Theoretically, there are the following three possible modes of DNA replication:
(1) Dispersive,
(2) Conservative and
(3) Semi-conservative.
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1. Dispersive Replication:
In dispersive replication, the parent DNA molecule is broken into several pieces, and the ‘new’ molecules will consist of both old and newly synthesized segments.
In the conservative scheme, the two ‘old’ strands would be present in one daughter molecule while the two newly produced strands would form the second daughter molecule. But in semiconservative mode of replication, each daughter DNA molecule consists of one ‘old’ and one ‘new’ strand.
All the available evidence clearly indicates that DNA replication is semiconservative. In semiconservative DNA replication, each newly synthesized strand of DNA remains associated with the old strand against which it was synthesized. Thus each progeny DNA molecule would consist of one ‘old’ and one ‘newly synthesized strand.
The semi-conservative mode of DNA replication was postulated by Watson and Crick along with their double-helix model of DNA. The DNA molecule that undergoes replication may be termed as parent molecule’ or ‘template molecule’, while the two molecules produced by replication may be called progeny molecules’ or ‘daughter molecules’.
The main features of this model of DNA replication are as follows:
(1) Progressive separation of the two strands of the DNA molecule undergoing replication,
(2) Complementary base-pairing of the bases located in the single-stranded regions thus produced with the appropriate free deoxyribonucleotides, and
(3) Formation of phosphodiester linkages between the neighbouring deoxyribo-nucleotides that have base-paired with the single strands, thereby producing the new strand.
(4) This ensures that the base sequences of the new strands are strictly complementary to those of the old strands and that
(5) Each DNA molecule produced by replication has one ‘old’ and one ‘new’ strand. Clearly, the old strands of a DNA molecule serve as templates or molds for the synthesis of new strands (Fig. 28.1).
Evidence for Semiconservative Replication:
The evidence for semiconservative replication of DNA was first presented by Meselson and Stahl in 1958. They grew E. coli on 15N (a heavy isotope of 14N) so that the nitrogen present in DNA bases of these cells was 15N. DNA having 15N has a detectable higher density than that having 14N; therefore, they are called heavy and light DNA, respectively.
Heavy and light DNAs can be readily separated through equilibrium density gradient centrifugation; they form distinct bands in the centrifuge tube (Fig. 28.2). In density gradient centrifugation, heavy salt, e.g., Cs Cl2, is centrifuged at high speed (30,000- 50,000 rpm) for 40 – 72 hr.
This leads to the formation of a linear gradient of increasing density from the lop to the bottom of the centrifuge tube. When molecules with small differences in density are now centrifuged in this solution, they form separate bands as shown in Fig. 28.2.
Meselson and Stahl transferred the E. coli cells grown on 15N medium to a medium containing normal 14N. They withdrew samples from these E. coli cells after approximately one, two and three cell generations. One cell generation represents the time during which all the cells present in a culture would undergo, on an average, one cell division. DNA from these cell samples was isolated and subjected to density gradient centrifugation.
The results obtained from their study may be summarized as follows (Fig. 28.2). After one cell generation, the DNA formed a single band ‘intermediate’ between the ‘heavy’ (containing 15N) and ‘light’ (containing 14N) DNAs.
The DNA obtained after two cell generations formed two bands: one of the bands was ‘intermediate’, while the other was Might’ in density. The same two bands were recovered in the DNA isolated after three cell generations, although the ‘intermediate’ band was relatively lower in intensity than the ‘light’ band.
These findings can be readily explained on the basis of semiconservative replication of DNA (Fig. 28.2).
The DNA from E. coli cells grown on 15N had 15N in both the strands; therefore it was heavier than the normal DNA having 14N. When these E. coli cells were allowed one semiconservative replication of their DNA on the 14N medium, each of the resultant DNA molecules would have one ‘heavy’ (having 15N) and one ‘light’ (having14N) strand. Therefore, these DNA molecules would have ‘intermediate’ density.
One more semiconservative replication of these DNA molecules in the 14N medium would generate two types of DNA molecules:
(1) Half of the molecules would have one ‘heavy’ and ‘one light’ strand (‘intermediate’ density), while
(2) The remaining half would have both ‘light’ strand (‘light’ density).
These molecules would obviously form one intermediate’, and one ‘light’ band. Following the third round of DNA replication on 14N, the ‘intermediate’ density DNA molecules would yield half ‘intermediate’ and half ‘light’ molecules, while ail the molecules obtained from replication of the ‘light’ DNA would be ‘light’; this is the reason for the lower intensity of the ‘intermediate’ band after three cell generations.
Autoradiograph of Replicating E. coli Chromosome:
One of the consequences of semiconservative replication is the presence of a replication fork in a DNA molecule undergoing replication (Fig. 28.3). At the replication fork, one DNA molecule appears to be branched into two molecules.
Such a fork is formed due to a progressive separation of the two strands of a replicating DNA molecule and the concomitant synthesis of the new complementary strands on these ‘old’ strands. The presence of replication forks in E. coli chromosomes was first shown by J. Cairns in 1963 using the technique of autoradiography (Fig. 28.3).
E. coli chromosomes were shown to exist as θ-shaped structures during replication (Fig. 28.3). It was also clear that the unwinding of the two strands of a replicating DNA molecule and the synthesis of the new complementary strands occur almost simultaneously. The semiconservative replication of DNA begins at a definite point called ‘origin’ and proceeds in both the directions; thus both the Y-shaped structures in Fig. 28.3 are replication forks.
Replicon:
A replicon is that segment of DNA that is capable of DNA replication independent of other segments of DNA. Therefore, each replicon has an origin of replication (or simply, origin) at which DNA replication begins, and may have a terminus at which replication stops.
The bacterial and viral chromosomes usually contain a single replicon per chromosome. But in eukaryotes, each chromosome is made up of several replicons, e.g., Total of 3,500 replicons in the 4 chromosomes of Drosophila. The replicon size varies from 40 kb in Drosophila to 300 kb in Vicia. However, usually only a proportion (~ 15%) of replicons function during replication in S phase.
Origin of Replicon:
The sequence of a replicon that supports initiation of DNA replication is called origin. Origin also controls the frequency of replication of the replicon.
In E. coli, origin is identified as the oriC locus, which is 245 bp long. In general, origins are A.T. rich, which may be important for easy unwinding of the two strands during replication. Origins function in a species-specific manner in that origin of one species functions well in that species or a group of related species.
Terminus of Replicon:
Several prokaryotic replicons have specific sites called terminus, which stop replication fork movement and, thereby, terminate DNA replication. E. coli chromosome has two termini, one on each strand; these termini are termed as terE, D, A and terC, B. The ter sequences contain a short ~ 23 bp sequence that functions in only one direction.
Termination requires tus gene product, which recognizes and binds to the ter sequence and stop the progress of replication fork. In E. coli, complete replication of chromosome takes ~ 40 min; this gives to rate of DNA replication as ~ 50,000 bp/min. The rate of DNA replication appears ~10-times as faster transcription by RNA polymerase.
Bidirectional Replication:
Studies with small viruses have shown that DNA replication is bidirectional, that is, it progresses in both the directions from the origin. The chromosome of E. coli phage T7 replicates in a linear form, and DNA replication begins near one end of the chromosome. Replication produces the so-called eye-structure in the T7 chromosome (Fig. 28.4).
If the replication were bidirectional, the replication fork will reach one end of the chromosome much sooner than the other end. This will give rise to a Y-shaped chromosome. Both eye-shaped and Y-shaped chromosomes of T7 chromosome have been seen under electron microscope. This confirms the DNA replication to be bidirectional. DNA replication in eukaryotes is also bidirectional.
