Initiation and Reinitiation of DNA Synthesis during Replication of Bacteriophage T7*

Transcription

1 Proc. Nat. Acad. Sci. USA Vol. 69, No. 4, pp , April 1972 Initiation and Reinitiation of DNA Synthesis during Replication of Bacteriophage T7* (E. coli/electron microscopy of DNA/origin of replication/direction of replication) DAVID DRESSLER, JOHN WOLFSON, AND MARILYN MAGAZIN The Biological Laboratories, Harvard University, Cambridge, Massachusetts Communicated by J. D. Watson, February 14, 1972 ABSTRACT In its first round of replication, the T7 chromosome follows a simple pattern, as viewed in the electron microscope. The initiation of DNA synthesis occurs about 17% from the genetic left end of the viral DNA rod. Bidirectional DNA synthesis from this origin then generates a replicating intermediate that we call an "eye form." In the eye form, when synthesis in the leftward direction reaches the left end of the viral chromosome, the molecule is converted into a Y-shaped replicating rod. The remaining growing point continues synthesis rightward, until presumably it runs off the right end of the DNA rod, thus terminating replication. Numerous T7 chromosomes were found in which a second round of replication had begun before the first round had finished. Analysis of these reinitiated DNA molecules showed that the second round of replication, like the first, began 17% from the end of the chromosome and involved bidirectional DNA synthesis. The replication of coliphage T7 DNA appears to be both simple and novel. Whereas previously observed chromosomes replicate as circles, the parental T7 chromosome has been shown by electron microscopy to replicate as a Y-shaped rod (1) (Fig. 1A). Partial denaturation of the Y-shaped DNA molecules showed that they contained duplicate copies of the left end of the viral chromosome. This indicated an overall direction of replication from left to right, and confined the origin of replication to the left half of the T7 chromosome. This paper locates the origin of T7 DNA synthesis and defines the events leading to the formation of the Y-shaped intermediate. Evidence will be presented that T7 DNA synthesis is initiated at a point about 17% from the left end of the viral chromosome. Bidirectional DNA synthesis from this origin generates an "eye form" (Fig. 1B). When DNA synthesis in the leftward direction reaches the left end of the DNA rod, the eye form is converted into a Y-shaped replicating intermediate (Fig. 2). This pattern of replication is confirmed by the observation of viral chromosomes that have initiated a second round of DNA replication before the completion of the first round. In these molecules there is a secondary eye opening in the region 17% from the end of one of the daughter portions of the partially replicated chromosome. To see the T7 initiation pattern, the multiplicity of infection must be near one phage per host cell. In our previous experiments, where cells were infected at a high multiplicity, the initiation pattern was obscured. We found numerous eye forms in which the eye was located in the center or right * This is paper II in a series, "T7 DNA Replication." 998 portion of the T7 DNA molecule, rather than in the left portion. As the data of this paper show, these randomlylocated eyes disappear when the multiplicity of infection is decreased to one. Since low multiplicity is a condition that is expected to suppress recombination involving parental chromosomes, we believe that the randomly-located eyes found at a high multiplicity are candidates for intermediates in T7 recombination. FIG. 1. T7 Chromosomes during replication. (A) During the first round of DNA replication, the T7 chromosome appears as a Y-shaped rod. Thus, T7 differs from previously observed bacterial, viral, and organelle chromosomes which, during replication, are circular (1). (B) This paper indicates that the "eye form" is the precursor of the Y-shaped replicating rod. DNA synthesis in the eye form is bidirectional. When the leftward growing point runs off the left end of the DNA rod, the eye form is converted to a Y-shaped molecule. 1B

2 Proc. Nat. Acad. Sci. USA 69 (1972) Experimental design The experimental procedure has been designed to provide actively replicating T7 chromosomes so that their structure can be determined by electron microscopy. The basic protocol (see Fig. 3) is an extension of the Meselson and Stahl experiment (2), and was developed by Ogawa, Tomizawa, and Fuke (3) for their studies of bacteriophage lambda DNA replication. One infects Escherichia coli B/r growing in heavy ('5N2H) medium with phage particles containing light ("4N1H) isotopes. As the isotopically light viral chromosomes begin to replicate, they incorporate heavy nucleotide precursors from the medium and shift in density from light (LL) toward hybrid (HL). Because viral DNA molecules in their first round of replication have a density between light and hybrid, they can be readily separated from unreplicated T7 DNA (LL) and host DNA (HH) when the intracellular DNA forms are centrifuged to equilibrium in a CsCI gradient. This purification scheme allows one to recover partially replicated viral chromosomes for a quantitative electron microscopic analysis. In the experiments described in this paper, the multiplicity of infection is near one T7 per cell. The low multiplicity is designed to suppress DNA recombination, a very active process in T7 that might be expected to interfere with a structural analysis of replicating DNA. Results Fig. 3 shows a CsCl gradient that contains the intracellular DNA forms recovered from cells grown in heavy medium and infected for 10 min with isotopically light T7 phage. Parental B.,z C E--- -_i, PARENTAL STRANDS -NEWLY SYNTHESIZED STRANDS FIG. 2. A schematic representation of the first round of T7 DNA replication. DNA synthesis on the T7 rod (A) is initiated 17% from the left end of the viral chromosome. Bidirectional DNA synthesis from this origin generates an eye form (B). As bidirectional replication continues, the eye increases in size (C). Because the origin of replication is relatively near the left end, the leftward growing point reaches the left end of the DNA rod soon after initiation, and the eye form is converted into a Y- shaped molecule (D). The remaining growing point moves rightward (E), until presumably it runs off the right end of the DNA rod, thus producing two progency chromosomes (F). By the time we see a partially replicated T7 chromosome, the specific events of initiation have already occurred, and daughter DNA strands are being elongated to the left and to the right. It would appear simplest to imagine that the act of initiation itself involves nothing more than the site-specific separation of the parental DNA strands (perhaps under the influence of a sequence-recognizing denaturation protein), followed by the de novo initiation of daughter polynucleotide strands on the exposed parental DNA templates. However, we have no evidence to indicate that the initiation events might not involve other mechanisms such as the nicking and elongation of parental DNA strands (26). z Li 200, -j D x CI HH HL LL T ~~A-1 T7 DNA Replication 999,-P FRACTION OF CsCI GRADIENT FIG. 3. Infection and isolation of actively replicating T7 J)NA. E. coli B/r growing in isotopically heavy (15N2H) medium was infected at a multiplicity of one with isotopically light ("4N- 1H) T7 phage particles. The details of the infection and the processing of the DNA are precisely as described in Paper I. Continuous assay for progeny phage showed a normal, biphasic growth curve. Each of the two cycles of growth took about 22 min and produced about 100 progeny phage per cell. The infected cells were harvested at 7, 10, and 13 min after infection as a source of actively replicating viral chromosomes. After lysis, the intracellular DNA forms from each harvest were resolved on individual CsCl gradients. In each gradient, the material in the region from light to hybrid is expected to contain T7 chromosomes engaged in the first round of D)NA replication. The light (LL) position of the gradient has been marked by the addition of tritium-labeled bacteriophage OX-174 duplex rings, and the heavy (HH) position was identified by the visible viscosity of the bacterial DNA. Most often, the 10-min time point contained the highest percentage of forked molecules in the HLL region. By 13 min, few forked forms were found in this region, presumably because the infection had proceeded past the first round of DNA replication. The CsCl gradient fractions between light and hybrid were individually dialyzed (two 40-min changes against 0.1 M Tris (ph 8.5)-0.01 M EDTA-0.1 M NH4OAc-10%, formamide) and examined in the electron microscope. T7 chromosomes recovered from the gradient between the positions of light (LL) and hybrid (HL) represent viral DNA molecules engaged in the first round of DNA replication. The HLL region of the gradient contains Y-shaped replicating rods (Fig. 1A). In addition, this region of the gradient contains viral DNA molecules with the topology shown in Fig. 1B. Here, a unit-length T7 chromosome possesses an internal region of duplication. The internal duplication takes the form of a bubble interrupting the linear DNA double helix. We refer to T7 chromosomes in this configuration as 'eye forms." Each fraction of the CsCl gradient between light and hybrid has been analyzed separately for its content of eye forms and Y-shaped molecules. The material from fraction A (near LL) consists primarily of unit-length T7 rods. However, about 2% of the rods are eye forms (Fig. 4A). The average size of the eye in this set of molecules corresponds to a 5.5% duplication of the T7 DNA molecule. In Fig. 4, the eye forms have been aligned so that their interior openings are all located in the left end of the viral chromosome. This orientation is verified below.

3 1000 Biochemistry: Dressler et al. 17%:. Jvv5.5% duplicated ( LEFT 17%7,. qv--19-7%duplicated Fraction A Fraction B RIGHT LOCATION OF EYE FIG. 4. Electron microscopy of density-shifted parental T7 DNA molecules. Fractions A-D of the CsCl gradient shown in Fig. 3 were individually analyzed for the presence of T7 eye forms and Y-shaped rods. The DNA was prepared for viewing in the electron microscope by the Davis, Simon, and Davidson modification (4) of the basic protein film technique of Kleinschmidt and Zahn (5). The exact procedure has been detailed previously (1). Panel A shows line diagrams of representative eye forms from fraction A of the CsCl gradient. The molecules of this fraction show the least degree of density shift from the LL position. The eye forms were photographed, measured, and normalized to a scale of 100 units. The molecules were then aligned on the assumption (see Fig. 5) that the eyes all occur in the same end of the viral chromosome. The eye forms of fraction B are shown in Panel B; here, the degree of density shift is greater and the eyes are larger. In fraction B, about 5% of the molecules contained eyes. The histograms beneath Panels A and B show that, on the average, the eyes are opened equally to the right and to the left of the 17% point. After alignment, the eye overlaps a point that is 17% from the left end of the DNA rod in all but one of the molecules. Fraction B contains T7 chromosomes that have been shifted from the LL position to a greater extent. The representative eye forms of this fraction (Fig. 4B) contain interior openings that span 20% of the viral chromosome and are, thus, about 4-times larger than the openings in the molecules of fraction A. In each molecule, except one, the eye overlaps the 17% region. We interpret the eye forms to be T7 chromosomes in the process of DNA replication. The point 17% from the left end of the chromosome would then correspond to the origin of replication. The increase in the size of the eyes of fraction B relative to fraction A is interpreted to be evidence that the eye forms represent a continuum of actively replicating viral chromosomes: the greater the extent of replication, the greater the degree of density shift. The molecules and the histograms of Fig. 4 show that, on the average, the eyes are extended equally to the right and Proc. Nat. Acad. Sci. USA 69 (1972) to the left of the 17% point. This result is interpreted as evidence for bidirectional DNA synthesis. A few eyes have one fork at the 17% point and, thus, seem to have engaged only in unidirectional DNA synthesis, either leftward or rightward. The very limited overlap of the eyes that grow unidirectionally, but in opposite directions, provides a rather precise way to locate the origin of replication. Fig. 5 presents evidence that the interior openings in the eye forms are located in the left arm of the T7 chromosome. This is shown by preparing the eye forms for electron microscopy in the presence of a high concentration of formamide. In 86% formamide, the AT-rich regions of T7 DNA melt out and can be observed in the electron microscope as singlestranded blisters interrupting the linear double helix (1). For T7, the AT-rich regions are known to be located in the regions 0-5, 15-30, 60-65, and % from the genetic leftend of the DNA molecule. It is seen in Fig. 5 that, when the partially denatured eye forms are aligned to conform with this pattern, the eyes appear in the left arm of the T7 chromosome. In addition to eye forms, the more-dense fractions of the CsCl gradient (fractions B, C, and D) contain Y-shaped molecules (Fig. 1A). As is the case for the eye forms, the Y-shaped molecules show a greater degree of duplication as the more dense fractions are examined. The presence of the Y-shaped molecules only in the more dense fractions of the gradient argues that these structures are derived by extended replication of the eye forms. Reinitiation In fractions C and D there are additional forked species, namely Y-shaped rods and eye forms with an additional eye. The second eye occurs in the region 17% from the end of the viral chromosome. We interpret these structures to be T7 DNA molecules that have initiated a second round of replication before the completion of the first round. For instance, Fig. 6A is interpreted as a 58% replicated Y form that has reinitiated. Line diagrams of this and 14 other reinitiated T7 chromosomes are shown in Fig. 6B. In each DNA molecule, the secondary eye overlaps the region 17%o from the end of one of the daughter arms. The pattern indicates that the first and second rounds of T7 DNA replication are identical in terms of the origin of replication and the involvement of bidirectional DNA synthesis. Discussion The data given here and in our previous paper (1) lead to the description of the first two rounds of T7 DNA replication given in Fig. 2. Replication is initiated at an interior point on the viral chromosome. Bidirectional DNA synthesis then generates an eye-form which, by continued growth, is converted first into a Y-shaped intermediate and then into two progeny rods. The evidence presented in this paper indicates that the origin of T7 DNA replication is located 17% from the genetic left end of the DNA rod. This places the site for the initiation of viral DNA synthesis at the beginning of a series of sixeight genes, most or all of which are involved in DNA metabolism. These genes, which are translated as a block, include a polymerase (7), ligase (8), two nucleases that digest the bacterial chromosome (9, 27), and four genes of unknown function.

4 Proc. Nat. Acad. Sci. USA 69 (1972) T7 DNA Replication 1001 T I =.7772v - - A G D C LuLJ 5, Z~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ LEF RIGHT,. a - F T -- LOCATION OF DENATURED REGION FIG. 5. The eyes are located in the left end of the viral chromosome. Fig. 5A is an example of a partially denatured eye form. Fig. 5B shows a linear representation of this molecule after it had been traced, measured, and normalized to a scale of 100 units. The AT-rich regions are shown as horizontal black bars. Fig. 5C shows 12 additional partially denatured eye forms. Fig. 5D is a histogram constructed by summing the denatured regions of the molecules in Fig. 5C. The pattern of partial denaturation in the histogram corresponds to the partial-denaturation map obtained for unit-length T7 DNA rods (1). In the construction of the histogram, since some regions of each molecule were present in duplicate, the denatured segments in the nonduplicated regions were counted twice. After orientation of the partially denatured eye forms, the eyes are seen to be opening on the left. A paradox arises because of the existence of a T7 mutant in which the region of the chromosome from 15.2 to 22.5% (and thus the origin) is deleted (6). Possibly, in the deletion mutant, the original origin has been translocated. Or, perhaps, secondary origins are used, and this is reflected in the poor growth of these p)hage mutants. The region of the origin of T7 DNA synthesis is also interesting because of its involvement in RNA metabolism. It is at or near the 17% point that the viral RNA polymerase begins its transcription of the late T7 genes. The coincidence of initiation sites for DNA and RNA synthesis might reflect a direct involvement of the viral RNA polymerase in the initiation of T7 DNA synthesis. Recent studies with bacteriophage M13 (10) have demonstrated that E. coli RNA polymerase is directly required for the conversion of the infecting positive strand circle to the double-stranded ring form. Experiments with phage lambda (11, 12) have also demonstrated an involvement of RNA synthesis in DNA replication. The possible involvement of the T7 RNA polymerase in viral DNA synthesis is under study. The replicating T7 DNA molecule is unusual because it is linear. However, if the two ends of a replicating T7 eye-form were to be linked to each other, the T7 chromosome would then assume a topology that is actually quite common-the Cairns form. This circular configuration, first observed for E. coli (13), is now known to represent the replicating chromosomes of pleuropneumonia-like organisms (14), lambda (early in its life cycle) (3, 15), mitochondria (16, 17), polyoma (18, 19), SV40 (20, 21), and colicinigenic factors (22). If one disregards the overall circularity of the Cairns forms, then it becomes apparent that these replicating DNA molecules are similar to T7 in what seems to be a fundamental way; both share the eye as the internal replicating structure. Topologically, each eye contains two points at which the parental DNA strands could be separating from each other to serve as templates for the synthesis of new DNA. That such bidirectional DNA synthesis does in fact occur was first demonstrated for HeLa cell DNA replication by Huberman and Riggs (23) and for lambda DNA replication by Schnos and Inman (15). Recent studies have extended the finding of bidirectional DNA synthesis to B. subtilis (28), E. coli (25), phage T4 (24), and, as reported here, phage T7. Eyes appear to be involved not only in the initiation, but also in the reinitiation, of DNA synthesis. Thus, eyes within eyes have been observed not only for intact T7 chromosomes (Fig. 6), but also for fragments of the B. subtilis (28) and T4 chromosomes (24). In addition to their involvement in DNA replication, the eye structures may also be intermediates in genetic recombination. In our experiments with T7, eyes in parental chromosomes virtually always overlap the region 17% from the left end of the viral DNA molecule, when the multiplicity of infection is low. But, when the multiplicity of infection is high, and recombination involving parental (LL) and progeny

5 1002 Biochemistry: Dressler et al. Proc. Nat. Acad. Sci. USA 69 (1972) A Froctions C+D : LEFT RiGHT LOCATION OF EYE B FIG. 6. Reinitiated T7 DNA molecules. Panel A shows a Y-shaped molecule that contains an eye in one of its daughter arms. Panel B shows line diagrams of the molecule in Panel A, and those of 14 additional forked molecules that contain a secondary eye in one of their duplicated regions. In each case, the secondary eye overlaps the region 17% from the end of the T7 chromosome, and thus appears to represent an act of initiation.- (HL and HH) chromosomes can occur, we find parental molecules with eyes located in the center and right portions of the T7 chromosomes, as well as molecules with eyes in the left portion. Since the existence of the additional, randomlylocated eyes is multiplicity-dependent, we are now investigating them as candidates for structural intermediates in T7 recombination. We thank Drs. James Watson, John Cairns, and Walter Gilbert for their thoughtful readings of the manuscript. This work was supported by the National Institutes of Health (GM 17088) and the American Cancer Society (NP-57A). D. 1). is a fellow of the Helen Hay Whitney Foundation. J. W. is a trainee under the National Institute of General Medical Sciences Training Grant GM , and a medical student on leave from The Johns Hopkins Medical School. 1. Wolfson, J., Dressler, D. & Magazin, M. (1972) Proc. Nat. Acad. Sci. USA 69, Meselson, M. & Stahl, F. (1958) Proc. Nat. Acad. Sci. USA 44, Ogawa, T., Tomizawa, J. & Fuke, M. (1968) Proc. Nat. Acad. Sci. USA 60, Davis, R., Simon, M. & Davidson, N. (1971) in Methods in Enzymology, eds. Grossman, L. & Moldave, K. (Academic Press, New York), Vol. XXI, pp Kleinschmidt, A. & Zahn, R. (1959) Naturforscher B14, Studier, F. (1972) Science, in press. 7. Grippo, P. & Richardson, C. (1971) J. Biol. Chem. 246, Masamune, Y., Frenkel, G. & Richardson, C. (1971) J. Biol. Chem. 246, Center, M., Studier, F. & Richardson, C. (1970) Proc. Nat. Acad. Sci. USA 65, Brutlag, D., Schekman, R. & Kornberg, A. (1971) Proc. Nat. Acad. Sci. USA 68, Dove, W., Inokuchi, H. & Stevens, W. (1971) in The Bacteriophage Lambda, ed. Hershey, A. (Cold Spring Harbor Laboratory, New York). 12. Dove, W., Hargrove, E., Ohashi, M., Hangli, F. & Gisha, A. (1969) Japan J. Genetics 44 Suppl. 1, Cairns, J. (1963) Cold Spring Harbor Symp. Quant. Biol. 28, Bode, H. & Morowitz, H. (1967) J. Mol. Biol. 23, Schnos, M. & Inman, R. (1971) J. Mol. Biol. 51, Kasamatsu, H., Robberson, D. & Vinograd, J. (1971) Proc. Nat. Acad. Sci. USA 68, Kirschner, R., Wolstenholme, D. & Gross, N. (1968) Proc. Nat. Acad. Sci. USA 60, Hirt, B. (1969) J. Mol. Biol. 40, Bourgaux, P. & Bourgaux-Ramoisy, D. (1971) J. Mol. Biol. 62, Sebring, E., Kelly, T., Thoren, M. & Salzman, N. (1971) J. Virol. 8, Jaenisch, R., Mayer, A. & Levine, A. (1971) Nature New Biol. 233, Inselburg, J. & Fuke, M. (1972) Proc. Nat. Acad. Sci., USA 69, Huberman, J. & Riggs, A. (1968) J. Mot. Biol. 32, Delius, H., Howe, C. & Kozinski, A. (1971) Proc. Nat. Acad. Sci. USA 68, Masters, M. & Broda, P. (1971) Nature New Biol. 232, Gilbert, W., & Dressler, D. (1968) Cold Spring Harbor Symp. Quant. Biol. 33, Sadowski, P. & Kerr, C. (1972) J. Biol. Chem., in press. 28. Wake, R. (1972) J. Mol. Biol., in press.