The replication of DNA in Escherichia coli*

Studies of bacterial transformation and bacteriaphage infection^1–5 strongly indicate that deoxyribonucleic acid (DNA) can carry and transmit hereditary information and can direct its own replication. Hypotheses for the mechanism of DNA replication differ in the predictions they make concerning the distribution among progeny molecules of atoms derived from parental molecules.⁶

Radioisotopic labels have been employed in experiments bearing on the distribution of parental atoms among progeny molecules in several organisms.^6–9 We anticipated that a label which imparts to the DNA molecule an increased density might permit an analysis of this distribution by sedimentation techniques. To this end, a method was developed for the detection of small density differences among macromolecules.¹⁰ By use of this method, we have observed the distribution of the heavy nitrogen isotope N¹⁵ among molecules of DNA following the transfer of a uniformly N¹⁵-labeled, exponentially growing bacterial population to a growth medium containing the ordinary nitrogen isotope N¹⁴.

A small amount of DNA in a concentrated solution of cesium chloride is centrifuged until equilibrium is closely approached. The opposing processes of sedimentation and diffusion have then produced a stable concentration gradient of the cesium chloride. The concentration and pressure gradients result in a continuous increase of density along the direction of centrifugal force. The macromolecules of DNA present in this density gradient are driven by the centrifugal field into the region where the solution density is equal to their own buoyant density.¹¹ This concentrating tendency is opposed by diffusion, with the result that at equilibrium a single species of DNA is distributed over a band whose width is inversely related to the molecular weight of that species (Fig. 1).

If several different density species of DNA are present, each will form a band at the position where the density of the CsCl solution is equal to the buoyant density of that species. In this way DNA labeled with heavy nitrogen (N¹⁵) may be resolved from unlabeled DNA. Figure 2 shows the two bands formed as a result of centrifuging a mixture of approximately equal amounts of N¹⁴ and N¹⁵ Escherichia coli DNA.

In this paper reference will be made to the apparent molecular weight of DNA samples determined by means of density-gradient centrifugation. A discussion has been given¹⁰ of the considerations upon which such determinations are based, as well as of several possible sources of error.¹²

Escherichia coli B was grown at 36° C. with aeration in a glucose salts medium containing ammonium chloride as the sole nitrogen source.¹³ The growth of the bacterial population was followed by microscopic cell counts and by colony assays (Fig. 3).

Bacteria uniformly labeled with N¹⁵ were prepared by growing washed cells for 14 generations (to a titer of 2 × 10⁸/ml) in medium containing 100 μg/ml of N¹⁵H₄Cl of 96.5 per cent isotopic purity. An abrupt change to N¹⁴ medium was then accomplished by adding to the growing culture a tenfold excess of N¹⁴H₄Cl, along with ribosides of adenine and uracil in experiment 1 and ribosides of adenine. guanine, uracil, and cytosine in experiment 2, to give a concentration of 10 μg/ml of each riboside. During subsequent growth the bacterial titer was kept between 1 and 2 × 10⁸/ml by appropriate additions of fresh N¹⁴ medium containing ribosides.

Samples containing about 4 × 10⁹ bacteria were withdrawn from the culture just before the addition of N¹⁴ and afterward at intervals for several generations. Each sample was immediately chilled and centrifuged in the cold for 5 minutes at 1,800 × g. After resuspension in 0.40 ml. of a cold solution 0.01 M in NaCl and 0.01 M in ethylenediaminetetra-acetate (EDTA) at pH 6, the cells were lysed by the addition of 0.10 ml. of 15 per cent sodium dodecyl sulfate and stored in the cold.

For density-gradient centrifugation, 0.010 ml. of the dodecyl sulfate lysate was added to 0.70 ml. of CsCl solution buffered at pH 8.5 with 0.01 M tris(hydroxymethyl)aminomethane. The density of the resulting solution was 1.71 gm. cm.⁻³ This was centrifuged at 140,000 × g. (44,770 rpm) in a Spinco model E ultracentrifuge at 25° for 20 hours, at which time the DNA had essentially attained sedimentation equilibrium. Bands of DNA were then found in the region of density 1.71 gm. cm.⁻³, well isolated from all other macromolecular components of the bacterial lysate. Ultraviolet absorption photographs taken during the course of each centrifugation were scanned with a recording microdensitometer (Fig. 4).

The buoyant density of a DNA molecule may be expected to vary directly with the fraction of N¹⁵ label it contains. The density gradient is constant in the region between fully labeled and unlabeled DNA bands. Therefore, the degree of labeling of a partially labeled species of DNA may be determined directly from the relative position of its band between the band of fully labeled DNA and the band of unlabeled DNA. The error in this procedure for the determination of the degree of labeling is estimated to be about 2 per cent.

Figure 4 shows the results of density-gradient centrifugation of lysates of bacteria sampled at various times after the addition of an excess of N¹⁴-containing substrates to a growing N¹⁵-labeled culture.

It may be seen in Figure 4 that, until one generation time has elapsed, half-labeled molecules accumulate, while fully labeled DNA is depleted. One generation time after the addition of N¹⁴, these half-labeled or “hybrid” molecules alone are observed. Subsequently, only half-labeled DNA and completely unlabeled DNA are found. When two generation times have elapsed after the addition of N¹⁴, half-labeled and unlabeled DNA are present in equal amounts.

These results permit the following conclusions to be drawn regarding DNA replication under the conditions of the present experiment.

1. The nitrogen of a DNA molecule is divided equally between two subunits which remain intact through many generations.

The observation that parental nitrogen is found only in half-labeled molecules at all times after the passage of one generation time demonstrates the existence in each DNA molecule of two subunits containing equal amounts of nitrogen. The finding that at the second generation half-labeled and unlabeled molecules are found in equal amounts shows that the number of surviving parental subunits is twice the number of parent molecules initially present. That is, the subunits are conserved.

2. Following replication, each daughter molecule has received one parental subunit.

The finding that all DNA molecules are half-labeled one generation time after the addition of N¹⁴ shows that each daughter molecule receives one parental subunit.¹⁴ If the parental subunits had segregated in any other way among the daughter molecules, there would have been found at the first generation some fully labeled and some unlabeled DNA molecules, representing those daughters which received two or no parental subunits, respectively.

3. The replicative act results in a molecular doubling.

This statement is a corollary of conclusions 1 and 2 above, according to which each parent molecule passes on two subunits to progeny molecules and each progeny molecule receives just one parental subunit. It follows that each single molecular reproductive act results in a doubling of the number of molecules entering into that act.

The above conclusions are represented schematically in Figure 5.

A molecular structure for DNA has been proposed by Watson and Crick.¹⁵ It has undergone preliminary refinement¹⁶ without alteration of its main features and is supported by physical and chemical studies.¹⁷ The structure consists of two polynucleotide chains wound helically about a common axis. The nitrogen base (adenine, guanine, thymine, or cytosine) at each level on one chain is hydrogen-bonded to the base at the same level on the other chain. Structural requirements allow the occurrence of only the hydrogen-bonded base pairs adenine-thymine and guanine-cytosine, resulting in a detailed complementariness between the two chains. This suggested to Watson and Crick¹⁸ a definite and structurally plausible hypothesis for the duplication of the DNA molecule. According to this idea, the two chains separate, exposing the hydrogen-bonding sites of the bases. Then, in accord with the base-pairing restrictions, each chain serves as a template for the synthesis of its complement. Accordingly, each daughter molecule contains one of the parental chains paired with a newly synthesized chain (Fig. 6).

The results of the present experiment are in exact accord with the expectations of the Watson-Crick model for DNA duplication. However, it must be emphasized that it has not been shown that the molecular subunits found in the present experiment are single polynucleotide chains or even that the DNA molecules studied here correspond to single DNA molecules possessing the structure proposed by Watson and Crick. However, some information has been obtained about the molecules and their subunits; it is summarized below.

The DNA molecules derived from E. coli by detergent-induced lysis have a buoyant density in CsCl of 1.71 gm. cm.⁻³, in the region of densities found for T2 and T4 bacteriophage DNA, and for purified calf-thymus and salmon-sperm DNA. A highly viscous and elastic solution of N¹⁴ DNA was prepared from a dodecyl sulfate lysate of E. coli by the method of Simmons¹⁹ followed by deproteinization with chloroform. Further purification was accomplished by two cycles of preparative density-gradient centrifugation in CsCl solution. This purified bacterial DNA was found to have the same buoyant density and apparent molecular weight, 7 × 10⁶, as the DNA of the whole bacterial lysates (Figs. 7, 8).

It has been found that DNA from E. coli differs importantly from purified salmon-sperm DNA in its behavior upon heat denaturation.

Exposure to elevated temperatures is known to bring about an abrupt collapse of the relatively rigid and extended native DNA molecule and to make available for acid-base titration a large fraction of the functional groups presumed to be blocked by hydrogen-bond formation in the native structure.^{19, 20, 21, 22} Rice and Doty²² have reported that this collapse is not accompanied by a reduction in molecular weight as determined from light-scattering. These findings are corroborated by density-gradient centrifugation of salmon-sperm DNA.²³ When this material is kept at 100° for 30 minutes either under the conditions employed by Rice and Doty or in the CsCl centrifuging medium, there results a density increase of 0.014 gm. cm.⁻³ with no change in apparent molecular weight. The same results are obtained if the salmon-sperm DNA is pre-treated at pH 6 with EDTA and sodium dodecyl sulfate. Along with the density increase, heating brings about a sharp reduction in the time required for band formation in the CsCl gradient. In the absence of an increase in molecular weight, the decrease in banding time must be ascribed¹⁰ to an increase in the diffusion coefficient, indicating an extensive collapse of the native structure.

The decrease in banding time and a density increase close to that found upon heating salmon-sperm DNA are observed (Fig. 9, A) when a bacterial lysate containing uniformly labeled N¹⁵ or N¹⁴ E. coli DNA is kept at 100° C. for 30 minutes in the CsCl centrifuging medium; but the apparent molecular weight of the heated bacterial DNA is reduced to approximately half that of the unheated material.

Half-labeled DNA contained in a detergent lysate of N¹⁵ E. coli cells grown for one generation in N¹⁴ medium was heated at 100° C. for 30 minutes in the CsCl centrifuging medium. This treatment results in the loss of the original half-labeled material and in the appearance in equal amounts of two new density species, each with approximately half the initial apparent molecular weight (Fig. 9, B). The density difference between the two species is 0.015 gm. cm.⁻³, close to the increment produced by the N¹⁵ labeling of the unheated DNA.

This behavior suggests that heating the hybrid molecule brings about the dissociation of the N¹⁵-containing subunit from the N¹⁴ subunit. This possibility was tested by a density-gradient examination of a mixture of heated N¹⁵ DNA and heated N¹⁴ DNA (Fig. 9, C). The close resemblance between the products of heating hybrid DNA (Fig. 9 B) and the mixture of products obtained from heating N¹⁴ and N¹⁵ DNA separately (Fig. 9, C) leads to the conclusion that the two molecular subunits have indeed dissociated upon heating. Since the apparent molecular weight of the subunits so obtained is found to be close to half that of the intact molecule, it may be further concluded that the subunits of the DNA molecule which are conserved at duplication are single, continuous structures. The scheme for DNA duplication proposed by Delbrück²⁴ is thereby ruled out.

To recapitulate, both salmon-sperm and E. coli DNA heated under similar conditions collapse and undergo a similar density increase, but the salmon DNA retains its initial molecular weight, while the bacterial DNA dissociates into the two subunits which are conserved during duplication. These findings allow two different interpretations. On the one hand, if we assume that salmon DNA contains subunits analogous to those found in E. coli DNA, then we must suppose that the subunits of salmon DNA are bound together more tightly than those of the bacterial DNA. On the other hand, if we assume that the molecules of salmon DNA do not contain these subunits, then we must concede that the bacterial DNA molecule is a more complex structure than is the molecule of salmon DNA. The latter interpretation challenges the sufficiency of the Watson-Crick DNA model to explain the observed distribution of parental nitrogen atoms among progeny molecules.

The structure for DNA proposed by Watson and Crick brought forth a number of proposals as to how such a molecule might replicate. These proposals⁶ make specific predictions concerning the distribution of parental atoms among progeny molecules. The results presented here give a detailed answer to the question of this distribution and simultaneously direct our attention to other problems whose solution must be the next step in progress toward a complete understanding of the molecular basis of DNA duplication. What are the molecular structures of the subunits of E. coli DNA which are passed on intact to each daughter molecule? What is the relationship of these subunits to each other in a DNA molecule? What is the mechanism of the synthesis and dissociation of the subunits in vivo?

By means of density-gradient centrifugation, we have observed the distribution of N¹⁵ among molecules of bacterial DNA following the transfer of a uniformly N¹⁵-substituted exponentially growing E. coli population to N¹⁴ medium.

We find that the nitrogen of a DNA molecule is divided equally between two physically continuous subunits; that, following duplication, each daughter molecule receives one of these; and that the subunits are conserved through many duplications.