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In this essay we will discuss about:- 1. Definition of DNA Replication 2. Mechanism of DNA Replication 3. Evidences for Semi-Conservative DNA Replication 4. Models for Replication of Prokaryotic DNA.
Essay # Definition of DNA Replication:
DNA replicates by “unzipping” along the two strands, breaking the hydrogen bonds which link the pairs of nucleotides. Each half then serves as a template for nucleotides available in the cells which are joined together by DNA polymerase. The nucleotides are guanine, cytosine, adenine and thymine. DNA replication or DNA synthesis is the process of copying a double-stranded DNA molecule.
This process is important in all known forms of life and the general mechanisms of DNA replication are the same in prokaryotic and eukaryotic organisms. The process by which a DNA molecule makes its identical copies is referred to as DNA replication. In other words, it is the process of duplicating the DNA to make two identical copies. The main points related to DNA replication are briefly presented below.
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1. Time of Replication:
The process of DNA replication takes place during cell division. The DNA replication takes place during S sub stage of interphase. In prokaryotes, DNA replication is initiated before the end of the cell cycle. Eukaryotic cells can only initiate DNA replication at the beginning of S phase.
2. Replication Site:
In humans and other eukaryotes, replication occurs in the cell nucleus, whereas in prokaryotes it occurs in the cytoplasm. Prokaryotes have only one active replication site, but eukaryotes have many.
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3. Template Used:
The existing DNA is used as a template for the synthesis of new DNA strands. It is possible that during replication on strand of DNA can replicate continuously and the other discontinuously or in piece. 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 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.
Short segments of nucleotides are synthesized in the lagging strand of DNA as a result of discontinuous replication. These are called Okazaki after the name of discoverer. Okazaki fragments are about 1,500 bases in length in prokaryotes, and 150 bases in eukaryotes
4. Enzymes Involved:
The process of DNA replication takes place under the control of DNA polymerase. In other words, the process is catalized by the polymerase enzyme. In eukaryotes, four types of polymerase enzymes, viz. alpha, delta, gamma and epsilon are used.
DNA Polymerase alpha and delta replicate the DNA. The alpha is associated with initiation, and delta extends the nascent strands. DNA polymerase epsilon and beta are used for repair. DNA polymerase gamma is used for replication of mitochondrial DNA
In prokaryotes [E. coli], there are three major DNA polymerases: DNA polymerase I, II and III. DNA poly I is found in the highest concentration of all DNA polymerases; it is involved in DNA repair and assists with primary DNA replication. DNA poly II is exclusively involved in repair. DNA poly III is the major DNA polymerase. All DNA polymerases add to the 3′ OH of the existing polynucleotide.
Currently, six families of polymerases (A, B, C. D, X, Y) have been discovered. At least four different types of DNA polymerases are involved in the replication of DNA in animal cells (POLA, POLG, POLD and POLE).
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5. Direction of Replication:
The synthesis of one new strand takes place in 5-3 and that of other in opposite (3-5) direction. The replication may take place either in one direction or in both the directions from the point of origin. When replication proceeds in one direction only, it is called unidirectional replication. When the replication proceeds in both the directions, it is called bidirectional replication.
6. Replication Type:
Based on the direction, the replication may be unidirectional or bidirectional. On the basis of continuity, the replication may be continuous or discontinuous.
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7. Origin of Replication:
The point of initiation of DNA replication is known as origin. The progress of replication process is measured from the point of origin.
8. Rate of Replication:
In prokaryotic cells the rate of replication is 500 bases per second. In eukaryotic cells the rate of replication- is 50 bases per second. Eukaryotes have 100 to 3,000 times more DNA than prokaryotes.
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9. Replication Models:
There are three models which explain the accurate replication of DNA. These are: (i) dispersive replication, (ii) conservative replication, and (iii) semiconservative replication (Fig. 17.1).
These are explained as follows:
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(i) Dispersive Replication:
According to this model of replication the two strands of parental DNA break at several points resulting in several pieces of DNA. Each piece replicates and pieces are reunited randomly, resulting in formation of two copies of DNA from single copy. The new DNA molecules are hybrids which have new and DNA in patches (Fig. 17.2). This method of DNA replication is not accepted as it could not be proved experimentally.
(ii) Conservative Replication:
According to this model of DNA replication two DNA molecules are formed from parental DNA. One copy has both parental strands and the other contains both newly synthesized strands (Fig. 17.2). This method is also not accepted as there is no experimental proof in support of this model.
(iii) Semiconservative Replication:
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This model of DNA replication was proposed by Watson and Crick. According to this model of DNA replication, both strands of parental DNA separate from each other. Each old strand synthesizes a new strand. Thus each of the two resulting DNA molecules has one parental and one new strand (Fig. 17.3). This model of DNA replication is universally accepted because there are several evidences in support of this mode.
Essay # Mechanism of DNA Replication:
The semi-conservative model (mechanism) of DNA replication consists of six important steps, viz:
(1) Unwinding,
(2) Binding of RNA primase,
(3) Elongation,
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(4) Removal of primers,
(5) Termination, and
(6) DNA repair.
These are briefly discussed as follows:
1. Unwinding:
The first major step in the process of DNA, replication is the breaking of hydrogen bonds between bases of the two anti-parallel strands. The unwinding of the two strands is the starting point. The splitting happens in places of the chains which are rich in A-T.
That is because there are only two bonds between Adenine and Thymine, whereas there are three hydrogen bonds between Cytosine and Guanine. The Helicase enzyme splits the two strands. The initiation point where the splitting starts is called “origin of replication”. The structure that is created is known as “Replication Fork”.
2. Binding of RNA Primase:
Synthesis of RNA primer is essential for initiation of DNA replication. RNA primer is synthesized by DNA template near the origin with the help of RNA Primase. RNA Primase can attract RNA nucleotides which bind to the DNA nucleotides of the 3′-5′ strand due to the hydrogen bonds between the bases. RNA nucleotides are the primers (starters) for the binding of DNA nucleotides.
3. Elongation:
The elongation proceeds in both directions, viz. 5′-3′ and 3′-5′ template. The 3′-5′ proceeding daughter strand that uses a 5′-3′ template— is called leading strand because DNA Polymerase ‘a’ can “read” the template and continuously add nucleotides. The 3′-5′ template cannot be “read” by DNA Polymerase a. The replication of this template is complicated and the new strand is called lagging strand.
In the lagging strand the RNA Primase adds more RNA Primers. DNA polymerase a reads the template and lengthens the bubbles. The gap between two RNA primers is called “Okazaki” Fragments. The RNA Primers are necessary for DNA Polymerase a to bind Nucleotides to the 3′ end of them. The daughter strand is elongated with the binding of more DNA nucleotides.
4. Removal of Primers:
The RNA Primers are removed or degraded by DNA polymerase I. This enzyme also catalyzes the synthesis of short DNA segments to replace the primers. The gaps are filled with the action of DNA Polymerase which adds complementary nucleotides to the gaps.
The DNA Ligase enzyme adds phosphate in the remaining gaps of the phosphate-sugar backbone. Each new double helix is consisted of one old and one new chain. This is called semi-conservative replication.
5. Termination:
The termination takes place when the DNA Polymerase reaches to an end of the strands. In other words, it is the separation of replicated linear DNA. After removal of the RNA primer, it is not possible for the DNA Polymerase to seal the gap because there is no primer.
Hence, the end of the parental strand where the last primer binds is not replicated. These ends of linear (chromosomal) DNA consist of noncoding DNA that contains repeat sequences and are called telomeres. A part of the telomere is removed in every cycle of DNA Replication.
6. DNA Repair:
The DNA replication is not completed without DNA repair. The possible errors caused during the DNA replication are repaired by DNA repair mechanism. Enzymes like nucleases remove the wrong nucleotides and the DNA Polymerase fills the gaps. Similar processes also happen during the steps of DNA Replication of prokaryotes though there are some differences.
Essay # Evidences for Semi-Conservative DNA 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.
These are briefly discussed as follows:
1. Meselson and Stahl Experiment [1958]:
Organism Used:
Meselson and Stahl conducted their experiment with common bacteria of human intestine i.e. Escherichia coli.
Procedure:
They used heavy isotope of nitrogen for labelling DNA. The bacteria were grown on culture medium containing heavy isotope of Nitrogen [N15] for 14 generations (30 minutes per generation) to replace the normal nitrogen [N14] of E. coli with heavy nitrogen.
Then the bacteria were transferred to normal nitrogen medium. The density of DNA was determined after one, two and three generations. Principle Involved. It is possible to detect minute differences in density through density gradient centrifugation. District bands are formed in centrifuge tube for different density DNA.
Results:
2. Cairns Experiment [1963]:
Organism Used:
He also conducted his experiment with human intestine bacteria E. coli.
Procedure:
He used heavy isotope of hydrogen [H3] for labelling of DNA. It replaces thymine of DNA with tritiated thymidine. The chromosome of E. coli was used to prepare slides. Slides were coated with photographic emulsion or film and stored in dark place.
Principle Involved:
The tritiated thymidine emits particle 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 autoradiograph, it suggests labelling of one strand of DNA, which indicates semi- conservative replication.
Results and Conclusion:
Cairns observed light film exposure in the chromosome slides of E. coli, which demonstrated that DNA replication is semi-conservative.
3. Taylor’s Experiment [1969]:
Organism Used:
Taylor conducted his experiment with root tip cells of Viciafaba plant.
Procedure:
He treated root tips of Viciafaba with radioactive thymidine to label the DNA. Then root tips were grown in normal medium.
Principle Involved:
The treated root tips will produce hybrid cells containing normal and labelled chromosomes in subsequent generation cell division when grown on normal medium. The DNA replication is associated with chromosome replication.
Results:
Conclusion:
The above results demonstrated semi-conservative mode of chromosome replication in Viciafaba.
Essay # Models for Replication of Prokaryotic DNA:
In prokaryotes (bacteria and viruses), the DNA is circular. Hence, replication of DNA in these organisms differs from that of eukaryotes, where DNA is linear. Several models of replication of circular DNA have been suggested.
There are two most commonly known models of circular DNA replication, viz:
(1) Cairns Model, and
(2) Rolling circle model.
These are briefly discussed as follows:
1. Cairns model of DNA Replication:
This model of DNA replication in prokaryotes was proposed by Cairns in 1963.It explains the mechanism of DNA replication in double stranded circular DNA of bacteria. According to this model the DNA replication consists of following important steps.
(i) Unwinding of DNA:
The double stranded circular DNA starts unwinding or separation at a specific point called origin. Two growing points are established. As the growing points move apart, unwinding of the DNA double strand takes place. This unwinding creates torque since the parental DNA strands cannot unwind freely.
(ii) Initiation of Replication:
At the point of origin, bidirectional replication is initiated. Both strands of DNA are replicated. This leads to further unwinding of DNA double strand, resulting in formation of torque.
(iii) Super Twisting:
The torque leads to super twisting of DNA strand. As a result of super twisting, one of the strand is cut (nicked) which makes the parental strand to rotate freely. The cut is made by a swiveling protein, which relieves the strain.
(iv) Sealing of Broken Points:
The breaks are sealed by swiveling protein and thus the replication is over. The replication process continues in this way. Cairns type replication has been demonstrated in the bacteria E. coli and Bacillus subtilis, in several viral and plasmid chromosomes and in DNA synthesis of mitochondria and chloroplasts.
2. Rolling Circle Model of DNA Replication:
This model of circular DNA replication was proposed in 1968. This model explains mechanism of DNA replication in single stranded circular DNA of viruses, e.g. ɸX174, and the transfer of E. coli sex factor (plasmid). The ϕX174 chromosome consists of a single stranded DNA ring (Positive Strand). This model is most widely accepted.
The mechanism of replication consists of following important steps:
(i) Synthesis of New Strand:
First the chromosome becomes double stranded by synthesis of a negative strand. The original strand is positive. The negative strand is synthesized in side of parental positive strand.
(ii) Cut in Outer Strand:
The negative or inner strand remains a close circle and the positive strand is nicked at a specific site by endonuclease enzyme. This enzyme recognizes a particular sequence at this point. Thus a. linear strand with 3′- and 5′-ends is created.
(iii) Formation of Tail:
The original positive strand comes out in the form of a tail of a single linear strand as a consequence of rolling circle. The 5′-end of the broken strand becomes attached to the plasma membrane of the host bacterium.
Such replicating phage DNA is commonly found associated with bacterial membranes. The unbroken parental strand rolls and unwinds as synthesis proceeds, leaving a ‘tail’ which is attached to the membrane.
(iv) Synthesis of New Strand:
The synthesis of new strand takes place along the parental strand at the tail end in a 3-5 direction. The 3′-end serves as a primer for the synthesis of a new DNA strand under the catalytic action of DNA polymerase. The unbroken strand is used as the template for this purpose, and a complementary strand is synthesized. Thus the parental molecule itself is used as a primer for initiating replication.
New DNA is also synthesized in the tail region in discontinuous segments in the 5-3 direction. This synthesis is presumably preceded by the synthesis of an RNA primer under the catalytic action of RNA polymerase. The tail is cut-off by a specific endonuclease into a unit length progeny rod.
(v) Cutting of Tail:
Now the tail is cut-off into a linear segment by endonuclease. The linear segment becomes circular by joining two ends with the help of ligage enzyme. Thus a new circular molecule is formed which can become new rolling circle and replicate further.
Genetic information is preserved in the single stranded template ring which remains circular and serves as an endless template. There is no swiveling problem or creation of torque in the rolling circle model. As the strands unwind the 3′-end is free to rotate on the unbroken strand. The growing point itself thus serves as a swivel.
Evidence for the rolling circle model has been obtained from the replication of several viruses (M13, P2, T4, λ), replication resulting in transfer of genetic material during mating of bacteria, and the special DNA synthesis during oogenesis in Xenopus.
