Initiation of the DNA replication cycle in Escherichia coli linkage of origin daughter DNA to parental DNA?

J. Nol. Biol. (1972) 64, 393497

Initiation of the DNA Replication Cycle in Escherichia cd: Linkage of Origin Daughter DNA to Parental DNA? GRETCHEN H. STEIN? AND PHILIP

C.

HANAWALT

Deprtment of Biological Sciences Stanford University, Stanford, Calif. 94305, U.S.A. (Received 26 July 1971, and in revised form 25 November 1971) Many models for the mechanism of initiation of DNA replication in bacteria include the idee that DNA synthesis is initiated at the 3’-OH end of a p8m&al DNA strand. We have tried to determine whether the initiation of DNA replication in Ewhmkhia co& involves covalent joining of origin daughter DNA to pre-existing parental DNA. The technique used to visualize such a linkage ~8s e density shift from &bromoum& to thymine at the time of initiation of a cycle of DNA replication. When such 8 density shift is made during DNA ohain elongation, intermediate density DNA fragments due to transition points 8re observed, but when the density shift is made et the time of initiation of new rounds of DNA replication, intermediate density DNA fragments due to linkage of origin daughter DNA to parental DNA are not found. These results indicate that if DNA replicetion in E. ~0% is initiated by linkage to parental DNA, such linkage persists for less than one to two minutes.

1. Introduction B8eteri81 DNA replic8tion is semi-conservative and proceeds aequentielly from 8 unique origin (cf. Wolf, Newman & Glaser, 1968; Cerd&Olmedo, Hmawelt & Guerola, 1968). Although both strands of the DNA duplex appear to be rephcsted simultaneously in the gross topological sense (Cairns, 1963), there is no known DNA polymerase which c8n synthesize DNA in the 3’ to 5’ direction (Richardson, 1969; Moses, Fleischmen, Campbell, Frenkel & Rich8rdson, 1971; Kornberg $ Gefter, 1971). Thus there is no known mechanism for the continuous replication of the 5’ perental DNA strands. One mechanism which has been proposed to resolve this dilemme is the discontinuous synthesis of short fragments of DNA which are subsequently joined by the polynucleotide ligase (Okazaki et al., 1968). These smell fr8gments of DNA can be synthesised in the 5’ to 3’ direction for the DNA strand being elongated in the 3’ to 5’ direction over-all. Studies on the initiation of the DNA replication cycle in bacteria indicate that it requires protein synthesis (Maaloe & Hanswelt, 1961; Lark, Repko & Hoffman, 1963), is independent of the presence or location of existing points of replication (Stein & Hanawalt, 1969) and has a key role in the co-ordination of DNA duphcation

with

cell growth

mechanism of initiation t Present address: cine, Stanford, Calif. $ The deeignation replicated over-all in

and division

(Cooper

& Helmstetter,

1968). The molecular

is not understood although a number of models have been

Department of Medical Microbiology, Stanford University School of Medi94306, U.S.A. 3’ or 5’ strand refers to the complementary strands of the DNA which are the 5’ to 3’ and 3’ to 5’ direction, respectively. 393

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proposed. One important unanswered question is whether initiation of DNA synthesis in vivo can take place de novo along a template strand or whether a “primer” is required. The Escherichia coli DNA polymerases I, II, and III all require the 3’-OH end of a polydeoxyribonucleotide as a primer in vitro (A. Kornberg, 1969; T. Kornberg & Gefter, 1971). We sought to determine the validity of the primer requirement in vivo by examining whether or not in E. coli the DNA strands synthesized immediately after initiation are covalently joined to parental DNA strands. The results of our experiments indicate that if the DNA made immediately after initiation in E. co& is covalently linked to pre-existing DNA, such linkage is transient and is maintained for less than a few minutes.

2. Materials and Methods (a) Bacterial strains, growth conditions and isotqk labeling Escherichia co.% TAU-bar, thy-, ura-, arg-, met-, pro-, and trp- was grown with aeration in a Tris-glucose-salts minimal medium (Maalm & Hanawalt, 1961) supplemented with 2 pg thymine/ml., 8 pg uracil/ml., and 20 pg amino acids/ml. Balanced exponential growth in this medium at 37°C exhibits a 45-min generation time. Changes of media were accdmplished by rapid filtration of 150 ml. cultures on g-cm B6 (pore size 0.65 pm) Schleicher & Schuell membrane filters. The elapsed time for filtration was 10 to 15 sec. The cells were rinsed on the filters with 50 ml. Tris-salts solution without glucose and supplements at 37°C and then resuspended in fresh medium. 5-[14C]bromouracil at a specific activity of 2.1 mCi/m-mole was used at 5.5 pg/ml. with O-2 rg thymine supplement/ml. to density-label the DNA. [3H]thymine at a specific activity of approximately 0.5 Ci/m-mole was used at 1 pg/ml. as pulse-label. Following administration of the pulse-label, the cells were immediately harvested on a filter and one-third of the culture was resuspended in 50 ml. ice-cold NaCl-EDTA-Tris buffer (0.1 M-N&~, 0.01 M-EDTA, 0.01 M-Tris, pH 8). Simultaneously, the other twothirds of the culture was resuspended in fresh medium without radioactive label to chase the pulse. The observed amount of pulse-label incorporated relative to the amount of prelabel was the same for both the pulse and chase time points. (b) Synchronization

of DNA replication

cycles and den&y shift8

Linkage of origin daughter DNA to parental DNA was first looked for in cells which had been synchronized by ammo-acid starvation (Hanawalt & Wax, 1964) in thyminecontaining medium and then allowed to initiate new rounds of replication in 5-[3H]bromouracil for 2 min. However, the isolation and characterization of DNA fragments containing a transition point from thymine to 5-bromouracil was obscured by the presence of DNA fragments synthesized from a mixture of residual thymine in the intracellular nuoleotide pools and the 5-[3H]bromouracil incorporated during the pulse label (Stem, 1971). By virtue of the selectivity for thymine over 5-bromouracil (Hackett & Hanawalt, 1966) it was hoped that a density shift from 5-bromouracil to thymine would produce sharper transition points and therefore this method w&s used in the experiments described below. In order to examine a density shift from 5-bromouracil to thymine at the time of initiation, the DNA replication cycles must be aligned in 5bromouracil-containing medium. It was found that DNA synthesis stops after approximately 130 to 150 mm of amino-acid starvation in the presence of 5-bromouracil. When ammo acids were added back replication resumed at the same location as the origin defined by amino-acid starvation in the presence of thymine, which has been shown to be the same as the vegetative origin (Wolf et al., 1968; Caro & Berg, 1969). The method of Pritchard & Lark (1964) was used for this control experiment. No loss of viability was observed after 120 min of ammo-acid starvation in the presence of 5-bromouracil, but after 160 min the viability had decreased by approximately 30%.

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(c) Density-gradient analysis 50-ml. samples of cultures at approximately 10s cells/ml. were taken for density-gradient analysis. The cells were harvested and resuspended in 2 ml. NaC%EDTA-Tris/lO (a tenfold dilution of the buffer in section (a) above). The DNA was extracted by the following procedure : (1) 20 min at 37°C in the presence of 0.4 mg lysozyme/ml. (Boehringer Mannheim Corp.); (2) addition of Calbiochem grade B pronase to O-1 mg/ml. ; (3) 2 min vortexing on a Clay-Adams Cyclomixer ; (4) addition of Geigy NL9’7 Sarkosyl to a final concentration of 0.5%; (5) incubation at 60°C for 30 min. Usually the DNA in the resulting lysate was denatured by the addition of 0.6 ml. of 0.4 M-K~HPO~ at pH 12.5 (Vinograd, Morris, Davidson & Dove, 1963). The lysate was diluted to a 6nal volume of 6 ml. and mixed with 8.90 g CsCl (Harshaw optical grade). The samples were centrifuged in polyallomer tubes in a Spinco no. 40 rotor for 36 hr at 37,000 rev./min. The centrifuge tubes were punctured at the bottom and 15-drop fractions were collected. When selected fractions were to be rebanded, 20 4. samples were taken from each fraction for counting. The DNA was precipitated with 5 ml. of ice-cold 5% trichloroacetic acid and collected on HA 0.45 pm Millipore filters. The dried titers were counted in 5 ml. toluene containing 17.3 mg PPO and 0.43 mg dimethyl POPOP in the Tricarb liquid scintillation spectrometer. (d) Sucrose-gradient analysis 50-d. samples of the lysates prepared for CsCl gradient analysis were taken before the DNA was denatured. Each sample was layered on top of 4.8 ml. of a linear 5 to 20% sucrose gradient in 0.9 M-Nacl, 0.1 M-NaOH buffer. Centrifugation was carried out at 35,000 rev./min for 3 to 4 hr at 20°C in the SW39 swinging bucket rotor of the Spinco model L2-65B ultracentrifuge. Approximately 0.2 ml. fractions were collected through a pinhole punched in the bottom of the tube. The DNA was precipitated and counted in the same manner described for the CsCl gradients. Approximate molecular weights were calculated on the basis of the calibrations of Abelson & Thomas (1966) and Studier

(1965). (e) Shearing of DNA DNA was sheared to approximately 106 molecular weight fragments by subjecting the lysate at 4°C to a total of 4 min of discontinuous sonication at power setting 8 of a Branson Sonifier, model 575. (Discontinuous sonication involved giving the lysate 20 to 30 short sonication pulses of 0.5 to 1 set each and then shaking and cooling the lysate briefly before the next set of short pulses.)

3. Results If replication in Escherichia coli begins at the origin with the addition of nucleotides to the 3’-OH ends of parental DNA strands, then there should exist stretches of DNA in which daughter strands are covalently linked to parental strands. Such stretches upon isolation should exhibit densities intermediate between those of 5-bromouracil-labeled parental-strand fragments and thymine-labeled daughterstrand fragments if a transition from Lbromouracil medium to thymine medium is made at the time of initiation. DNA stretches containing transition points are clearly identifiable when the density transition is made in the middle of the replication cycle in E. coli (Pettijohn & Hanawalt, 1964). Our initial results seemed to indicate that such transition-point fragments also existed when the density transition was made as the replication cycle began. However, further characterization of the intermediate density DNA led us to question that interpretation and to conclude that if the parental strand does serve a priming function in the initiation of

396

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replication then it must involve a transient bond persisting for less than several minutes. In what follows we describe in chronological sequence the experiments and controls that led to our conclusion. (a) Density shift from 5-bromouracil to thymdne The DNA replication cycles of a culture of E. coli were aligned in Sbromouraciloontaining medium and initiation of DNA replication was allowed to take place in medium containing [3H]thymiue for 30 seconds. When the DNA was denatured and subjected to equilibrium sedimentation in an alkaline C&l density gradient, some of the 3H-labeled DNA banded at a density intermediate between fully heavy and fully light single-stranded DNA. Figure l(a) shows the typical result of such an

800

-1600 Omin Chase

Light

T

400-

- 800

‘; c \ 20 ;i p_

-2 .-5 Y) 20 min Chase

Light

2

I 400

m 800

Reband ?. ,

0M

0

5

IO 15 20 25 Fraction

FIG. 1. Initial banding after initiation. An exponential culture (1) 180 min amino-acid starvation (+ a.e.), (3) The DNA was denatured DNA strands is indioeted

in allreline

0

no.

CsCl density-gradients

of DNA

pulse-labeled

immediately

of TAU-bar was synchronized and labeled as follows: starvation in the presence of 5-[14C]bromouracil, (2) 40 min thymine 30 seoonds [3H]thymine, (4) 0 or 20 min chase in unlabeled thymine. and subjeated to density-gradient centrifugstion. The position of light -@0 -, l*C; - A - A -, 3H.

experiment. By selecting and rebanding the intermediate density fractions from the alkaline CsCl banding, it was possible to resolve the intermediate density DNA from the fully light- and fully heavy-DNA strand peaks, as shown in Figure 2, zero-time curve. The synthesis of intermediate density DNA strands labeled by the [3H]thymine pulse, which is presumably incorporated at the origin of the daughter DNA strands, is consistent with the hypothesis that the DNA made immediately after initiation ie linked to the pie-existing DNA. However, there are two trivial ways that 3H-labeled intermediate density DNA could have been formed. If there were still firat

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Heavy

397

Light

Fraction

no.

FIG. 2. Density distribution of pulse-labeled DNA made immediately &er initiation. An exponentictl culture of TAU-bar w&s synohronized and labeled as follows: (1) 140 min amino-acid starvation in the presence of 6-[14C]bromouraoil, (2) 40 mm thymine starvation (+ &.a.), (3) 30 sea [3H]thymine, (4) 0, 3 and 60 min chase in unlabeled thymine. The DNA was denatured and subjected to density-gradient centrifugation. The intermediate density fractions were selected and rebanded. The Figure is a composite of the distribution of the pulse-labeled DNA from the second banding of the 0-, 3-, and 60-min samples. The data are presented in this fashion in all subsequent Figures. The positions of light and heavy DNA strands are indicated.

some cells containing growing points at positions other than the origin, then a density shift would produce ordinary intermediate density transition points in these elongating chains. Second, if some B-bromouracil were still available in the intracellular nucleotide pools of the bacteria, then the first DNA fragments synthesized after the density shift would be synthesized using a mixture of ELbromouracil and thymine. These DNA fragments would band in the intermediate density region whether or not they were attached to the pre-existing DNA. In order to distinguish between the alternative explanations for the presence of intermediate density DNA, the effect of a chase with unlabeled thymine was examined (Fig. l(b) shows the typical result of the first banding in an alkaline C&l density gradient). Intermediate density DNA strands that represent ordinary transition points or that were synthesized from a mixture of 5-bromouracil and thymine should persist during subsequent growth. In contrast, any covalent linkage between origin daughter DNA and parental DNA would ultimately have to be broken to allow segregation of the daughter chromosomes. Figure 2 shows the results obtained from the second banding of the intermediate density DNA from a 5-bromouracil-labeled oulture which was allowed to initiate DNA replication for 30 seconds in [3H]thymine and then was grown in unlabeled thymine-containing medium for both a short and a long chase. Intermediate density

398

G. H.

STEIN

AND

P. C. HANAWALT Light

Heavy 4oc

3oc E \ 2 0 mI

200

100

Fraction

no

FIQ. 3. Density

distribution of pulse-labeled DNA made at randomly distributed growing points. An exponential culture of TAU-bar was grown in 5-[14C]bromouracil for 60 mm and then pulse-labeled with [sH]thymine for 30 sec. Subsequent growth in unlabeled thymine-containing medium was allowed for 0, 10, and 60 min. The DNA was denatured and subjected to denaitygradient centrifugation. The intermediate density fractions were selected and rebanded. The positions of light and heavy DNA strands are indicated.

Fraction no

4. Size of the DNA fragments pulse-labeled immediately after initiation. A 150-ml. culture of TAU-bar was synchronized and labeled according to the following protocol: (1) 140 mm amino-acid starvation in the presence of 5-[l%]bromouracil, (2) 40 min thymine starvation ( f &.a.), (3) 30 set [3H]thymine, (4) 0, 3, and 60 min chase (thymine). The cells were lysed and SO-p]. samples were analyzed on 5 to 20% alkaline sucrose gradients oentrifuged for 3 hr at 35,000 rev./min in the Spinco SW39 rotor. The direction of sedimentation is from right to left. The 0-, 3-, and 60-min curves show the distribution of the 3H pulselabel, and the 5-[1*C]bromouraoil curve shows the position of the 5bromouraoil-containing parental strands, FIU.

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399

no.

Fm. 6. Effect of shearing on the density distribution of the DNA pulse-labeled immediately after initiation. A 150-ml. culture of TAU-bar was synchronized and IabeIed in the following manner: (1) 150 min amino-aaid starvation in the presence of 5-[W]bromouracil, (2) 40 min thymine starvation (+ ea.), (3) 30 set [3H]thymine, (4) 0 and 60 min chase (thymine). The cells were lysed and half of each sample was sheared by 6 min of discontinuous sonioation at power setting no. 8. The DNA was denatured and banded in alkaline CsCl gradients. The intermediate density fractions were selected and rebanded. The positions of the light and heavy DNA strands are indicated. The zero time and zero-time sheared curves are identical.

DNA fragments are observed at the zero-time point taken immediately after the pulse. After as little as a three-minute chase, most of this intermediate density DNA has been chased out of the intermediate density region. By way of comparison, Figure 3 shows the results of a control experiment in which the cells were unsynchronized. In this case the transition points were at random locations in the chromosomes and hence should not be affected by a chase. (b) Effect of DNA f r q ment size on the density distribution of the pulse label Analysis of the molecular weight of the pulse-labeled DNA both before and after the chase (Fig. 4) indicated that immediately after the pulse is given, the label is present in small pieces of DNA comparable in size to Okazaki fragments (Okazaki et al., 1968). The chase of these small fragments of DNA into the bulk DNA was coincident in time with the chase of the intermediate density DNA into the fully light position (compare Figs 2 and 4). Furthermore, sonication of the DNA after the chase recreated the intermediate density DNA (Fig. 5). One possible explanation for these results was that the effect of the chase was due to the joining of small pulse-labeled fragments of DNA synthesized from a mixture of 5-bromouracil and thymine to fully light DNA synthesized during the chase. An experiment was designed to eliminate the problem of residual Sbromouracil in the intracellular nucleotide pools by letting the cells grow in thymine for a period of time between the termination of DNA replication in 5bromouracil and the pulselabeling of the DNA made immediately after initiation. This experimental procedure required aligning the replication cycles a second time in thymine before the pulse was given. Consequently, only one quarter of the available chromosomal termini would be labeled with 5-bromouracil, but linkage of the origin daughter DNA to

400

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Heavy

Light I

I

200 I2

E \ 2 ” IQ=

0

5

IO

15 Fraction

20

25

no.

FIG. 6. Effect of a period of growth in thymine between termination of DNA replication in 6-bromouracil and initiation of DNA replication in [3H]thymine. A 16Oml. culture of TAU-bar was synchronized and labeled in the following manner: (1) 150 min amino-acid starvation in the presence of S-[‘*C]bromouraoil, (2) 30 min growth in fully supplemented thymine-containing medium, (3) 115 min amino-acid starvation (thymine), (4) 40 min thymine starvation (+ a.a.), (6) 30 sea [sH]thymine, (6) 0, 1, and 30 min chase (thymine). The DNA was denatured and analyzed by density-gradient centrifugation. The intermediate density fractions were selected and rebanded. The positions of light and heavy DNA strands are indicated.

the pre-existing DNA should still be indicated by the existence of intermediate density DNA fragments. Figure 6 gives the exact protocol and a typical result for this type of experiment. As in Figure 2, intermediate density DNA is found after the pulse and is subsequently chased out of the intermediate density fractions. This result indicates that the appearance of chaseable intermediate density DNA is not due to residual 5-bromouracil in the intracellular nucleotide pool. A control experiment with unsynchronized cells was performed with an interval of growth in thymine interposed between the period of 5-bromouracil labeling and the administration of the pulse. In this experiment, no intermediate density DNA was expected but the results, shown in Figure 7, were contrary to expectation. The likely explanation for this result seemed to be that there is a heterogeneity in the G+C composition of the small pieces of nascent DNA. By isolating and rebanding the DNA from the intermediate density region, one selects those small fragments of DNA with above average G+ C content. To test this hypothesis, DNA was isolated from cells which had been grown only in thymine-containing medium, sheared into fragments of approximately one million daltons, and then banded in an alkaline CsCl gradient. Figure 8 shows the result obtained when the intermediate density fractions were banded a second time with unsheared DNA as a marker for the position of fully light DNA strands. Small pieces of DNA selected from the heavy side of the

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Lit

HE

T .-5 ” 2 mx 100

VOmr \ I \ \ \ \ \ \

I I I

\

/ /

0

5

:

1

I

‘...... 2

..: _....’

IO 15 Froct~on no

FIU. 7. Effeot of a period of growth in unlabeled thymine between unsynchronized DNA replioation in S-bromouraoil and pulse-labeling of the DNA with [3H]thymine. An exponential culture of TAU-bar was labeled as follows: (1) 60 min 6-[14C]bromouracil, (2) 35 min normal growth (thymine), (3) 30 set [eH]thymine, (4) 0 and 60 min chase (thymine). The DNA was denatured and analyzed by density-gradient centrifugation. The intermediate density fractions were selected and rebanded. The positions of light and heavy DNA strands are indicated.

I

Light

/ 1 Fraction no FIQ. 8. Effect of shearing on the density distribution of thymine-containing DNA. A oulture of TAU-bar was labeled with [sH]thymine for 2 generations. The cells were lysed and the DNA was sheared by sonic&ion to fragments with a molecular weight of approximately 1 million daltons. The sheared DNA was banded in an alkaline CsCl gradient. The intermediate density fraotions were seleoted and rebanded. The position of light DNA strands is indicated.

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G. H.

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fully light DNA peak do have higher buoyant densities than the DNA as a whole. Yamagishi (1970) has shown that fragments of E. coli DNA of approximately one million molecular weight show sufficient heterogeneity in G+C content to account for our result. Consequently, s, valid estimate of the amount of intermediate density DNA due to linkage of origin daughter DNA to pre-existing DNA cannot be made when the pulse-labeled DNA is in small pieces. Thus this experimental approach cannot detect very transient (< 1 to 2 min) linkage of daughter DNA to parental DNA. (c) Quantitative comparison of the effect of a density shift made at the time of initiation to a density shift made during chain elongation When a culture is synchronized in the presence of 5-bromouracil and allowed to initiate DNA replication in [3H]thymine for 30 seconds, some intermediate density DNA is still observed after the pulse-labeled DNA has been incorporated into high molecular weight DNA (Pig. 2, 3-min curve). This intermediate density DNA persists during a 60-minute chase (Fig. 2, 60-min curve). In other experiments the effect of longer chase periods up to 150 minutes have been examined and no significant chase of the pulse-label into the fully light strand position was found. Hence the intermediate density DNA observed does not behave in the msnner predicted for DNA fragments containing origin daughter DNA and parental DNA. However, if the intermediate density DNA observed does not signify linkage of origin daughter DNA to the pre-existing parental DNA, then the question remains, what is the source of this material ? The relative amount of the persistent pulse-labeled intermediate density DNA obtained following a density shift made when the growing point was at the origin was compared to the amount obtained following a density shift made when the growing point was at random locations in the chromosome. Table 1 shows the raw data on the percentage of a 30-second pulse of [3H]thymine incorporated into intermediate density DNA in a series of experiments on both synchronized and random cultures. Because approximately twice as much DNA synthesis took place during the 30-second pulse in unsynchronized cultures, the relative amount of intermediate density DNA synthesized in the random cultures is approximately (2 x 22x)/2*45% = 18 times greater than that in the synchronized cultures. A ratio of 2 would have been expected if the intermediate density DNA in the synchronized cultures represented linkage of origin daughter DNA to parenbal DNA, because initiation of DNA synthesis presumably took place along both parental DNA strands, only one of which was density labeled, whereas in the random cultures both daughter strands were density labeled at the growing point. The results in Table 1 suggest that the intermediate density DNA synthesized immediately after initiation does not represent linkage of origin-daughter DNA to pre-existing DNA, but rather is an artifact of the experimental procedure. However, it is still important to rule out the possibility that the synchronization procedure itself in some unknown way artificially reduces the amount of intermediate density DNA produced. (d) ZCffectof thymine starvation on the synthesis of intermediate density DNA A fundamental difference between the experiments in synchronized and unsynchronized cultures is that in the former case the pulse-label is incorporated

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TABLE 1 Relative amounts of intermediate density DNA Synchronized Thymine o/O3H in concn intermediate density DNA b&-u

Unsynchronized Thymine '$/o3H in ooncn intermediate density DNA b+W.

1.02 1.02 1.02 1.02 1.02 0,62 0.52 0.26 Average

1.03 l-02 l-02 0.26 Average

2.1 2.4 1.3 1.5 3.2 1.6 5.7 1.3 -zc

22 15 23 28 22

immediately after a 40-minute period of thymine starvation, and in the latter case the pulse-label is incorporated during exponential growth. An experiment was done to determine the effect of a 40-minute period of thymine starvation preceding the incorporation of the pulse-label in an otherwise unsynchronized culture. Specifically, the cells were grown for 60 minutes in 5j1*C]bromouracil, deprived of thymine for 40 minutes, labeled for 30 seconds with [3H]thymine and then grown in unlabeled thymine-containing medium for 0, 10, and 60 minutes. Following a 40-minute period of thymine starvation, DNA synthesis takes place at both the pre-existing growing point and as a premature initiation at the origin (Pritchard $ Lark, 1964). The amount of pulse-label observed in intermediate density DNA was 11% and this did not decrease during the chase. This value is four times greater than the amount of intermediate density DNA observed in synchronized cultures. It is less than the amount of intermediate density DNA observed in unsynchronized cultures because the pulse-label is incorporated at the origin (where it is presumably not linked to 5-bromouracil-labeled DNA) as well as at the pre-existing growing point, (where linkage has been observed). Hence thymine starvation per se does not prevent the synthesis of DNA containing a transition point between 5-bromouracil and thymine. It is possible that the small amount of pulse-labeled intermediate density DNA observed in the synchronized cultures is due to synthesis of DNA fragments containing a mixture of 5-bromouracil and thymine. The problem of residual 5-bromouraoil in the intracellular nucleotide pool was considered earlier in the experiment illustrated in Figure 6. In that experiment, the cells were grown in thymine-containing medium for a period of time between termination of DNA replication in 5-bromouracil and initiation in the presence of the pulse-label. Nevertheless a small amount (2+30,!) of intermediate density DNA was present. However, it is possible that a small amount of DNA degradation takes place during the period of thymine starvation immediately preceding the addition of the pulse-label and thus releases some 5-bromouracil into the intracellular nucleotide pool. Alternatively, the pulse-label might be incorporated in a repair-type synthesis of gaps produced during thymine starvation (cf. Pauling t Hanawalt, 1965). This is considered unlikely because essentially no pulse-label is found in the parental heavy-strand position as would be expected if repair replication were taking place.

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G. II.

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TABLE 2

Protocol for examining the effeectof a period of thymine sturvation on the synthesis of intermediate density DNA 1. 100min --&.a., +thymine ......... 2. 40 min -thymine ............... 3. 30 min 5-[f*Cjbromouraoil. ........ 4. 16 min +thymine

...............

6. 40 min - thymine

...............

6, 30 set [sH]thymine

..............

terminate DNA replication. synthesize initiator components. synthesize 6-bromouraeil-containing DNA that does not in&de the chromosome terminus. produce growing points in thymine-containing DNA only.

‘7. 0,6and76min +thymine.. ...... 8. Alkahne CsCl gradients ...........

synthesize initiator components (possibly produce some DNA degradation). pulse-label DNA at new growing points at the origin and at preexisting growing points. compare pulse, short chase, and long chase. determine density distribution of the pulse-label.

The suggestion that some DNA breakdown can take place during thymine starvation is consistent with the results of the experiment outlined in Table 2. At the time of the second period of thymine starvation, the cells contain some B-bromouraoillabeled DNA, but neither the termini nor the growing points are density labeled. Consequently, the pulse-label given after the period of thymine starvation should not be linked to 5-bromouracil-labeled DNA at either the origin growing point or the pre-existing growing point. Nevertheless, 2% of the 30-second pulse-label is found in intermediate density DNA. This result supports the hypothesis that the small amount of intermediate density DNA observed after a density shift at the time of initiation is the result of the presence of a limited amount of 5-bromouracil in the intracellular nucleotide pool. Recent evidence suggests that DNA replication in E. coli may be bidirectional (Masters & Broda, 1971). If this is the case, then the incorporation of 5-bromouracil in the terminus of the chromosome does not density label the parental DNA at the site of initiation of the subsequent round of replication. The experiment described in Table 2 would result in density labeling the parental DNA on both sides of the origin if replication is bidirectional. The data show that when the next round of replication is initiated in the presence of [3H]thymine for 30 seconds, only 2% of the pulse-label appears in intermediate density DNA (once the nascent DNA fragments have been joined). Hence our results point to the same conclusion whether we assume uni- or bidirectional replication. In conclusion, these studies indicate that origin-daughter DNA is not covalently linked to parental DNA once the nascent DNA fragments have been joined. The fact that shearing the DNA into small pieces after a 60-minute chase of the pulselabel recreates the same amount of intermediate density DNA as existed immediately after the pulse (Fig. 5) suggests that within 30 seconds after initiation there is no intermediate density DNA due to linkage of origin daughter DNA to parental DNA. 4. Discussion The four models illustrated in Figure 9 represent alternative mechanisms by which DNA replication can be initiated. Our results are consist,ent with the Cairns model (Cairns, 1963; Cairns & Davern, 1966) which proposes that initiation takes

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FIG. 9. Four models for the replication of circular DNA (modified from Dressier & Wolfson, 1970). A, The Cairns model; the stippled rectangle represents the swivel to which the two daughter origins and the terminus of the partially replicated ohromosome are attaohed. B, The rolling circle model. C, The Yoshikaws model. D, The Haskell t Davern model. The solid lines indicate parental polynuoleotide strands (+ is the 6’-strand: - is the 3’-strand). The dashed lines indiaate daughter polynucleotide strands (for A,C,D). The jagged lines indicate the nascent DNA.

place de nova along the parental DNA strands which have unwound at the origin. They are also consistent with the Haskell & Davern (1969) model which proposes that DNA synthesis is initiated serially at 3’-OH termini made available by singlestrand nicks made in the parental DNA ahead of the replicating fork. The linkage of nascent DNA and parental DNA is severed by a second nick, the integrity of the parental strand is restored, end the fragments of replicated DNA are joined together. According to this model then, the linkage between the parental DNA and the replicating DNA is very transient and is broken before the nascent DNA fragments are joined. Hence, it would not be detected as intermediate density DNA in our experiments. Our results are not consistent with the rolling circle model (Gilbert t Dressier, 1968) or the Yoshikawa (1967,197O) model, both of which predict, respectively, that one or both of the origin daughter DNA strands will be linked for some time to the parental DNA. There is evidence from density-shift experiments done with germinating spores (Yoshikawa, 1967,1970), for linkage of both strands in .Ba.&Uus subtilis. However, if a 3’-OH primer is an absolute requirement for the initiation of DNA synthesis, then the Yoshikawa model ignores the question of how DNA synthesis along the 5’strand is initiated and attached to the 5’-terminus of the parental DNA.

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Similarly, the rolling circle model proposes that DNA synthesis is initiated along the 3’-strand of a circular DNA duplex using as a primer the 3’-OH terminus produced by nicking the 5’-strand at the origin, but does not specify how DNA synthesis is initiated along the #-strand. Nevertheless, there is good evidence in $X174 for the existence of the rolling circle intermediate characterized by the possession of 5’-strands that are longer than the length of a mature viral genome, and 3’-strands that are covalently closed single-stranded circles. Moreover, the 3’-end of the long 5’-strand lies upon the circular template while the 5’-end is free in solution (Dressler, 1970). The Cairns model and the Haskell & Davern model are also both compatible with the evidence that initiation of dichotomous replication is symmetric in E. wli (Caro, 1970; Fritsch & Woroel, 1971) and in B. subtilis (Quinn & Sueoka, 1970). Symmetric multifork replication is not consistent with the asymmetric rolling circle model nor is it in simple agreement with the Yoshikawa model. There cannot be a simultaneous symmetric initiation of dichotomous replication according to the Yoshikawa model because there are only half as many parental strand ends available for initiation as there are daughter strands to be initiated. This work was supported by grant no. GM09901 from the Institute of General Medical Sciences, U.S. Public Health Service. One of us (G.H. S.) was a Predoctoral Fellow of the National Science Foundation. REFERENCES Abelson, J. t Thomas, C. A., Jr. (1966). J. Mol. Biol. 18, 262. Cairns, J. (1963). Cold Sp. Hark Symp. &want. Biol. 28, 43. Cairns, J. & Davern, C. (1966). J. Mol. BioZ. 17, 418. Care, L. (1970). J. Mol. Biol. 48, 329. Caro, L. & Berg, C. M. (1969). J. Mol. BioZ. 45, 325. C&da-Olmedo, E., Hanawdt, P.C. & Guerola, N. (1968). J. Mol. BioZ. 33, 705. Cooper, S. & Helmstetter, C. E. (1968). J. Mol. BioZ. 31, 519. Dressier, D. (1970). Proc. Nat. Ad. Sci., Wwh. 67, 1934. Dressler, D. & Wolfson, J. (1970). Proc. Nat. Acad. Sci., Wash. 67, 456. Fritsoh, A. & Worcel, A. (1971). J. Mol. BioZ. 59, 20’7. Gilbert, W. & Dressier, D. (1968). Cold Spr. Harb. Symp. Quant. Biol. 33, 473. Hackett, P. & Hanawalt, P. C. (1966). Biochim. biophys. Acta, 123, 356. Hanawalt, P. & Wax, R. (1964). Science, 145, 1061. Haskell, E. H. & Davern, C. I. (1969). Proc. Nat. Acad. Sk., Wash. 64, 1065. Kornberg, A. (1969). Science, 163, 1410. Kornberg, T. & Gefter, M. (1971). Proc. Nut. Acad. Sci., Wash. 68, 761. Lark, K. G., Repko, T. & Hoffman, E. J. (1963). Biochim. biophys. Acta, 76, 9. Maalee, 0. & Hanawalt, P. C. (1961). J. Mol. BioZ. 3, 144. Masters, M. & Broda, P. (1971). Nature New BioZ. 232, 137. Moses, R. E., Fleiachman, R. A., Campbell, J. L., Frenkel, G. D. & Richardson, C. C. (1971). Fed. Proc. 38, 1027. Okazaki, R., Okazaki, T., Sakabe, K., Sugimoto, K., Kainumu, R. Sugino, A. & Iwatsuki, N. (1968). CoZd Sp. Harb. Symp. Quad. Biol. 33, 129. Pauling, C. & Hanawalt, P. C. (1965). Proc. Nat. Acad. Sci., Wash. 54, 1728. Pettijohn, D. E. & Hanawalt, P. C. (1964). J. Mol. BioZ. 8, 170. Pritchard, R. H. & Lark, K. G. (1964). J. MOE. BioZ. 9, 288. Quinn, W. G. t Sueoka, N. (1970). Proc. Nat. Acad. Sci., Wash. 67, 717. Richardson, C. C. (1969). Ana. Rev. Biochem. 37, 795. Stein, G. H. (1971). Ph.D. Thesis, Stanford University. Stein, G. H. & Hanawalt, P. (1969). J. Mol. BioZ. 46, 135.

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Studier, F. W. (1965). J. Mol. BioZ. 11, 373. Vinogrd, J., Morris, J. Davidson, N. t Dove, W. F. (1963). Proc. Nat. Acad. Sci., Wush. 49, 12. Wolf, B., Newman, A. & Glsser, D. A. (1968). J. Mol. BioZ. 32, 611. Yamagishi, H. (1970). J. Mol. BioZ. 49, 603. Yoshikawa, H. (1967). Proc. Nat. Acad. Sci., Wash. 58, 312. Yoshikawa, H. (1970). J. Mol. BioZ. 47, 403.