DNA replication steps | 3 steps of DNA Replication explained in simple terms

In this article, we will get you to know about What is DNA replication, and the 3 steps of DNA replication in simple terms.

DNA replication

DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication is the longest phase in the whole cell cycle.

E. coli that has only 4.6 ×10⁶ bp completes the process of replication within 18 minutes; with an amazing speed of 2000 bp per second.

How DNA replication occurs?

There are three proposed approaches: –

• Conservative Replication – It assumes that after duplication of DNA, parent DNA stays together while daughter DNA is a separate one.

• Semiconservative Replication – It assumes that post replication, both strands consist of parent DNA complementary to daughter DNA.

• Dispersive Replication – It assumes that DNA molecules exist as a hybrid of parent and daughter DNA post replication.

Fig2. Three models of DNA replication (Figure by Parker, N., et.al. (2019) Openstax)

The experimental proof to test DNA replication / Matthew Meselson and Franklin Stahl’s experiment

• In 1958, Matthew Meselson and Franklin Stahl performed the experiment to test the model of DNA replication out of conservative, semiconservative and dispersive.

• They grew E.coli in a medium containing ¹⁵NH₄Cl (a heavy isotope of nitrogen i.e. ¹⁵N) as the only nitrogen source for several generations (doubling time of E.coli is 20 minutes).

•  This led to the incorporation of ¹⁵N into their nitrogenous based and eventually into their DNA.

• After that the medium was provided with a normal nitrogen source containing ¹⁴NH₄Cl (¹⁴N) as only nitrogen source and DNA was extracted with doubling time of E.coli i.e. 20 minutes and one after 40 minutes.

• The DNA separation was done using density gradient centrifugation with Caesium chloride (CsCl) as a density gradient.

• The DNA extracted from E.coli, one generation after transfer from ¹⁵N to ¹⁴N (after 20 minutes) has hybrid DNA (¹⁵N¹⁴N) that indicated probable chances of either semiconservative or dispersive.

• Further, after another generation (after 40 minutes,) extracted DNA has hybrid DNA (¹⁵N¹⁴N) and light DNA (¹⁴N). This ruled out the dispersive model.

• Hence, it was experimentally proved that DNA replication occurs in semiconservative mode where new strands are formed complementary to the parental strands.

DNA replication steps

DNA replication is bidirectional, semiconservative, and semi-discontinuous.

The bidirectional replication results in the formation of a replication fork where one strand is synthesized in opposite directions.

E. coli that has only 4.6 ×10⁶ bp completes the process of replication within 18 minutes; with an amazing speed of 2000 bp per second.

3 steps of DNA replication

Initiation of DNA replication

DNA replication starts at the origin of replication (ori).

DNA double strands are opened by enzyme helicase forming a replication fork (RF).

Further, to these opened strands Primase binds followed by DNA dependent DNA polymerase resulting in replisome.

Replisome formation indicates the initiation of replication.

Elongation

After primase forms a short strand of polynucleotide and prime the synthesis. Then, DNA polymerase III starts adding nucleotide bases at 3’ OH end complement to parent DNA (A base pairs with T and G pairs with C and vice versa).

DNA synthesis is bidirectional and occurs in the 3’ to 5’ direction.

Therefore, in the replication forms two different strands are formed one in the direction of opening of DNA called leading strand (i.e., continuous synthesis) and the other one lagging strand (i.e., discontinuous synthesis).

The fragments formed as results of discontinuous synthesis are called Okazaki fragments.

DNA replication termination

During termination, primers are removed, replaced with new DNA nucleotides and, the backbone is sealed by enzyme DNA ligase.

Fig5. DNA replication (source LadyofHats Mariana Ruiz)

DNA replication machinery and enzymes in prokaryotes

These are the following enzymes and machinery involved in prokaryotic replication.

S.No.	Enzyme involved	Function
1.	Helicase	This enzyme opens up the double helix of DNA by breaking the hydrogen bonds.
2.	Primase	It synthesizes an RNA primer (five to ten nucleotides) complementary to the DNA and, helps in the initiation of DNA synthesis.
3.	Sliding clamp	It holds the DNA polymerase during the elongation phase.
4.	Topoisomerases	It helps to release tension in opening DNA to avoid supercoiling of DNA.
5.	DNA ligase	It seals the gaps by forming phosphodiester bonds in lagging strands.
7.	Single-strand binding proteins (SSB)	It binds to the single-stranded near the replication fork to prevent them from winding back to form a double helix.
8.	DNA polymerase I	It has both 5’-3’ & 3’-5’ exonuclease activity, &has 5’-3’ DNA synthesis activity. It helps in DNA repair & gap filling.
9.	DNA polymerase II	It has 3’-5’ exonuclease activity&5’-3’ DNA synthesis activity. It helps in DNA repair.
10.	DNA polymerase III	It has 3’-5’ exonuclease activity & 5’-3’ DNA synthesis activity. It is the main enzyme involved in DNA replication.

Author: Supriya Bhatt

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