forms of viral DNA (Table 1) attests to the precision of the enzymatic operation. It should now be possible to apply the techniques used in this work to the syn- thesis of the duplex circular genomes of other viruses, such as phage A and animal viruses, and DNA molecules of comparable structure from cellular organelles. Such synthetic efforts will permit the insertion of base and nucleoside analogues in a manner and variety not attainable with in vivo systems. In addition, base changes generated by replication of the DNA with defective polymerases can now easily be studied in combination with standard genetic tools. It is of interest that DNA of approximately normal specific infectivity has been synthesized here without the use of any methylated nucleotide. This result may be related to the lack of host modification or restriction in the E. coli C-K22 pair and might not be applicable to other viral DNA's. Since the conversion of phage DNA to RF-form I is accomplished in vivo by VOL. 58, 1967 BlOCHEMISTh'Y: GOULIAN ET AL. 2327 ' 5 10 15 20 25 30 35 40 Froction number FIG. 8.-Identification of synthetic, (+) circles by alkaline sucrose-gradient sedimenta- tion of synthetic RF exposed to limited DNase action. The ra idly sedimenting fractions of synthetic Rf' in the alkaline sucrose gradient (Fie;. 6) were pooled, dialyzed against 10 mM Trls HCl (pH 7.6)-1 mM EDTA and incubated for 20 min at 20" (final volume of 2 ml) at a concentration of 50 ppmoles/ml in 10 mM Tris HC1 (pH 7.6)-5 mM MgClrO.1 mg/ml bovine plasma al- bumin-1.2 mpg/ml pancreatic DNase. The mixture was then made 15 mM in EDTA, re- duced in volume to 0.15 ml by rotary evap oration under reduced pressure, brought to pH 12 with .NaOH, and centrifu ed in a sucrose gradient in 0.2 M NaOk-0.8 M NaCl-1 mM EDTA, at 60,000 rpm and 10' for 240 min. The bottom of the tube was punc- tured with a hollow needle and the contents were displaced by saturated CsCl solution (containing Blue Dextran from Pharmacia) using a peristaltic pump. The sucrose-gradient fractions were collected from the top of the tube via a fine polyethylene tube in a stopper at the to . The fractions were neutralized (as in Fig. 6) prior to assays. The fractions were numberd in the reverse order of their collection, in order that the direction of sedimeAation conform to the illustration of velocity sedimentations in the other figures. Pa* in the BU, template strand was not measurable due to radioactive decay and the small amounts of DNA employed. host enzymes, and since the DriA polymerase and polynucleotide-joining enzyme are so effective in converting phage DNA to RF-form I in vitro, it appears likely that these enzymes are used by infected E. coli cells to carry out this conversion in vivo. Although the predominant pathway of phage replication appears to involve the open RF-form II,n the two forms are in fact interconvertible in vivo. Questions of the roles of these enzymes and the replicative forms in the production of (+) circles for progeny phage require further study. The fact that E. coli DNA polymerase can synthesize biologically active DNA does not establish its function in the replication of the bacterial chromosome. However, the effectiveness of the combined action of the polymerase and the polynucleotide-joining enzyme in forming infective DNA may have considerable significance for chromosomal replication. In an earlier paper,4 a mechanism was suggested whereby polymerase, with a then hypothetical polynucleotide-joining enzyme, might function in the simultaneous replication of both strands of helical DNA. The subsequent discovery of this joining enzyme, the requirement for it in phage T4 DNA synthesis,* its persistence in the most purified E. coli and phage T4 DNA polymerase preparation^,^ as well as the current demonstration of its conjoint action with polymerase, all strengthen the suggestion of this replication mechani~m.~ Summary.-A partially synthetic, closed replicative form (RF) of $X174 DNA, consisting of phage DNA as the (+) circle and a bromouracil-containing comple- ment synthesized by DNA polymerase as the (-) circle, was used as the source of synthetic (-) circles. The latter were separated from template strands by limited DNase action on the RF followed by denaturation and density-gradient equilibrium sedimentation. The isolated (-) circles were infectious and had the buoyant density, sedimentation velocity, and radiation sensitivity expected for DKA containing bromouracil. These (-) circles served as templates for a second round of replication which produced a fully synthetic RF with the specific infectivity 2328 BIOCHEMISTRY: GOULIAN ET AL. PROC. N. A. S. of natural RF. Infective synthetic (+) circles, corresponding to the original phage DNA, were isolated from the synthetic RF after DNase treatment, as in the previous isolation of synthetic (-) circles. These results imply a relatively error-free synthesis of the 4x174 genome by DNA polymerase. Note added in proof: A study by Okaraki, It., T. Okazaki, K. Sakabe, and K. Sugimotu (Jap. J. Med. Sn'. Biol., 20, 255 (1967)) of DNA replicatioil in E. coli supportv a mechanism of dir- continuous 5' + 3' chaiu growth on the 5' template xtraiid (see Discussion). We gratefully acknowledge the cxpert assistance of Mrs. Gloria Davis of the Division of Biology at the California Institute of Technology in performing the spheroplast assays for infectivity. * This research wassupported by grants from the National Institutes of Health and the Natiorial t USPHS special fellow. Present address: Department of Medicine, University of Chicago. 1 Litman, R. M., and W. Szybalski, Biochem. Biophys. Res. Commun., 10, 473 (1963). 2 Richardson, C. C., C. L. Schildkraut, H. V. Aposhian, A. Kornberg, W. Bodmer, and J. Lederberg, in Injonnatid Mamonwlecules, ed. H. J. Vogel, V. Bryson, and J. 0. Lampen (New York: Academic Press, 1963), p. 13. Science Foundation. *Richardson, C. C., R. B. Inman, and A. Kornberg, J. Mol. Biol., 9, 46 (1964). 4 Mitra, S., P. Reichard, R. B. Inman, L. L. Bertsch, and A. Kornberg, J. Mol. Biol., 24, 429 6 Goulian, M., and A. Kornberg, these PROCEEDINGS, 58, 1723 (1967). 6Abbreviations used are: RF for replicative form; T for thymine; (1967). for bromouracil; dCTP, dmTP, and dTTP for the deoxyribonucleoside triphosphates of cytosine, SU, and T, respectively; (+) circle for phage DNA; (-) circle for complementary copy of (+) circle. 7 Rbt, P., and R. L. Sinsheimer, J. Mol. Biol., 23, 545 (1967). 'Siegel, J. E. D., and M. Hayashi, J. Mol. Biol., 27, 443 (1967). 9 Experimental procedures were as described previously6 or as detailed in the figure legends. 10 In this figure, and in all succeeding ones, the ordinate values repreaent the total moles of The fractions, except where indicated otherwise, are num- nucleotide or plaques per fraction. bered in the order of their collection from the bottom of the tube. 11 Elson, E. L., thesis, Stanford University, Stanford (W). I* Guthrie, G. D., and R. L. Sinsheimer, Bwchim. Biophys. Acta, 72, 290 (1963). 13 Denhardt, D. T., and R. L. Sinsheimer, J. Mol. Biol., 12, 647 (1965). 14 The p values for BU-containing DNA were calculnted from the base composition (Sinsheimer, R. L., J. Mol. Biol., 1,43 (1959)), and the figure of 0.2 gm/ml determined by Baldwin and Shooter (J. Mol. Biol., 7, 511 (1963)) for the difference in p between dAT and dAm. 16Sinsheimer, R. L., M. Lawrence, and C. Nagler, J. Mol. Biol., 14, 348 (1965). 16 Burton, A., and R. L. Sinsheimer, J. Mol. Bwl., 14, 327 (1965). 17 The possibility of a facilitative effect of the synthetic DNA upon the infectivity of a small contaminant of natural DNA waa tested by mixing synthetic DNA molecules (Pn-m; Fig. 2; amber mutant) and natural DNA (yh, temperature-sensitive mutant).la The plaque count for the amher mutant was 398 for the DNA alone and 459 for the mixture, whereas the corre- sponding figures at similar dilutions for the temperature-sensitive mutant, alone and mixed, were 88 and 126, thus indicating the lack of interaction. 18 Denhardt, D. T., and R. L. Sinsheimer, J. Mol. Bwl., 12, 674 (1965). 19 This relatively large amount is unexplained and surprising in view of the low level to which infectivity dips between the Pn-m peak and the denatured hybrid duplex region of the CsCl gradient (Fig. 2). *OWang, J. C., D. Baumgarten, and B. M. Olivera, in preparation. 21 Sinsheimer, R. L., C. Hutchison, and B. H. Lindqvist, in The Molecular Biobgu of Viruses a Lindqvist, B. H., and R. L. Sinsheimer, in preparation. W Fareed, G. C., and C. C. Richardson, these PROCEEDINGS, 58, 665 (1967). ed. J. S. Colter (New York: Academic Press, in press.)