DNA Replication : General Features Semi-conservative DNA replication in E. coli Both DNA as well as genetic RNA are capable of undergoing self-replication. Theoretically, replication of double stranded. DNA, could be (i) conservative, (ii) dispersive, or (iii) semi-conservative. The conservative replication would mean that double stranded molecule is conserved as such and that a new copy is synthesized from old molecule. In dispersive replication, the old molecule should disintegrate and two new molecules would be synthesized. In the semi-conservative replication the two strands would separate from one another, maintain their integrity and each will synthesize, from the pool of nucleotides, its complementary strand. The result would be, that the newly synthesized molecule would carry or conserve one of the two strands from the parent molecule and the other strand would be newly assembled (Fig. 26.1). There is sufficient evidence to prove that double stranded DNA really replicates by semi-conservative method.

Fig. 26.1. Basic plan of the structure of douhic helical DNA and the semi-conservative replication.

Meselson and Stahl's experiment (using N¹⁵). M. Meselson and F.W. Stahl in 1958 reported the results of an experiment which was designed to test whether double stranded DNA replicates in a semi-conservative manner. The following two basic principles were involved :

(1) DNA was labelled with heavy nitrogen (N¹⁵) and was then allowed to replicate in a medium containing N¹⁴. If replication is semi-conservative, then after first generation of replication, one of the two strands would have heavy nitrogen (N¹⁵) and the other strand would have normal nitrogen (N¹⁴). The resulting molecule would have a density which is intermediate between N¹⁵ DNA and N¹⁴ DNA. This density will gradually fall and in the following generations of replication, density would approach that of N¹⁴ DNA

(2) Another principle utilized was the preparation of a caesium chloride density gradient. This density gradient is prepared by gradual dilution of a heavy salt solution. When this is subjected to ultracentrifugation, with a substance having density within the range of this gradient, this substance will find place at its own level of density. This enables detection of very slight differences in density.

Meselson and Stahl (1958) allowed Escherichia coli cells to grow on N15 culture medium for about 14 cell generations, so that almost all nitrogen (N14) in DNA is replaced by N15. Then the cells were abruptly transferred to N14 culture medium. Since the time required for one cell generation was determined to be about 30 minutes, it was possible to remove the cells after one or more known number of generations of replication. Their DNA could then be analysed and the following results were obtained. DNA sample obtained one cell generation after the transfer to N14 culture medium showed only one density band as observed through ultraviolet absorption pattern. This band indicated uniform homogeneous density of DNA after one cell generation. Moreover, the band was exactly between the bands formed by N15 DNA and N14 DNA, indicating that all DNA found after first generation had intermediate density. This is what one would expect if DNA replicates in a semi-conservative manner. Density of DNA was studied in the following generations also. After two generations, when DNA was analysed, two bands were observed, as one would expect according to semi-conservative method of replication. These two bands were of equal intensity after the second generation. In the subsequent generations, although the same two bands appeared, the intensity of hybrid density band gradually decreased, while the intensity of light density band gradually increased (Fig. 26.2). Meselson and Stahl's experiment is, thus, a classical experiment which was the first to show that DNA replicates in a semi-conservative manner.

Fig. 26.2. Experiment of Meselson and Stahl demonstrating semi-Conservative replication.

Cairns' autoradiography experiment. Semi-conservative mode of replication of bacterial chromosome was also demonstrated by-J. Cairns, using the technique of autoradiography. In autoradiography technique, the cells are first supplied with a suitable radioactive material like tritiated thymidine (H³-TdR; H³ is heavy isotope of hydrogen and it replaces normal hydrogen in thymidine to give rise to tritiated thymidine). Tritiated thymidine is used since this will selectively label only DNA and will not label RNA, since thymine base is absent in RNA. The tritiated thymidine gets incorporated into DNA and replaces ordinary thymidine. The cellular material is then sectioned or else the cells may be broken down to release the intact bacterial chromosomes on slides. These slides are then covered by photographic emulsion and stored in dark. During this storage the particles emitted by tritiated thymidine will expose the film, which can be developed. This photograph will then show the regions of the presence of tritium and thus indirectly show the presence of labelled DNA.

Using the above technique, replication of DNA could be easily followed by J. Cairns and the results were reported in 1963. During incorporation of tritiated thymidine, autoradiographs could be prepared at regular known intervals. Autoradiographs from this replicating material prepared at regular known intervals demonstrated semi-conservative mode of replication. Lighter density of dots is considered to indicate that only one of the two strands is labelled, while a heavier density of dots will indicate that both strands are labelled. Such a situation was actually observed. The rate at which the replication proceeds could also be worked out by measuring the length of DNA undergoing replication in a known interval of time. The generation time was worked out by Cairns as 30 minutes in E. coli. The length of the chromosome was worked out to about 1 mm. The rate of replication thus would be approximately 30μ - 40μ per minute (1 mm = 1000μ). Figure 26.3 indicates that after replication, one of the two strands in the daughter DNA molecules is derived from the parent molecule and the other is synthesized afresh. In θ shaped figure, which is obtained in the second cycle of replication in presence of label, two arcs in the split region would never be equally labelled. For instance, one arc would be twice as heavily labelled as the other arc. This is what was actually observed by Cairns. The observations thus clearly supported the semi-conservative nature of replication.

Fig. 26.3. Cairns' autoradiography experiment, showing (A) an autoradiograph and (B) its interpretation in terms of DNA.

In the above set of two experiments, it was demonstrated that DNA replicates in a semi-conservative manner. Subsequent experiments of different kinds proved the validity of this mechanism.