DNA Replication: It is a unique and complex process that takes place in both prokaryotes and eukaryotes. Do you know how many chromosomes you have? How does the number of chromosomes in a particular organism remain constant? It is because of the DNA Replication process that takes place during the S-phase (synthetic phase) of the cell division (mitosis or meiosis) in each and every cell.

Replication occurs in three major steps: the opening of the double helix and separation of the DNA strands, the priming of the template strand, and the assembly of the new DNA segment. During separation, the two strands of the DNA double helix uncoil at a specific location called the origin. Several enzymes and proteins then work together to prepare, or prime, the strands for duplication. Finally, a special enzyme called DNA polymerase organizes the assembly of the new DNA strands.

Mechanism of DNA Replication: DNA Replication Process

DNA Replication is a very unique and complex multistep biological process of producing two identical replicas from one original DNA molecule. It occurs in all living organisms (both prokaryotes and eukaryotes) because it forms an essential part of biological inheritance. It requires a number of enzymes, protein factors, and metal ions.

DNA Replication Diagram

Check the diagram to understand DNA replication better:

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In 1953, Watson and Crick proposed the mechanisms for DNA replication while proposing the double-helical structure of DNA.

DNA Replication is Semiconservative

In 1953, Watson and Crick suggested that the two strands of DNA would separate and act as templates for the synthesis of new complementary strands. After the completion of replication, each DNA molecule would have one parental and one newly synthesised strand. This shows that the replication of DNA is a semiconservative process.

The Experimental Proof

Mathew Meselson and Franklin Stahl 1958 performed the following experiment using heavy nitrogen (¹⁵N) in E. coli as follows:

1. They grew the bacterium E. coli in a medium containing \(^{15}N{H_4}Cl\) as the only source of heavy nitrogen for many generations. \(^{15}N\) was incorporated into newly synthesised DNA as well as other nitrogen-containing compounds.
2. This heavy DNA molecule could be distinguished from the normal DNA by centrifugation in a cesium chloride \(\left( {CsCl} \right)\) density gradient. Then they transferred the cells into a medium with normal \(^{14}N{H_4}Cl\) and took samples at various definite time intervals.
3. DNA that remained as a double-stranded helix was isolated by high speed and evaluated for its density on \(CsCl\) gradient.
4. Thus, the DNA that was extracted from the culture after the \({1^{st}}\) generation, i.e., just after 20 minutes had a hybrid or intermediate density. DNA extracted from the culture after another generation, i.e., \({2^{nd}}\) generation or \(40\) minutes was composed of equal amounts of this hybrid DNA \(\left( {{N^{14}}{N^{15}}} \right)\) and of light DNA \(\left( {{N^{14}}{N^{14}}} \right).\)
5. An increase in the amount of light DNA and a decrease in the amount of hybrid DNA can be possible due to the semiconservative mode of replication.

Enzymes of DNA Replication

The enzymes involved in the process of DNA replication are as follows:

1. Helicases: These are special unwinding enzymes that help in breaking the weak hydrogen bonds which hold the two strands together.
2. Topoisomerases: These are the enzymes that can break and reseal one strand of DNA.
3. Primase: This enzyme helps in the formation of a primer (primer is a short RNA segment that is formed on the DNA template before the replication can begin which is absolutely essential). It polymerises the complementary RNA building blocks A, U, G, and C in the primer.
4. DNA polymerase: This enzyme is the main enzyme required for DNA replication. It can link free DNA nucleotides to form the complementary strand of DNA. It polymerises nucleotides in 5’→3’ direction only. It is also known as a DNA-dependent enzyme as it uses a DNA template for polymerisation of deoxynucleotides.
5. DNA ligase: This enzyme can join together the short sections of newly synthesised polynucleotide chains. It puts the short pieces together after replacing the RNA primer with DNA.
6. Repair enzymes: These enzymes can cut off the wrong bases and replace them with the correct ones. They help in rectifying the errors that may have occurred during replication.

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DNA Replication Steps

The process of DNA replication occurs by the following three steps:

1. Initiation
2. Elongation
3. Termination

Here we are providing you step-wise process of DNA Replication through the use of the diagram below:

Initiation of DNA Replication

In prokaryotes, DNA replication takes place in the cytoplasm whereas, in eukaryotes, DNA replication occurs in the nucleus during the S-phase of the cell cycle. During initiation, the DNA is made accessible to the proteins and enzymes that are involved in the replication process. There are a number of specific chromosomal locations called initiation points or origin of replication (ori), from where the replication of DNA begins.

There are no specific sequences for the origin of replication. But there are certain proteins that recognise and bind to it, and also allow other proteins necessary for DNA replication to bind to the same region. DNA replication is semi discontinuous in eukaryotes.
Bacterial and viral DNA has a single origin of replication. It functions as a single replication unit called a replicon. But in eukaryotic DNA, there are a number of origins of replications or replicons. So, they are multirepliconic. In the absence of ori, replication will not occur.

Elongation of DNA Replication

Following are the steps followed during the elongation of DNA replication:

1. Activation of Deoxyribonucleotides: Deoxyribonucleotides occur freely inside the nucleoplasm in the form of monophosphates. So, they are first phosphorylated and changed to active forms of triphosphates with the help of the enzyme phosphorylase along with the energy.
2. Exposure of parent DNA strands: Enzyme helicase acts over the ori site and unwinds the two strands of DNA by destroying the hydrogen bonds. It results in the formation of a replication fork (Y-fork). Due to unwinding, supercoiling gets developed on the end of DNA opposite to the replication fork which is released by the enzyme topoisomerase.

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3. Formation of RNA Primer: At the free \(3’\) end of one strand and fork end of the second strand, a small strand of RNA called RNA primer is synthesised with the help of enzyme RNA polymerase or primase. RNA primer functions like \(5’\) end of the new strand to be synthesised.

4. Base Pairing: The two separated DNA strands in the area of the replication fork now function as their nitrogenous bases attract complementary phosphorylated nucleotides. Enzyme pyrophosphatase removes two phosphates from phosphorylated nucleotides and changes them into a monophosphate state. The energy released in this process is used in forming hydrogen bonds.

5. New Strand Formation: In the presence of \({\bf{M}}{{\bf{g}}^{{\bf{2}} +,}}\)ATP (GTP), TPP and DNA polymerase enzyme, the adjacent nucleotides found attached to nitrogenous bases of each template of DNA strand establish phosphodiester bonds and get linked to form replicated DNA strand. As replication proceeds, new areas of the parent DNA duplex unwind and separate so that replication proceeds rapidly from the place of origin towards the other end. RNA primer is removed and the gap is filled with complementary nucleotides by means of DNA polymerase.

Since the two strands of DNA run in antiparallel directions, the two templates provide different ends for replication. Thus, replication over the two templates proceeds in opposite directions. One strand with polarity \(3′ \to 5’\) forms its complementary strand continuously and is called the leading strand.

But replication is discontinuous on the other template with polarity \(5′ \to 3’\) because DNA polymerase enzymes can add nucleotides in \(5′ \to 3’\) direction only. So, short segments of replicated DNA are formed by DNA polymerase known as Okazaki fragments which are joined together by means of DNA ligase enzyme. So, this DNA strand built up of Okazaki fragments is called a lagging strand.

6. Proofreading and DNA repair: A wrong base is sometimes introduced during replication (one in ten thousand). DNA polymerase goes back, removes the wrong base, allows the addition of the proper base, and then proceeds forward. The newly formed segment is sealed by DNA ligase.

Termination of DNA Replication

The termination of DNA replication takes place by telomere replication. The ends of the eukaryotic linear chromosomes are known as telomeres, which have repetitive sequences that do not code for a particular gene. As the enzyme DNA polymerase adds nucleotides in only one direction, the synthesis of the leading strand is continuous until the end of the chromosome is reached.

But there is no place for a primer on the lagging strand to be made for the DNA fragment to be copied at the end of the chromosome. So, the enzyme telomerase attaches to the end of the chromosome, and the complementary bases are added to the RNA template at the end of the DNA strand. After the elongation of the lagging strand template, the DNA polymerase enzyme adds nucleotides that are complementary to the ends of the chromosome. Thus, the ends of the chromosome are replicated.

DNA Replication Significance

The significance of DNA replication is as follows:

1. To produce two identical copies of the parental DNA so that each daughter cell receives its own copy of DNA.
2. To maintain the original chromosome number of an individual.
3. Essential for the cell division process during the growth or repair of an individual.
4. Essential for coding of proteins.
5. Essential for providing instructions for life and its processes.

DNA Replication Notes

We can conclude that DNA replication is a semiconservative method in which each of the two parental DNA strands acts as the template for new DNA to be synthesised. After the completion of the DNA replication, each DNA has one parental (or old) strand and one daughter (or new) strand.

FAQs on DNA Replication

Some of the frequently asked questions about DNA replication are mentioned below:

Q.1: Why is DNA replicated?
Ans: DNA replication takes place due to the following reasons:
a. To maintain the original chromosome number of an individual.
b. Essential for the cell division process during the growth or repair of an individual.
c. Essential for coding of proteins.
d. Essential for providing instructions for life and its processes. 

Q.2: What are the four steps of DNA replication?
Ans: The four steps of DNA replication are:
a. Formation of Replication Fork.
b. Binding of RNA primer to the DNA template.
c. Elongation of the new DNA strand by DNA polymerase.
d. Proofreading and DNA repair.

Q.3: In which stage does the DNA replication occur?
Ans: DNA replication takes place during the S-phase (synthetic phase) of the cell cycle. 

Q.4: Where does DNA replication begin?
Ans: DNA replication begins at a specific point known as the initiation point or origin of replication (ori).

Q.5: Where does DNA replication occur?
Ans: DNA replication takes place in the cytoplasm of prokaryotes and inside the nucleus of eukaryotes.

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