Formation of the parental replicative form DNA of bacteriophage φX174 and initial events in its replication

J. Mol. Biol.

(1971) 61, 565-586

Formation of the Parental Replicative Form DNA of Bacteriophage #X174 and Initial Events in its Replication BERTOLD

FRANCKE AND DAN

S.RAY

Molecular Biology Institute and Department of Zoology University of California, Los Angeles, Calif. 90024, U.H.A. (Received 30 December

2970, and in revised

form

28

May 1971)

Intracellular 4X174 DNA ww studied under & variety of conditions that prevent the replication of the parental replicative form DNA. These conditions included treatment with 150 rg of chloramphenicol per ml., the use of the rep 3 mutation of the host cell, amber mutation (am 8) in the viral gene responsible for RFt replication (gene A)? and combinations thereof. In all cases the majority of the parental RF was in the covalently closed form (RFI). The relative amount of RF with a discontinuity in one strand (RFII) in these cases was between 2 and 10% of the total RF and independent of the multiplicity of infection. The only exception was seen in infections of reps cells with 4X am 3 (a mutant in the lysis gene, gene E, used as a wild-type representative). In this case a fairly constant absolute amount of RF11 (1 to 4 per cell), independent of the multiplicity of infection, was formed, consisting almost exclusively of a closed complementary and an open parental viral strand. Since the formation of this type of RF11 was dependent on protein synthesis and the presence of the product of 4X gene A, it is concluded that the discontinuity in the parental viral strand represents the result of the action of the gene A product on the DNA. Possible mechanisms for the mode of action of the gene A product are discussed. Intermediates during the synthesis of the first complementary strand were isolated from cells infected with ultraviolet light-irradiated phages. Such intermediates contained incomplete linear complementary strands and circular parental viral strands. It is therefore concluded that the virus-specified discontinuity in RFII is introduced after the first complementary strand is completed and closed. Some residual virus-specific DNA synthesis beyond the formation of the parental RF was seen in rep, cells in infeotions where the viral gene for RF replication was functioning. This residual synthesis resulted in replicative intermediate structures containing circular complementary strands and elongated linear viral strands. The signiflcsnce of this type of RI for the existing models of 4X174 RF replication is discussed. t Abbreviations used: Cam, ohlorampheniool; E3H]dThd, tritiated thymidine; RFI, replicative form DNA with both strands olosed; RFII, replioative form DNA with one or more discontinuities in either strand; RI, replioative intermediate DNA; SDS, sodium dodeoyl sulfate. $ Hayashi, Sinsheixner, Tessman & Tessman (personal communication) have suggested that a uniform nomenclature for the eight known genes of 4X174 and 813 bo adopted. During the work presented here we used amber mutants in two genes. We will use the new nomenclature for these genes, but to avoid any oonfusion, we will also give their previous nomenolature here. 4X am3 is a mutant of the lysis gene of 4X (gene I, Sineheimer; gene CJ, Hayaabi; and 813 gene V, Tessmsn) which now is gene E. 4X am8 is a mutant in the viral funotion necessary for RF rep&&ion (gene VI, Sinsheimer; gene C, Heyashi: and 513 gene IV, Tesaman) which ia now gene A. 565

600

B. FRANCKE

AND

D. S. RAY

1. Introduction The replication of the single-stranded circular DNA of the bacteriophage 4x174 has been sub-divided into three steps (review article: Sinsheimer, 1968): (1) synthesis of the first complementary strand resulting in the double-stranded parental RF, (2) RF replication, yielding 20 to 30 progeny RF molecules per cell and (3) synthesis of progeny viral single-stranded DNA. In this paper results will be described which were obtained under various conditions that are non-permissive for replication of the parental RF. RF replication can be prevented by a variety of means: mutations of the viral gene A (Hutchison & Sinsheimer, 1966), mutations of the host cell genome (Denhardt, Dressier & Hathaway, 1967), high dosesof chloramphenicol (Tessman, 1966; Levine & Sinsheimer, 19693; Greenlee & Sinsheimer, 1968), and amino-acid starvation of an amino acid-requiring host cell (Snippers & Miiller-Wecker, 1970). If viral DNA replication can proceed uninhibited, the parental RF immediately enters into a replicative intermediate structure. RI is thought to be a rolling-circle type molecule (Gilbert & Dressler, 1968) containing one circular strand and one linear strand, which is reeled off while being elongated using the circular strand as a template. Currently a controversy exists in the literature as to which of the two strands remains closed during RF replication. Knippers, Whalley & Sinsheimer (1969) have described circular parental viral strands in RI, whereasDressler& Wolfson (1970) have reported the complementary strand to be the circular one. Whichever of the two mechanisms operates, they both require an opening in one of the strands of the parental RF as a prerequisite. Studies on the structure of parental RF under the various conditions mentioned above could potentially provide some insight into this problem. Such studies have been aimed at determinin g the relative amounts of RF1 (covalently closedin both strands) and RF11 (with one or more discontinuities in either strand) and the specificity of the discontinuity in RF11 (Kuippers & Miiller-Wecker, 1970). Interpretation and comparison of such results have been complicated by the various treatments usedby the different groups of investigators (starvation, chloramphenicol, mitomycin C, etc.). In particular, the role of the viral gene A during RF replication, for which it is required, remained unknown. For the present studies, we chosethe host cell mutant rep;, which only allows the synthesis of the parental RF but is unable to support its replication. Denhardt et al. (1967) have shown that viral genescan be expressedin rep 3. This cell, therefore, was the suitable host to study the effect of a viral gene A mutation under presumably physiological conditions without the complication of replication.

2. Materials

and Methods

(a) Phuge and bacterial strains The source of most of the strains has been described previously (Francke & Ray, 1971). +X am 3: lysis-defective mutant in gene E (Hutchison & Sinsheimer, 1966). 4X am 8: mutant in the RF replication gene A (kindly provided by Dr D. T. Denhardt). Emherichia coli HF4714 (SU x): permissive host for +X174 amber mutants used for plating of mutant phage and for growing am 8. E. co&i HF4704 (her-, thy-): non-permissive host for $X174 amber mutants used for growing am 3. E. coli rep, : derivative of HF4704, unable to replicete (6X174 RF. E. coli K12-W6: used for spheroplasts.

PARENTAL (b)

+X174

Media

and

A67

RF

solutions

TPA and TPA with low phosphate content (Sinsheimor, Starman, Nagler & Gut,hrie, 1962) were prepared as described previously (Francke & Ray, 1971). Tris-EDTA was 0.01 M - Tris, 0.001 M - EDTA, pH 8.0, unless otherwise indicated. Borate-EDTA was 0.05 M - tetrasodium borate, 0.006 M - EDTA. Chloramphenicol was stored as a 3 mg/ml. solution in 0.05 M - Tris, pH 7.5. (c) Biologicnl

assays

Phage titers were determined by the usual top agar overlayer technique. Wild-typo revertants in amber mutant preparations were dctrrmined by plating on Sz*. x and on HF4704 in parallel. The infectivity of viral DNA was assayed as described by Guthrie & Sinsheimer (1963) on spheroplasts prepared from E. coli K12-W6. Generally, two parallel incubations of the DNA with the spheroplasts were carried out at different DNA dilutions. Plating for progeny phage after lysis of the spheroplasts was on two parallel plates for each dilution. Specific infectivities of viral DNA samples were calculated from the plating efficionca of a standard DNA preparation tested with each experiment. (d) Preparation

of 3?P-labeled

phages

Labeling and purification of +X am. 3 has been described previously (Francke & Ray, 1971). The procedure was similar for +X mn 8, with t,he following modifications: HF47 14 (Su x) was used instead of HF4704. A 16-ml. culture in TPA with low phosphate cont,ent (2. 5 i: 10 -* M) was infected with a multiplicity of infection of 5 at a density of 3 X 10s cells/ml. Five minutes after infection, the isotope (5 mCi of carrier-free [32P]phosphate), 4 ml. of 50% sucrose and 0.6 ml. of 1 M - MgSO, were added. Aeration was reduced to about, 1 bubble/see. After 2 hr the cells were pelleted, leaving 10 to 15% of the tot,al plaque-forming units in the supernatant. The cells were washed twice with IO ml. of 0.05 M-borate in 10% sucrose and resuspended in 1 ml. borate-EDTA. Lysis, nucleaso treatment, and sucrose gradient centrifugation were as described previously for am 3. The am 8 preparation used for the experiments described in this paper contained 5.0 x 1011 plaque-forming units/ml. on Su x, 3.4 x lo7 plaque-forming units/ml. on 4704 and 2.1 x lo6 cts/min/ml. at the t,irno of preparation. (0) Ultraviolet

irmdidion

Drops of up to 0.2 ml. volume of phage (< 2 x lOI2 plaque-forming units/ml.) werp exposed to a Westinghouse Sterilamp G36TSL at 100 cm distance. At the times desired. samples were removed with a pla&ic pipette. Larger volumes were stirred with a magnetic stirrer during irradiation. The phage suspension was in 1 M-NaCl, 0.01 M-Tris (pH 8.0). 0.001 M-EDTA, and approx. 15% sucrose. Under these conditions. t.he surviving plaqueforming units decreased by a factor of 10 within 20 sec. (f) Infection,

labeling

and

lpis

of cells

In a standard experiment, the cells were grown to a density of 2 x lO*/ml. in 20 ml TPA. If Cam was to be used, it W&B added 20 min before infection. For infection, the culture was pipetted into a fresh tube containing the desired amount of 32P-labeled phages and 0.4 ml. of [3H]dThd (Schwarz BioResearch, 16.4 Ci/m-mole, O-5 mCi/ml.). Aeration was resumed at 5 min, and at 20 min after infection the culture was chilled in an ice bath and centrifuged in the cold. The cells were washed 3 times with 10 ml. borate-EDTA and resuspended in 1 ml. of the same buffer. Lysozyme was added to a concentration of 0.2 mg/ml. and incubated at 37°C for 10 min. From this time on, any harsh shaking or pipetting of the lysate was avoided. SDS was added to a final concentration of 1% and pronase (predigested at 37°C for 30 min in 0.05 M-Tris, pH 7.5) to a final concentration of 200 pg/ml. Both were mixed with the lysate by gentle rolling of the tube, relying on diffusion during the following 2.5 hr of incubation at 37°C. After this time, the extremely viscous lysate was completely clear and was poured directly on top of a high-salt neutral 37

608

B.

FRANCKE

AND

D.

S. RAY

sucrose gradient which had been kept at 4°C before use. The gradient, consisted of 5 to 20% sucrose in 1 M-NaCl, Tris-EDTA of a total volurnc of 34 ml. Contrifugation was at 24,000 rev./min for 15.5 hr in a Spinco SW27 rotor at 5°C. Fractions were collected from the top of the tube by pumping 70% sucrose in from the bottom, thus avoiding the labeled E. coli DNA which formed a visible pellet. This method removed almost quantitatively the labeled E. coli DNA by pelleting, aided by the high NaCl concentration (1 M) in the gradient and the presence of I”/; SDS in the lysate. This procedure made mitomycin treatment, nf the hnst cells unnecessary.

b)

‘i

D-

FIG. 1. High-salt neutral sucrose gradients of lysates from infected cells: ana 3 in rep, (a); ana 3 in rep, with 150 pg Cam/ml. (b); am 8 in rep; (c); am 8 in HF4704 (d); and anz 3 in 4704 with 150 pg Cam/ml. (e). Cells were grown in TPA to 2 x lO*/ml., and to (b) and (e) Cam was added for 20 min Infection was with 23P-labeled phages at a multiplicity of infection of 10 (the am 3 preparation was 8.4 x 10-s saP cts/min/plaque-forming unit, and the am 8 preparation w&s 4.0 x 10-O 3aP cts/min plaque-forming unit). At the time of infection, 0.2 mCi[sH]dThd was added. After 20 min at 37°C with aeration, the cultures were chilled in an ice-bath and washed 3 times with 10 ml. borate-EDTA. After resuspension in 1 ml. of 0.05 M-borate, they were lysed with lysozyme (100 pg/ml.) and EDTA (0.003 M). SDS (1%) and pronaae (200 pg/ml.) were added and incubated for 2.5 hr at 37’C. Linear sucrose gradients (5 to 20%) (in 1 M-NaCl, Tris-EDTA, vol. 34 ml.) were formed and kept at 4°C. The lysates were poured directly on top of the gradients and centrifuged for 15.5 hr at 24,000 rev./min and 6°C in an SW27 rotor. Fraotions of approx. 0.8 ml. each were collected and 100 ~1. of each fraction assayed for radioactivity. Direction of sedimentation in this 3zP cts/min (parent,al label); --O--O--, and all other Figures was to the left. -a-----, 3H cts/min (post-infection label).

PARENTAL

+X174

(g) Centrifuquhon

569

RF

techniques

The various gradient centrifugation techniques (velocity sedimentation and equilibrium centrifugation) have been described in detail by Francke & Ray ( 197 1) and by Ray ( 1969). If only the radioactivity was to be measured, fractions were collected directly onto Whatman no. 3 cellulose filter discs. From preparative gradients, a portion of each fraction was spotted onto cellulose filter discs. Filters were dried and counted in a toluenebased liquid scintillator. For infectivity assays samples were diluted lo- or IOO-fold into 0.05 M-Tris (pH 7.5) and stored frozen until assayed. Concentration of DNA samples from preparative gradients was accomplished by ethanol precipitation (2 vol. of ethanol in the presence of 0.2 M-potassium acetate at -20°C for at least 4 hr, followed by centrifugation at 17,000 rev./min for 40 min).

3. Results (a) Viral DNA forms under non-replicating

conditions

If a $X174-sensitive host eel1 was infected under conditions that did not allow replication of the parental RF, two peaks of virus-specific DNA were observed after neutral sucrose gradient centrifugation of the lysate. Figure 1 shows five experiments of this type. In all five cases, the cells were infected with 32P-labeled phages in the presence of [3H]dThd. After 20 minutes the DNA was fractionated by neutral sucrose gradient sedimentation. The non-replicating conditions used included the rep, mutation of the host cell, the ana 8 mutation of the phage, the addition of 150 pg Cam/ml. 20 minutes before infection and combinations thereof. Of the two peaks recovered from the gradients, the slower one represented RF11 and the faster one mainly RFI. Under certain conditions the RF1 region contained material which in alkaline sucrose gradients did not sediment rapidly as does supercoiled DNA (a criterion for closed circular double-stranded DNA). This observation will be discussed in detail below. The forward trailing of the RF1 peaks in Figure 1 was probably caused by the high viscosity of the sample before centrifugation. The amount of 3H label found in both RF regions in all experiments was on the order of one phage DNA equivalent per infecting parental DNA molecule, indicating that indeed no DNA synthesis beyond the format’ion of the parental RF had taken place. The different 3H : 32P ratios seen in Figure 1 were due to t,he different specific 32P activities of t,he phage preparations used for infection. Similar experiments were performed at different multiplicities of infection. These data are summarized in Table 1. In all cases, except when rep 3 cells were infected with urn 3 phages in the absence of Cam, the relative amount of parental label in RFD was fairly constant, representing between 1.4 and 9.2% of the total intracellular 32P. Only in the case of am 3 infecting rep; did the relative amount of RF11 increase with decreasing multiplicity of infection, resulting in a fairly constant number of parental RF11 molecules per cell, independent of the multiplicity of infection (the data in Table 1 were not normalized for infected cells but calculated for the total number of cells present at the time of infection; therefore, the number of RF11 molecules per cell at low multiplicity of infection represented a minimum estimat,e). These data indicate that in case of am 3 in rep; a specific RF11 might have been formed, whereas in all other cases, the constant relat’ive amount of RF11 could have been a result of random nicking of RFI. One further observation in Table 1, which was not of direct relevance for the subject, of this paper, should be commented on. In all systems shown, the addition of Cam to the culture increased the recovery of parental label in RF DNA at high multiplicit)ies

570

B.

Parental Bacterial strain rep

viral DNA

Phage strain

found

150 pg Cam/ml.

am 3

(L,,L

HI!‘4704

FRANCKE

8

urn 8

CIWL 3

AND

in RFII

1).

S. RAY

TABLE 1 under various

Input (m.0.i.)

Recovered (m.0.i.)

non-replicating Label in RFII(

cor&itions RF11

Oh)

molecules/ cell

100 10 1

40 5.5 0.85

8.7 38.5 66.0

+

100 10 1

86 Y 038

7.2 8.4 0.2

6.1 0.67 0~088

-

100 10 I

“7 5 0.7

7.2 4.0 1.4

1.92 0.2 0.02

-I-

I00 10 1

38 7.5 0.02

3.5 3.6 2.0

1.35 0.27 0.018

-

100 10 1

25 4.5 0.6

5.7 2.85 4.85

1.42 0.13 0.020

-1.

100

35

-t

3.5 2.2 0.7i

8

” $

10 1

6 0.85

5.0 2.2

0.35 0.03

100 10 1

30 r . .;I.:6

3.35 4.0 4.6:

1.01 0.22 0.04

Experimental details were described in the legend to Fig. 1. The recovered multiplicity of was calculated from the sum of parental label found in the preparative high-salt, sucrose and the specific aaP radioactivity of the phage preparation used for infection. o/0 RF11 culated from the amount of parental label in the slower sedimenting peak as o/o of recovered parental label. The number of RF11 molecules was calculated per total numbor present at the time of infection. m.o.i., Multiplicity of infection.

infection gradient, MBS calthe tot,al cjf cells

of infection. It was very likely that in these unsynchronized infections the multiplicity of infection was decreased at high input multiplicity of infection by a superinfection exclusion mechanism, which was sensitive to Cam (Hutchison & Sinsheimer, 1966). (b) The discontinuity

in the viral strand of RFII in the rep 3 host as a function gene A of 95x174

of

The RF11 regions of the neutral sucrose gradients shown in Figure 1 were separately pooled and analyzed by alkaline velocity sedimentation. To the samples b through e an excess of unlabeled viral DNA extracted from mature phage was added as a marker for the position of intact circular DNA. Sedimentation in alkali at high speed allowed the separation of the circular strand from the slower sedimenting unit-length linear strand of denatured RFII. The results are shown in Figure 2. In all cases, except in Figure 2(a), parental [32P]DNA and newly synthesized L3H]DNA appeared in both circular and linear strands. The distribution of label between the two forms indicated that RF11 molecules with a closed parental viral

PARENTAL

+X174

RF

671

Cd)

Fraction

no.

Fra. 2. Long alkaline sucrose sedimentation of RF11 from rep, infected with am 3 without (a) and with (b) Cam; rep; infected with nna 8 (c); HF4704 infected with ana 8 (d); and HF4704 infected with am 3 with Cam (e). The fraotions indicated by (II) in Fig. 1 were pooled, concentrated by ethanol precipitation, mixed with an unlabeled viral DNA marker (except in (a)), and sedimented through 5 to 20% alkaline sucrose gradients (containing 0.25 an-NaOH and 0.025 M-EDTA, 3.5 ml. total vol.). Centrifugation was for 5.6 hr at 65,000 rev./min and 5°C in an SW56 rotor. Fractions of 3 drops each ((a) and (e)) or 6 drops each ((b), (c) and (d)) were collected into 100 ~1. Tris-EDTA. 100 ~1. of each fraction were used for the radioactivity assay. Infectivity assays were done after dilution into 0.06 M-Tris, pH 7.5. -O-a--, 3aP cts/min (parental label); 3H cts/min (post-infection label); -- A-- a--, plaque-forming units. The arrow --c---o--, indicates the position of the marker DNA in (b), (c), (d) and (e).

and an open complementary strand were more frequent than those in which the viral strand was open. In RF11 generated in rep; cells after infection with ana 3 (Fig. 2(a)), a different distribution was seen. In this case the 3H-labeled complementary strands were almost exclusively circular and the viral strands were open. The same sample as in Figure 2(a) was also analyzed by neutral and alkaline CsCl density-gradient centrifugation (Fig. 3). These da,ta allowed the following statements on this particular RF11 species. It was of RF density (Fig. 3(a)); it contained the parental 3aP label in DNA of viral i)ype and the 3H label in DNA of complementary type density (Fig. 3(b)). The alkaline sucrose gradient (Fig. 2(a)) did not contain an added marker DNA. The infectivity shown was that of the complementary strand itself, further proving its circularity (single-stranded 4X DNA is generally only infectious in the circular form (Guthrie & Sinsheimer, 1960) ; q5X complementary single strands are known to be infectious in a strand

b72

B.

FRANCKE

AND

D.

6. RAY

(a)

Fraction

no

FIG. 3. Neutral and alkaline C&l equilibrium gradients of RF11 from rep; infected with tcnb 3. The same sample aa used for the alkaline suorose gradient (Fig. 2(a)) was centrifuged to equilibrium in neutral (a) and alkaline (b) C&l with an added unlabeled infectivity marker (single-stranded viral DNA). Gradients were prepared by the two-layer method aa described by Ray (1969), centrifuged for 27 hr at 40,000 rev./min and 2O”C, and fractions of 10 drops each were collected. Density increased from right to left. -a----a-, 3aP cts/min (parental label); -O---O-,. 3H cts/min (post-infection label) ; -- A-- A--, plaque-forming units (single-stranded viral marker DNA).

spheroplast assay system (Rust & Sinsheimer, 1967)). The specific infectivity of these complementary strands was 100% when compared with a standard viral DNA preparation tested with the same spheroplast preparation (calculated from the specific radioactivity of the 3aP-labeled parental strands present in the RF11 sample and assuming that one complementary strand was present per parental strand). The two distinguishing features of RF11 from am 3-infected rep; were: (1) its relatively constant number per cell at different multiplicities of infection and (2) the restriction of the discontinuity to the viral strand. 150 pg Cam/ml. inhibited the formation of this type of RFII. Also, after infeotion of rep; with arn 8 in the absence of Cam, it was not seen, nor was it formed, by ana 8 in HF4704 in which am 3 can replicate normally. The specific discontinuity in the viral strand was therefore not a function of the host cell on the rep; mutation, but could only be attributed to gene ii of $X174. How gene A accomplished the formation of specific RF11 could not bc concluded from these data; however, it had to be a protein-mediated reaction rather than a property of the mutant DNA itself, as a substrate for endonucleolytic enzymes. This could be concluded from the type of mutant used (am) and from the fact that the addition of Cam prevented the reaction in any of the systems used.

PARENTAL

(c) Characterization

$X174

RF

573

of the discontinuity in the viral strand of RFII from rep; cells

To obtain some information on the functions of the +X174 gene A or the E. coli rep; gene, a characterization of the discontinuity in the viral strand in the specific RF11 was attempted. Schekman, Iwaya, Bromstrup & Denhardt (1971) demonstrated that RF11 during +X174 replication could have several nucleotides missing (gap), could result from a single endonucleolytic hit (nick), or could have additional nucleotides (tail). Schekman & Ray (1971) also demonstrated that RF11 generated in a ligase-deficient host cell contained infectious linear strands of viral type, indicating that RF11 molecules of the “nicked” type with the nick at a specific site generated linear strands that were infectious as single strands (7% the specific infectivity of circular viral strands). To test this possibility for the nick in RF11 from rep ; cells, the 3aP-labeled linear strands were purified by three successive alkaline sucrose gradients as shown in Figure 4(a), (b) and (c). The infectivity assay after the third gradient (Fig. 4(c))

Fraction

no.

FIG. 4. Repeated alkaline sucrose centrifugation and alkaline C&l equilibrium banding of the linear strands of RF11 from rep, infected with am 3. RF11 was prepared from a gradient similar to that shown in Fig. l(a). First alkaline sucrose sedimentation (a); second sedimentation (b) of the linear strands (indicated by the braoket in (a)). The fractions indicated by the bracket in (b) were pooled and divided into 2 portions. One portion was ueed for the third alkaline sedimentation (c) and the other banded in alkaline C&l (d). For the suoroee gradients &&ion, 15 to 42 of a total of 76 fraotiona oolleoted are shown. Of the equilibrium gradient, fraationa 19 to 36 of a total of 47 fraotions oolleoted are shown. The remainder of the gradients did not contain any radioactivity. Fractions were colleated into 100 ~1. Tris-EDTA and 25 4. used for the radioactivity assay in (a) and (b), 100 ~1. in (o), and 150 ~1. in (d). --O---O-, saP cts/min (parental label) ; -O-(1-, 3H cts/min (post-infection label); -A---A-, plaque-forming units.

574

B. FRANCKE

AND

D. 8. RAY

revealed a peak on the slow side of the 32P radioactivity. This peak of infectivity probably represented the infectious linear molecules observed by Schekman & Ray (1971), while the peak at fraction 20 representedthe residual infectivity from circular single strands. This observation was made with two independent RPII preparations from rep; cells. If the 32P-labeledinfectious single strands observed here were indeed linear, then these results indicated the non-infectious linear strands were slight,ly longer than unit length. After the second alkaline sucrosegradient shown in Fig. 4(b), the linear material was divided into two portions and half of it used for the third alkaline sedimentation and the other half centrifuged to equilibrium in alkaline CsCl. The 3H label that did not separate from the linear strands in alkaline sucroseformed a band partly at the density of complementary strands (Fig. 4(d)), indicating that a few linear complementary strands had been present in the original RF11 preparation, possibly generated by random nicking of RFI. But a considerable amount of 3H label was found at the density of the 32P-labeledviral strands. Therefore, somenew material must have been incorporated into the parental strand, possibly by a displacement type synthesis, resulting in an RF11 with a short tail. Some parental DNA might also have been degraded and been replaced by new material similar to the process of “nick t’ranslation” in vitro carried out by E. co& DNA polymerase and $X174 RFIT as a t)emplate at low temperatures (Kelly, Cozzarelli, Deutscher, Lehman & Kornberg, 1970). Which of the two processeshad causedthe observation could not be decided, but it was possible that both nick translation and displacement synthesis had cont,ributed to the result. We have repeatedly observed low-molecular weight material containing the parental label in lysates obtained under conditions where gene A was functioning (compare Fig. l(a)), supporting the possibility of nick translation. On the other hand, the infectivity peak on the slow side of the radioactivity peak in Figure 4(c) could indicate someelongation of the majority of the parental strands as a result of displacement synthesis. (d) Elongation

of the viral strand

in repi cells

So far, we have concentrated on RF11 which was isolated under various conditions that did not allow the replication of the parental RF, assuming that the faster peak in neutral sucrosegradients (Fig. 1) consistedonly of RFI. That this was not the casr under all the non-replicating conditions used is shown in Figure 5. For the alkaline sucrosegradients illustrated in this Figure, the RF1 regions from the gradients shown in Figure 1 were separately pooled, concentrated, and sedimented. In the alkaline sucrose gradients, denatured RF1 sediments rapidly as supercoiled DNA, whereas those forms that have not been covalently closed in both strands sediment much slower as single-stranded DNA. From Figure 5(b) through (e), it was evident that the newly synthesized [3H]DNA was almost quantitatively in RFI. Most of the parental [32P]DNA was also in this form except for 10 to 20% that sedimented slowly. When the slowly sedimenting parental DNA was recovered from the gradient (as indicated in Fig. 5(b)) and analyzed by long alkaline sucrosegradient sedimentation with an added 3H-labeled viral DNA marker, it contained the following components: 50% circular, 20% unit length linear, and 30% shorter than unit length viral DNA. The origin and significance of this material was not further investigated. It was unlikely, though, that it arose from any partially replicated structures since no 3H label was seenin that region of the gradient.

T’ARENTAL

.L: .: ”

3

I

$5174

675

RF

:e)

Cdl

I IO Fractlan

no.

FIG. 5. Short alkaline sucrose gradient. sedimentation of the RF1 regions of Fig. 1 (as indicated by [I] in Fig. 1). nm 3 in rep; without (a) and with (b) Cam: trm 8 in rep, (c); am 8 in HF 4704 ((1): and am 3 in HF4704 with Cam (e). Centrifugation was EM described in the legend to Fig. 2, except, that centrifugation time was reduwd to 110 min. 12 Drops per fraction were collected and 50 ~1. 32P cts/min (pawnt,al label): -o--((1)-, 3H c:tn/min assayed for radioactivity. --@--a-, (post-infection label).

Figure 5(a) shows a different result. This was the only condition under which gene A of the phage was active. In this case the “RFI” material from Figure l(a) contained a considerable amount of denaturable DNA. The relative amount of single-stranded DNA in short alkaline runs of this type varied from experiment to experiment, but the slower sedimenting peak always contained an excess of 3H label as compared to the peak of denatured RFI. This observation suggested that some DNA synthesis had gone on beyond the formation of the parental RF. For an initial characterization, this material was recovered from an alkaline velocity gradient (as indicated in Fig. 5(a)), mixed with an excess of unlabeled viral DNA as reference marker, and sedimented through a neutral sucrose gradient (Fig. 6(a)), an alkaline sucrose gradient (Fig. 6(h)), and centrifuged to equilibrium in s,n alkaline CsCl density gradient (Fig. 6(c)). Of the fractions from the neutral sucrose gradient, only 50 ~1. were used for the radioactivity assay (the data in Fig. 6(a) were then calculated for 100 ~1. to allow quantitative comparison with the other gradients) and 1 ~1. was used for the infectivity assay. The remainder of the fractions was then pooled from three different areas of the gradient (as indicated in Fig. S(a)) and used for further alkaline CsCl equilibrium centrifugations. The majority of the 3H label sedimented under neutral conditions as a uniform peak at approximately the same rate as the viral DNA marker. It also sediment4 with the infectious DNA marker under alkaline conditions, indicating its circular structure. AlkaJine density banding showed the majority of the 3H label at the position of complementary strands. Thus, the major component of newly synthesized DNA was single-stranded complementary circles. The parental 32P label gave a

676

B.

FRANCKE

AND

Fraction

D.

S. RAY

no

Fm. 6. Analysis of the single-stranded material after short alkaline sucrose sedimentetion of RF1 material generated in rep, by am 3 (as indicated by the bracket in Fig. 6(a)). The material used for these gradients had been obteined from a lOO-ml. culture, treated exactly as described in Figs l(a) and 5(a). Neutral sucrose gradient sedimentation (a) was in 6 to 20% sucrose, 1 M-NaCI, Tris-EDTA (tot81 vol. 3.6 ml.) for 2 hr at 65,000 rev./min at 6°C in an SW66 rotor. Fractions of 6 drops each were oollected into 100 4. Tris-EDTA and 60 (.J. assayed for radioaotivity. The remainder was pooled in 3 different portions ((a), (b) and (c)) and saved for alkaline equilibrium gradients. Long slkaline sucrose gradient sedimentation (b) was as desoribed in Fig. 2. Alkaline CsCl density banding (c) was as described in Fig. 3(b). All 3 gradients contained an excess of unlabeled viral DNA as a marker. The position of the marker in (b) 8nd (o) is indicated by the arrow. -a-----a-, 32P cts/min (parental label); -O---O-, 3H cts/min (post-infection label) ; -~-a-, plaque-forming units (viral marker DNA).

symmetrical band at the density of the viral DNA. In alkaline sucrose, the parental label sedimented as a broader band than the majority of the 3H label, covering the whole range from unit-length linear to circular DNA. Since in neutral sucrose it separated from the position of unit length viral DNA with a peak slightly faster than the infeativity marker and pronounced trailing to the faster side, it must have consisted of some unit length and mostly longer linear strands. The material that had been added to the infecting parental strands probably consisted of 3H-labeled DNA of viral type. Two observations in Figure 6 supported this conclusion. There was 3H label sedimenting ahead of the infectivity marker in neutral sucroseand there was a shoulder of 3H label at viral strand density in alkaline CsCl. In alkaline sucrosethe region that should have contained the 3H label in larger than unit-length linear DNA was masked by the excessof circular complementary strands. More direct evidence

PARENTAL

4X174

RF -

Fraction

577

-

no

7. Alkaline C&l density-gradients of portion a (a); portion b (b); and portion o (c); obtained f ram the neutral sucrose gradient shown in Fig. 6(a). Centrifugation was as described in Fig. 3(b). Fractions 1 to 16 are not shown and did not contain any radioactivity. All 3 gradients contained an excess unlabeled viral DNA as a marker, indicated by the arrows in (b) and (c). -@----a---, 3aP cts/min (parental label) ; -O-O-, 3H cts/min (post-infection label); -A-n-, plaque-forming units (viral DNA marker). Fra.

is presented in Figure 7. The material faster than viral DNA (a), the major 3H-labeled component (b), and the thus far unidentilied slower peak (c) were recovered from the neutral sucrose gradient (Fig. 6(a)) and separately centrifuged to equilibrium in alkaline CsCl density-gradients. The major 3H-labeled peak banded at complementary strand density with practically no shoulder at viral density (Fig. 7(b)). The 32P in this gradient would correspond to those parental strands that had been least elongated. Some complementary circles must have been carried over into the fast-sedimenting fractions (a), giving rise to the peak at complementary strand density in Figure 7(a). but here, a definite peak of 3H label at viral density was seen, demonstrating the elongation of the parental strand with newly synthesized DNA of viral type. In Figure 7(a) and (c), a peak at higher than viral strand density was seen containing both 3H and 3aP label. The density corresponded to that of denatured RFI. Some residual RF1 must have been carried over from the preparative alkaline sucrose gradient (Fig. 6(a)), had partly renatured and would be expected at two positions in the neutral sucrose gradient (Fig. 6(a)): (1) as denatured RF1 (faster than single strands) and (2) as renatured RF1 (slower than single strands in high salt concentration). This explained the absence of the dense peak in Figure 7(b), for which the DNA had been pooled from the single-strand region of the neutral sucrose gradient. The denatured RF1 was obscured in Figure 6(a) by the elongated strands, but renatured RF1 constituted the majority of the small peak (c) in Figure 6(a). The only material left unaccounted for was the shoulder of shorter than unit-length material in alkaline sucrose(Fig. 6(b)). Since this const,ituted a very minor component, we did not attempt to characterize it. RF1 would have sedimented to the bottom of the tube in this

578

B. FRANCKE

AND

I). 8. RAY

gradient, and indeed the 3H label was much reduced as compared to the slow peak in neutral sucrose. Whether the remainder (about twice the background in Fig. 6(b)) represented complementary DNA of shorter than unit length could not be decided. The 32P at this position could have a similar origin as the single-stranded partia,lly degraded parental DNA observed in Figure 5(b) through (d). The main point which was proved with these experiments was that, under conditions where gene A is functioning, some DNA synthesis beyond the formation of the first RF took place. This synthesis resulted in covalent elongation of the parental viral strand with DNA of viral density. We have also shown that circular complementary DNA was present and would like to conclude that both were derived from replicative intermediates consisting of a double-stranded circle containing a closed complementary template with a tail as part of an elongated viral strand. Whether the tail was single- or double-stranded (containing complementary strand pieces) could not be decided. This type of synthesis was seen only under conditions where the gene A-specific RF11 was present, arguing that the discontinuity in the viral strand was a prerequisitcb for the elongation. The RI molecules of this type represented only a minor fraction of the viral DNA in rep, cells and could therefore indicate a leakiness of the rep; mutation. If this was the case and the elongation represented an attempt at normal RF replication, it was not a very efficient process, as demonstrated by the high yield of RI despite the long infection time (20 min) with [3H]dThd present all t,he time. (e) Timing

of the gene A functiorL

The effect of gene A on the viral DNA \vas to create or maintain a discontinuit,y in the viral strand of RFII. One way to achieve this might have been the persistence of a specific nick in the infecting strand that had been introduced as part of the initiation events of complementary strand synthesis. To test, this possibility, the following experiments were performed. So far it has not been possible to isolate and characterize intermediates during the synthesis of the parental RF. The experimental difficulties in unsynchronized infections arose mainly from the short time of synthesis as compared to the tim:: required for the absorption, attachment, and eclipse steps preceding it (Newbold & Sinsheimer, 1970). On the other hand, cells that had been starved for synchronization gave mainly abortive infections, resulting in degradation of the parental DNA (Francke & Ray, 1971). We therefore used for infection phages which had bean damaged by ultraviolet light, hoping that irradiation products would be introduced t,hat would prevent the synthesis of the first complementary strand beyond the site of the damage, yielding partially formed RF’s. First, some of the properties of the DNA from u.v.-irradiated phages were studied. Figure 8 shows an alkaline sucrose gradient sedimentation of 32P-labeled DNA extracted from phages that had been inactivated to 0.5% surviving plaque-forming units. The recovery of 32P label after SDS-pronase treatment and repeated phenol ext,raction was 53%, compared to 86% in an unirradiated control, indicating that some protein-DNA cross-linking had caused trapping of some of the DNA in the DNA was mixed with 3H-labeled phenol-buffer interface. The recovered 32P-labeled DNA from unirradiated phages. In a neutral sucrose gradient, both labels sediment4 together; but in alkaline sucrose (Fig. 8), a considerable portion of the irradiated DNA sedimented faster. This suggested that some cross-linking had taken place

PARENTAL

4x174

Fraction

RF

57!J

no

ctm 3 irradiated with u.v. FIG. 8. Alkaline sucrose gradient of DNA ext,rected from 321’-labcled to 0.5% surviving plaque-forming units. The irradiated phages were trectted with pronase (2OCJ pg/ml.) in 1% SDS for 2.5 hr, extracted twice wit,h Trite-EDTA-saturated phenol and the DNA precipitated with ethanol. “H-labeled DNA from unirradiated CI~ 3 w&s added&s a marker. Centrifug&ion was as described in Fig. 2, except that the running time was reduced to 3.0 hr. Fractions of 5 drops each were collected. -O-O-, 321’ cts/min (DNA from irradiated awz 3) : --c:--(-, 3H cts/min (unirradiated am 3 DNA marker).

preventing the complete unfolding of the DNA at alkaline pH. Using irradiated 35S-labeled phages containing a 3H-labeled DNA under the same conditions, less than 29/o of the original 35S label was found associated with the phenol-extracted DNA in alkaline sucrose gradients. This did not rule out completely DNA-protein crosslinking as a reason for the faster sedimentation rate in alkali. But the fact that DNA from irradiated and m&radiated phages sedimented together at neutral pH supported the explanation that it was DNA-DNA cross-linking, possibly mediated by a residual polypeptide linked to two or more places in the viral DNA. Figure 9 shows an experiment in which rep; cells were infected with 32P-labeled urn 3 that had been inactivated to 7.3% surviving plaque-forming units. During the 20-minute infection period, [3H]dThd had been present. The lysate was centrifuged through a high concentration salt-sucrose gradient (Fig. 9(a)) and the fractions containing the parental label (as indicated by b in the Figure) were pooled and re-analyzed by long alkaline sucrose sedimentation along with an unlabeled viral DNA marker (Fig. 9(b)). Most of the 3H label in Figure 9(a) was associated with the slowest sedimenting parental DNA, indicating that some complete or almost complete RF11 molecules had been made. The next faster peak contained some RF1 (as verified by alkalintb sucrose gradient centrifugation, not shown here). A considerable amount of parental DNA was seen at the position of single strands containing the smallest amount of 3H label. Figure 9(b) demonstrates that a wide range of complementary strand fragments had been made from unit length linear strands down to very small pieces. These fragments separated from the parental DNA under alkaline conditions, indicating t.hat they had not been covalently attached to the infecting DNA. The parental DNA itself was found to be largely circular with some forward trailing cross-linked material that, has already been described in Figure 8 for irradiated DNA before infection. The

580

B. FRANCKE

AND

D. S. RAY

(b)

Fro. 9. Preparative neutral (a) and long alkaline sucrose sedimentation (b) of intracellular DNA from rep, cells infected with am 3 phages which had been u.v.-inactivated to 7.3% surviving plaque-forming units. Infection 8nd labeling conditions were as described in Fig. 1 except that 0.5 mCi[2H]dThd were added to the 20-ml. culture. The fractions indicated in the neutral gradient (a) by the bracket, b, were pooled, concentrated, and sedimented through a long alkaline gradient with an excess of unlabeled viral DNA added as 8 marker. Five drops per fraction were collected saP cts/min (parental label) ; -O-O--, 3H cts/min for the alkaline gradient. -@-----, (post-infection label) ; -AA-, pleque-forming units (viral DNA marker).

small amount of linear parental DNA was probably derived from completed RF molecules that were a substrate for the action of gene A. A corresponding peak of circular complementary strands was also seen in Figure 9(b). To substantiate this interpretation, a series of four cultures of rep; cells were infected with 32P-labeled am 3 that had been inactivated by U.V. for 0 seconds (100% plaque-forming units, Fig. 10(a)), 7.5 seconds (45% plaque-forming units, Fig. 10(b)), 15 seconds (18% plaque-forming units, Fig. 10(c)), and 30 seconds (1.4% plaque-forming units, Fig. 10(d)). The experimental details were as described in Figure 9 and t’he corresponding alkaline sucrose gradients are shown in Figure 10(e), (f), (g) and (h). With increasing U.V. dose the neutral gradients revealed a rapid reduction of the RF1 peak and a slower decrease in the peak of completed RF11 with a gradual appearance of still largely single-stranded parental DNA. Along with these changes a shift of the main peak of the 32P label from the position of linears (due to the gene A-specific RF11 in the irradiated sample) to the position of circles, was seen in the alkaline gradients. Because these alkaline gradients were of the total virus-specific DNA, the effect was obscured by structures with elongated parental strands in the O-second sample and by cross-linked DNA in the later samples. Therefore, only the RF11 regions of Figure IO(a), (b) and (c) (as indicated by {II} in the Figures) were analyzed by alkaline gradients and this result is shown in Figure 11. While in Figure 11(a) all the parental DNA was linnar n’nd thr: complementary strands were circular as already described in Figure Z(a), the situation progressively reversed with increasing U.V. dose. The linear complementary strands in Figure 11(c) were shorter than unit

PARENTAL

4x174

RF

(e)

“i 5

c)

I

,9 .

\

2

2

I

FIa. 10. Infection of rep; cells with 32P-labeled nrn, 3, u.v. irradiated for v8rying times. 0 see u.v., 100°/O (8); 7.5 SIX u.v., 45% (b); 15 SW u.v., 15% (c); 30 see u.v., 1.4% plaque-forming units (d). Experiment&l details were a~ described in Fig. 9 except that the usual 0.2 mCi of 3[H]dThd were added to each 20-ml. culture. 200 1.11. of the fractions indicated by the brackets in (8) through (d) were used for the dkeline gradients shown in (e) through (h). The position of thF: infectious viral DNA marker in the alkaline gradients is indicated by the RWOW. -o---e--, nzp cts/min (parental label): -o-\ c’-, 3H cts/min (post-infection label).

length, indicating that they had originated from not-fully synthesized RF11 molecules. These data strongly suggested that a complete RF had to be made before the gene A. specified discontinuity could he introduced intro the viral strand. Three explanations

882

B.

FRANCKE

AND

D.

6.

RAY

(b)

-II

Fraction

no

Fxa. 11. Long alkaline sucrose gradients of the RF11 regions obtained after infection of rep; with ana 3, u.v.-irradiated to 100% (a), 45% (b), and 18% (c) surviving plaque-forming units. The fractions indicated by {II} in Fig. 10(a), (b) and (c) were separately pooled and concentrated. Centrifugation was as described in Fig. 2. Five drops per fraction were collected directly onto filter paper discs and counted. Fractions 10 to 28 of a total of 44 fractions collected are shown. -•----a---, 32P ots/min (parental label); -O--O-, 3H cts/min (post-infection label).

for this observation seem possible : (1) only RF1 is a substrate for the gene A function ; (2) only a completed RF can be transcribed to yield the gene A product; (3) the irradiation had damaged the DNA such that no functional viral proteins could bo made. While the experiments with u.v.-irradiated phages indicate that the timing of the gene A function is after the completion of the first RF, the circularity of the complementary strand in the parental RF11 also argues for this sequenceof events, since a discontinuity in the template (infecting viral strand) before the completion of the first RF could not easily result in a circular product (first complementary strand).

4. Discussion The results presented in this communication bear on the formation of the parental RF of +X174 and the function of the viral gene A after infection of rep; host cells. The experimental details have been discussedin the Results section and will not be repeated here. We would rather discussour results in terms of a proposed sequence of events that take place at the DNA level after infection in this system. This proposed sequenceis presented in the left column of Figure 12, which also lists the different experimental conditions under which the DNA forms characterized (right column) have been obtained. (a) Synthesis

of the first complementary

strand

RI during complementary synthesis was obtained by using u.v.-irradiated phage for infection. The amount of DNA t,hat could be synthesized in the infected cells on

PARENTAL

4x174

infened sequence of events during inkction of a permissive host with

683

RF DNA forms isolated under various non-permissive conditions

$ Host polymerase

0 -‘\

\r t-0

\

I

/I

t Polynucleotide

ligase Gene A amber

t +X174 ,--\ f \ 0

\

\

mutant

gene A product

\

Excess

RF1

rep3 mutation

:

L.,’

I

RF- replication

residual DNAsynthesis in rep; ceils beyond the parental RF

RFII with open vim1 si ,rand fiv

RI with elongated FIG. 12. Schematic presentation tions. Thick line, viral strand; synthesized DNA.

thin

of DNA forma isolated line, oomplementary

viral strand

under various non-replioating strand; dashed line indicates

condinewly

this template was greatly reduced at high U.O. doses, and the size of complementary strands of such RI molecules covered a whole range from small pieces to unit-length linear strands, indicating that DNA synthesis had been discontinued at the site of U.V. damage. The type of U.V. photoproduct giving this effect could not be identiCed. It was likely that cross-linking, observed in the DNA from irradiated phage, was one of the reasons. However, thymine dimers or other photoproducts could have given the same effect. Relevant for the mechanism of RF synthesis was the observation that in this type of RI the viral strand wa,s circular. Only under relatively low U.V. doses, where a measurable amount of circular complementary strands had been made, was a corresponding amount of open parental strands seen. This indicated that the gene A-specih RF11 with an open viral strand could only be generated after the first RF had been completed and did not result from a persistent nick, possibly introduced during the initid events of the infection process. The separation of the complementary strand pieces in alkali from the parental strand indicated that they were not covalently attached to the infecting DNA. This observation does not exclude, though, that such a covalent bond might have existed as one of the initial stages of the initiation of complementary strand synthesis, since nicking of this bond and rejoining of the parental viral circle might not require the completion of the complementary strand. The question of the mechanism of initiation thus still remains open. Ono & Shimazu (1967) reported that the infecting DNA of u.v.-irradiated C/R phages was 38

684

B.

FRANCKE

AND

D.

S. RAY

subject to endonucleolytic degradation in E. coli C but to a lesser degree in E. coli a u.v.-sensitive derivative which was deficient in the host-cell reactivation sp-, mechanism (her-). rep; is also her-, and thusourresults are in agreementwith these findings. The isolation of the rep; strain (Denhardt et al., 1967) involved extensive mutagenesis of the parent strain (HI? 4704) and it is believed (Benbow, personal communication) that the rep 3 strain has acquired other deficiencies in its her and ret functions that map differently and can be separated by transduction. Therefore, the lack of endonucleolytic degradation of irradiated parental DNA in rep; could have been a result of one such additional lesion. We have compared the fate of irradiated parental +X am 8 DNA both in rep; and HF4704 under identical conditions and it remained circular in both cases. Thus, the original her- mutation in HP4704 was sufficient to prevent nicking of u.v.-damaged +X DNA and the circularity of the parental strand in RI, with incomplete complementary strands, was not caused by an additional deficiency of the rep; host. (bj RPI as the major product in the abseltce of viral gene fundions In accordance with the idea that the first complementary strand is synthesized and closed on a circular viral template by host-cell enzymes is the finding that in the absence of all viral functions or the A gene function, RF1 constitutes the majority of virus-specific DNA. Such conditions include infection of either RF4704 or rep; in the presence of 150 pg of Cam/ml. with am 3, and infections of both cell types with am 8 in the presence or absence of Cam. The constantly low relative amount of RFII found under these conditions was independent of the multiplicity of infection. It contained both closed and open viral and complementary strands with a preference for the complementary strand to be the open one. This RF11 could have been produced by random nicking of the RF1 during extraction, or it could represent an equilibrium between RF1 and RF11 in viwo. The predominance of RF1 in the absence of viral functions is iu conflict with observations by Knippers Q Miiller-Wecker (1970) and Levine & Sinsheimer (1969a) in mitomycin-treated cells. Although the yield of progeny phage appears to be approximately normal in mitomycin-treated cells (Lindqvist & Sinsheimer, 1967) the effect on replicating DNA haa not been examined in detail. Under conditions of amino-acid starvation of a host cell requiring an amino-acid, Knippers & Miiller-Wecker (1970) obtained a parental RF11 with an open complementary strand and postulated an enzymic activity for the introduction of the nick. They could not exclude, though, that the complementary strand had never been closed, possibly as a result either of the starvation procedure or the mitomycin treatment. Levine & Sinsheimer (1969a) found nicked parental RF using gene A mutants under non-permissive conditions. In their experiments no strand analysis was performed. In addition to the possible mitomycin effect, the gene A product could have acted on the viral DNA during extraction in the case of the temperature-sensitive mutant ts128 (Francke, unpublished observations). (c) Possible functions of the gene A product When rep; cells were infected with am 3, a specific type of RF11 was seen. It was found at a fairly constant number per infected cell (1 to 4) independent of the multiplicity of infection, and the discontinuity was almost exclusively in the viral strand. ana 8 (a mutant in gene A) did not yield this type of RF11 in rep ; or in HF4704, and the addition of 160 pg Cam/ml. also prevented its formation in a,ny combination of host cell and phage mutant used. Thus, the role of gene A (the viral function required

PARENTAL

+X174

RF

585

for RF repI&tion) is to nick specifically the viral strand of the RF and/or to prevent it from being closed again. A characterization of the discontinuity in RF11 from rep, cells was attempted. After successive purification of the linear parental strands by alkaline sucrose-gradient sedimentations, not all of the newly synthesized DNA could be removed. Apart from a low level of linear complementary strands in such a preparation, a peak of newly synthesized DNA of the density of viral strands was seen, indicating that some new material had been incorporated into the parental viral strand after infection. This can either be seen in analogy to nick-translation in vitro (Kelly et al., 1970), a process in which DNA polymerase degrades from the 5’ end of nicked RF, elongating at the same time at the 3’ end, or as the result of a beginning displacement-type synthesis. Whether one of these processes or both contributed to the observation, could not be decided. As to the gene A product itself, there are basically two possibilities for its mode of action: (1) this protein activates or is itself an endonuclease specific for the viral strand; (2) it prevents polynucleotide ligase closure specifically of the nicked viral strand. A model for the generation of specitlc nicks without a specific endonuclease has recently been put forward by Razin & Sinsheimer (1970). At present no decision can be made. But knowing the effect gene A has on the DNA should facilitate the isolation of the gene product. (d) Residual DNA synthesis in rep, cell.9 beyond the parental RF The type of RI found at low levels in rep; cells infected with am 3 consisted of a closed complementary strand and an elongated viral strand. Assuming that this structure was the result of an attempt at normal RF replication, which was only grossly impeded in the rep; host, our results would strongly support the model of RF replication put forward by Dressier & Denhardt (1968), Gilbert & Dressler (1968) and Dressler C%Wolfson (1970). They characterized rolling-circle type molecules with a circular complementary strand template and an elongated viral strand. The RI structure reported here is inconsistent, though, with the mechanism proposed by Knippers et al. (1969). These authors postulated RF replication to occur by elongation of the complementary strand on a circular viral template. The function of the rep, gene is not known. Therefore the possibility has to be considered that RF replication is completely blocked by the rep 3 mutation. Then the RI found in rep 3 cells could represent an attempt at single-strand synthesis, for which the elongation of the viral strand has been established by all investigators involved. We were not able to exclude this possibility directly because of the ambiguity regarding the presence of nascent complementary strands in our system. But Levine & Sinsheimer (1969a) have shown thatgene A wasnotrequiredforsingle-strandsynthesis. Theirresults, togetherwith the known phenotype of gene A mutants (no RF replication) and our data on the function and timing of the gene, make the model of Knippers et al. (1969) the less likely one. As long as the gene A product and the rep; protein have not been isolated in an active form, we can only speculate on their mechanisms of action. But it is evident from our experiments that these two proteins known to be involved in the initiation of viral DNA replication clearly serve different functions that can be distinguished at the DNA level. This work was supported in part by grants from the National Science Foundation (GB 18074), and the University of California Cancer Research Co-ordinating Committee. One of us (B. F.) W&B the recipient of a U.S. Public Health Service Postdoctoral Fellowship (no. 5 F05 TWO 1630-02).

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D.

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REFERENCES Denhardt, D. T., Dressier, D. H. & Hathaway, A. (1967). Proc. Nat. Acad. Sci., Wad. 57, 813. Dressier, D. & Denhardt, D. T. (1968). Nature, 219, 346. Dressler, D. & Wolfson, J. (1970). Proc. Nat. Acud. Sk., Wash. 67, 456. Francke, B. BE Ray, D. S. (1971). Virology, 44, 168. Gilbert, W. & Dressier, D. (1968). Cold Spr. Harb. Symp. Qwmt. Biol. 473. Greenlee, L. L. & Sinsheimer, R. L. (1968). J. Mol. BioZ. 32, 303. Guthrie, G. D. & Sinsheimer, R. L. (1960). J. Mol. BioZ. 2, 297. Guthrie, G. D. & Sinsheimer, R. L. (1963). B,iochim. biophys. Acta, 72, 290. Hutchison, C. A., III & Sinsheimer, R. L. (1966). J. Mol. BioZ. 18, 429. Kelly, R. B., Cozzarelli, N. R., Deutscher, M. P., Lehman, I. R. S: Kornberg, A. (1970). J. Biol.

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Knippers, R. & Miiller-Wecker, H. (1970). Europ. J. Biochem. 15, 146. Knippers, R., Whalley, J. M. & Sinsheimer, R. L. (1969). Proc. Nat. Acad. Sci., Wash. 64, 275. Levine, A. J. & Sinsheimer, R. L. (1969a). J. Mol. BioZ. 39, 619. Levine, A. J. & Sinsheimer, R. L. (19693). J. Mol. BioZ. 39, 655. Lindqvist, B. & Sinsheimer, R. L. (1967). J. Mol. BioZ. 30, 69. Newbold, J. E. & Sinsheimer, R. L. (1970). J. Mol. BioZ. 49, 49. Ono, J. & Shimazu, P. (1967). J. Mol. BioZ. 24, 491. Ray, D. 8. (1969). J. Mol. BioZ. 43, 631. Razin, A. & Sinsheimer, R. L. (1970). Proc. Nut. Acad. Sci., Wash. 66, 646. Riist, P. & Sinsheimer, R. L. (1967). J. Mol. BioZ. 23,545. Schekman, R. W., Iwaya, M., Bromstrup, K. & Denhardt, D. T. (1971). J. Mol. BioZ. 57, 177. Schekman, R. W. & Ray, D. S. (1971). Nature, New Biol. 231, 170. Sinsheimer, R. L. (1968). Prog. NucZeic Acid Res. & Mol. BioZ. 8, 115. Sinsheimer, R. L., Starman, B., Nagler, C. & Guthrie, S. (1962). J. Mol. BioZ. 4, 142. Tessman, E. S. (1966). J. Mol. BioZ. 17, 218.