The Watson-Crick model of DNA immediately suggested that the two strands of DNA would separate. Each separated or parent strand serves as a template (model or guide) for the formation of a new but complementary strand. Thus, the new or daughter DNA molecules formed would be made of one old or parental strand and another newly formed complementary strand. This method of formation of new daughter DNA molecules is called semi-conservative method of replication.
The following experiment suggests that DNA replication is semi-conservative.
Messelson and Stahl (1958) conducted experiments using heavy nitrogen (15N) to determine whether the concept of semi-conservative replication is correct. They used Cesium chloride (CsCl2) gradient centrifugation technique for this purpose. A dense solution of CsCl2, on centrifugation, forms density gradient-bands of a solution of lower density at the top that increases gradually towards bottom with highest density. If DNAs of different densities are mixed with CsC12 solution, these would separate from one another and would form a definite density band in the gradient along with CsC12 solution.
Meselson and Stahl created DNA molecules of different densities by using normal nitrogen 14N and its heavy isotope 15N. For this purpose, Escherichia coli was grown in 15NH4C1 containing culture medium for many generations, so that bacterial DNA become completely heavy. This radioactive or heavy DNA (incorporating 15N) had more density than DNA with normal nitrogen (14N). The bacteria were then transferred to culture medium containing only normal nitrogen (14NH4CI). The change in density was observed by taking DNA samples periodically.
(Separation of DNA by Centrifugation)
Messelson and Stahl's Experiment
If DNA replicates semi-conservatively, then each heavy (15N) DNA strands should separate and each separated strand should acquire a light (14N) partner after one round of replication. This should be a hybrid DNA made of two strands, i.e., 14N-15N. Meselson and Stahl observed that such DNA was actually half dense indicating the presence of hybrid DNA molecules. After second round of replication there would be four DNA molecules. Of these, two molecules would be hybrid (14N-15N) showing half density as earlier and the remaining two molecules would be made of light strands (14N-14N) Thus, after second generation, the same half dense band (14N-15N) was seen but the density of light bands (14N-14N) increased. Messelson and Stahl's work as such provided confirmation of Watson-Crick model of DNA and its semi-conservative replication.
Taylor has proved semiconservative mode of chromosome replication in eukaryotes using tritiated thymidine in root of Vicia faba.
Cairns proved semiconservative mode of replication in E. coli by using tritiated thymidine (H3- tdR) in autoradiography experiment. He proposed θ-model for replication in circular DNA.
Mechanism of DNA Replication
DNA replication involves following four major steps
(1) Initiation of DNA replication,
(2) Unwinding of helix,
(3) Formation of primer strand
(4) Elongation of new strand
1. Initiation of replication. Replication of DNA in E. coli always begins at a definite site called origin of replication. On the other hand, eukaryotes have several thousand origins of replication. It is called ori-c in E.coli.
2. Unwinding of helix. DNA replication requires that a double helical parental molecule is unwound so that its internal bases are available to the replication enzymes. Unwinding is brought about by enzyme `helicase', which is ATP dependent.
Unwinding of DNA molecule into two strands results in the formation of Y-shaped structure, called replication fork. These exposed single strands are stabilised by a protein known as single-strand binding protein (SSB). Due to unwinding a supercoiling get developed on the end of DNA opposite to replicating fork. This tension is released by enzyme topoisomerase.
3. Formation of primer strand. To a new strand is II now to be synthesized opposite to the parental strands. DNA polymerase III is the true replicase in E. coli, which is incapable of initiating DNA synthesis, i.e., it is unable to deposit the first nucleotide in a daughter (new) strand. Another enzyme, known as primase, synthesizes a short primer strand of RNA. The primer strand then serves as a stepping stone (to start errorless replication). Once the initiation of DNA synthesis is completed, this primer RNA strand is then removed enzymatically.
4. Elongation of new strand. Once the primer strand is formed, DNA replication occurs in 5' -> 3' direction, i.e., during synthesis of a new strand, deoxyribonucleotides (dATP, dGTP, dTTP, dCTP) are added only to the free 3'OH end. Thus, the nucleotide at 3' end is always the most recently added nucleotide to the chain.
As the DNA replication proceeds on the two parental strands, synthesis of daughter or new strand occurs continuously along the parent 3'-45' strand. It is now known as leading daughter strand. Synthesis of another daughter strand along the other parental strand, however, takes place in the form of short pieces. This is called lagging daughter strand. These short pieces of DNA are known as Okazaki fragments, these segments are about 1,000 -2,000 nucleotides long in prokaryotes. Hence DNA replication is semidiscontinuous. Discontinuous pieces of the lagging strand are joined together by the enzyme DNA ligase (after removal of primer) to form continuous daughter strand.
Thus two DNA molecules are now formed from one molecule. Each of these daughter DNA molecules is made of two strands, of which one is old (parental) and other one is new.
DNA polymerase is the most important enzyme of DNA replication. DNA polymerases are of three types i.e. DNA polymerase I, II and III.
|DNA Polymerase I : ||Important function of this enzyme is 5' 3' polymerization. It removes RNA primer and replace it with the nucleotide of DNA and can correct the thymidine dimer (T = T) formed under the influence of UV-rays. Also take part in repair replication. It has a specific 5'3' exonuclease activity. |
|DNA Polymerase II : ||It takes part in polymerization from 5'-> 3' direction. Its polymerization speed is less i.e., only 50 nucleotide per minute. It is having supporting role over the activity of other DNA polymerases. |
|DNA Polymerase III : ||It is the most important polymerizing enzyme having a great polymerization speed (about 2000 bp per second). It performs 5' 3' polymerization. |
All the three polymerases have 3' 5' exonuclease activity.
In eukaryotes DNA polymerases are of 5 type these are DNA polymerase and ε.
Kornberg (1956), succeeded in demonstrating the in vitro synthesis of DNA molecule using a single strand of DNA as a template. He extracted and purified an enzyme from E. coli which was capable of linking free DNA nucleotides, in presence of ATP as an energy source, to form complementary strand. He called it DNA polymerase. DNA polymerase-I is called Kornberg enzyme.