The process by which a DNA molecule makes its identical copies is known as DNA replication. It takes place in S-phase of interphase.
There are three possible ways of DNA replication. The three possible ways are: (1) Dispersive (2) Conservative and (3) Semi-conservative. It also discuss about the evidences for semi-conservative replication. There are three important experiments which support that DNA replication is semi-conservative.
These experiments include: (1) Meselson and Stahl Experiment (2) Cairns Autoradiography Experiment and (3)Taylor’s Experiment.
Three Possible Ways of DNA Replication:
1. Dispersive Replication:
In this method of replication, the two strands of mother DNA break at several points resulting in several pieces of DNA. Each piece replicates and pieces are reunited randomly. Thus, two copies of DNA molecules are formed from single copy. The new DNA molecules are hybrid which have old and new DNA in patches. This method of DNA replication is not accepted, because such replication could not be proved experimentally.
2. Conservative Replication:
In this method, two DNA molecules are formed. One molecule has both parental strands and the other contains both newly synthesized strands. This method is also not accepted as there is no experimental proof in support of this model.
3. Semi-conservative Replication:
Semi-conservative mode of DNA replication was proposed by Watson and Crick. According to this method, both the strands of parental DNA separate from one another. Each old strand synthesizes a new strand. Thus, each of the two resulting DNA molecules has one parental and one new strand (Figs. 6.16, 6.17, 6.18, 6.19, 6.20).
This method of DNA replication is universally accepted because there are several evidences in support of semi-conservative method. All the enzymes involved in DNA replication or duplication or synthesis are collectively known as Replisome. Enzymes are synthesized in G1 phase or Gap one phase of interphase. Enzymes involved in replication are DNA polymerase-I, II, III (in prokaryotes) and α, β, γ, δ, ε (in eukaryotes), Helicase or Unwindase, RNA primase, Topoisomerase, and DNA ligase.
The semi-conservative method of DNA replication consists of several steps which are briefly described below:
1. Initiation of Replication:
DNA replication starts at a specific point on the chromosome. This unique site is known as origin. (ORI) or Origin of replication. The site of initiation differs from organism to organism.
2. Unwinding of Strands:
The two strands of DNA double helix unwind. The opening of DNA strands takes place with the help of DNA unwinding proteins helicase or unwindase and SSBP (Single stranded Binding Protein). This protein unites together both the strands and hence this protein is also known as DNA binding protein.
3. Formation of RNA Primer:
Synthesis of RNA primer is essential for initiation of DNA synthesis. RNA primer is synthesized by the DNA template near the origin with the help of a special type of RNA polymerase (RNA Primase).
4. Synthesis of DNA on RNA Primer:
After formation of RNA primer, DNA synthesis starts on the RNA primer. Deoxyribose nucleotides are added to the 3′ end position of RNA primer. The main DNA strand is synthesized on the DNA template with the help of DNA polymerase-III. The DNA synthesis takes place in short pieces which are known as Okazaki fragments (after the discoverer). Synthesis of the new strand takes place in 5′ – 3′ (Fig. 6.20).
It is possible that during replication one strand of DNA can replicate continuously and the other discontinuously or in pieces. The continuously replicating strand is known as leading strand and the discontinuously replicating strand is known as lagging strand.
When one strand of DNA replicates continuously and the “other discontinuously, it is called semi-discontinuous replication. Earlier it was thought that DNA replicates discontinuously. But “now it is”believed that DNA replication is semi-discontinuous.
5. Removal of RNA Primer:
The RNA primer is degraded by DNA polymerase I. (Proof reader enzyme or Kornberg enzyme). This enzyme also catalyzes the synthesis of short DNA segment to replace the primer. The newly synthesized segment is joined to the main DNA strand with the help of DNA ligase enzyme.
6. Union of Okazaki Fragments:
The discontinuous fragments of Okazaki are joined to make continuous strand. The union of Okazaki fragments takes place with the help of a joining enzyme called polynucleotide ligase. The replication may take place either in one direction or in both the directions from the point of origin.
When the replication proceeds, from the point of origin, in one direction only, it is called unidirectional replication. When the replication proceeds in both the directions, it is called bidirectional replication. Bidirectional replication has been reported in prokaryotes and several eukaryotes like yeast, Drosophila and man.
7. Proof-reading and DNA Repair:
A wrong base is sometimes introduced during replication. The frequency is one in ten thousand. DNA polymerase is able to sense the same. It goes back, removes the wrong base, allows addition of proper base and then proceeds forward. However, even DNA polymerase III is unable to distinguish uracil from thymine so that it is often incorporated in place of thymine. Such a mismatching is corrected by means of DNA Pol-I.
Evidences for Semi-conservative Replication:
Various experiments have demonstrated the semi-conservative mode of DNA replication. Now it is universally accepted that DNA replicates in a semi-conservative manner.
There are three important experiments which support that DNA replication is semi-conservative. These experiments include:
(1) Meselson and Stahl experiment,
(2) Cairns experiment, and
(3) Taylor’s experiment.
1. Meselson and Stahl Experiment:
Meselson and Stahl (1958) conducted their experiment with common bacteria of human intestine, E. coli. This bacteria was grown on culture medium containing heavy isotope of Nitrogen (N15) for 14 generations (one generation is completed in about 30 minutes) to replace the normal nitrogen (N14) of E. coli with heavy nitrogen. The density of normal and heavy nitrogen differs. The normal nitrogen (N14) is lighter (1.710 g/cm3) than N15 nitrogen (1.724g/cm3).
It is possible to detect such minute differences in density through density gradient centrifugation. Distinct bands are formed in the centrifuge tube for different density DNA. After 14 generations of multiplication in N15 medium, the E. coli was transferred to normal (N14) medium and allowed to grow there for one generation.
If the DNA replicates in a semi-conservative manner, it will show intermediate density after culturing on N14 because one strand will be heavy (N15) and the other will be light (N14). The density of DNA was determined after multiplication of E. coli for one generation in normal nitrogen culture.
It was found intermediate between N15 and N14, which proved that DNA replicates in a semi-conservative manner. After two generations, half the DNA was with intermediate density and half with light bands which further confirms semi- conservative mode of DNA replication. After third generation 3/4 DNA was found with normal N14 and 1/4 with hybrid nitrogen [N14+N15] (Fig. 6.21 & 6.22)
2. Cairns Autoradiography Experiment:
Cairns (1963) also conducted his experiments with E. coli. He used heavy isotope of hydrogen [H3] to replace thymine of DNA with tritiated thymidine and thus labeled DNA. The chromosome of E. coli was used to prepare slides. The slides were coated with photographic emulsion or film and stored in a dark place.
The tritiated thymidine emits particles in dark due to its radioactive decay. These particles expose the film. These films are then developed and interpreted. If the exposure is light on the autoradiographs, it suggests labeling of one strand of DNA, which indicates semi- conservative replication. Cairns observed light film exposures in E. coli which demonstrated that DNA replication is semi-conservative.
3. Taylor’s Experiment:
Taylor (1969) conducted his experiments with root tip cells of Vicia faba. He treated root tips with radioactive thymidine to label the DNA. Then root tips were grown in the normal medium. In the first generation, both chromatids were labeled.
In the second generation of cell division, one chromatid of each chromosome was labeled and one was normal. This demonstrated semi- conservative mode of chromosome replication. The DNA replication is associated with chromosome replication (Fig. 6.23).