What is DNA Replication and Its Steps?

What is DNA Replication?

The biological process by which DNA makes an identical copy of itself during cell division is known as DNA replication. It is read in the 3 ‘to 5’ direction by the DNA polymerase, which means that the resulting strand is synthesized in the 5 ‘to 3’ direction.

As you already know, DNA is a nucleic acid that has three main components: deoxyribose sugar, a phosphate, and a nitrogenous base. Since the DNA contains the genetic material for an organism, it is important that it be copied when a cell divides into daughter cells.

Replication involves the production of identical DNA helices form a double-stranded DNA molecule. Enzymes are critical to DNA replication as they catalyze very important steps in this process. The entire DNA replication process is extremely important for both cell growth and reproduction in organisms. It’s also important in cell repair.

Starting of the DNA replication

How do DNA polymerases and other replication factors know where to start? Is there a specific region to initiate DNA replication?

The answer is yes. Replication always begins at a specific point on the DNA, which is known as the origin of replication and is recognized by its sequence. Most bacteria, such as E. coli, have the origin of replication at the OriC site. It consists of a 254 bp long DNA sequence and consists mainly of A = T base pairs. That makes them easier to separate.

Before we start DNA replication just watch the video below for a basic understanding. It gives you a brief idea of how DNA replication is done.

Steps of DNA replication

There are three main steps to DNA replication: initiation, elongation, and termination. These three main steps are broken down into another six steps:

  • Initiation of DNA Replication: Preparatory step
    • Step 1: Replication fork formation.
  • Elongation of DNA replication: DNA Synthesis Begins
    • Step 2: Primer Binding
    • Step 3: Synthesis of Leading and Lagging Strands
    • Step 4: Remove Primer and Gap Fill
    • Step 5: Proofreading
  • Termination of DNA replication:
    • Step 6: End Of the Replication

Initiation of DNA Replication: Preparatory Step

Step 1: Replication fork formation.

Before DNA can be replicated, the double-stranded molecule must be “unzipped” into two single strands. As we know, DNA has four bases called adenine (A), thymine (T), cytosine (C), and guanine (G) that form pairs between the two strands. Adenine only pairs with thymine and cytosine only binds to guanine.

In order to unwind DNA, these base-pair interactions must be broken. This is done by an enzyme known as DNA helicase.

However, there is a special initiator protein that is required to trigger DNA replication, namely DnaA. It binds regions at the oriC site throughout the cell cycle. In order to initiate the replication, however, the DnaA protein must bind to a few specific oriC sequences that have five repeats of the 9 bp sequence (also known as the R site).

When DnaA binds to the oriC site, it recruits a helicase enzyme (DnaB helicase). Now the DNA helicase breaks the hydrogen bond that holds complementary DNA bases together.

The separation of the two single strands of DNA creates a two Y-shaped structure called a replication fork. together they form a bubble-like structure called a replication bubble. These two separate strands serve as a template for the production of the new DNA strands.

How does the replication actually work on the replication forks?

Helicase is the first replication enzyme to be loaded at the origin of replication. Helicase’s job is to simply move the replication forks forward by “unwinding” the DNA. As we know, DNA is very unstable in the form of a single strand. In this way, cells can prevent them from coming back together in a double helix.

To do this, a specific protein called single-stranded DNA binding proteins (SSBs) coats and keeps the separated strands of DNA near the replication fork.

When the helicase quickly unwinds the double helix. It increases the tension on the remaining DNA molecule. Topoisomerase plays an important maintenance role during DNA replication. This enzyme prevents the DNA double helix in front of the replication fork from becoming too tight when the DNA is opened. It does this by making temporary nicks in the helix to release tension and then sealing the nicks to prevent permanent damage.

Elongation of DNA replication: DNA Synthesis Begins

Step 2: Primer Binding

Another enzyme was introduced in this step, which plays the most important role in the synthesis of DNA. which is DNA polymerase. it can only add nucleotides at the 3 ‘end of an existing DNA strand. (They use the free OH group found at the end of the 3′ as a “hook” by adding a nucleotide to this group in the polymerization reaction.) Then how does DNA polymerase add the first nucleotide with a new replication fork?

Alone, cannot. The problem is solved with the help of an enzyme called primase. Primase forms the RNA primer, or short nucleic acid strand, that completes the template, providing a 3 ‘end for working on DNA polymerase. A typical primer has about five to ten nucleotides. The primer begins the synthesis of DNA.

Once the RNA primer is in place, DNA polymerase “extends” it and sequentially adds nucleotides to create a new strand of DNA that is complementary to the template strand.

Note: In bacteria like E. coli, polymerase III is the main replication enzyme, while polymerase I, II, IV, and V are responsible for error checking and repair. DNA polymerase III binds to the strand at the site of the primer and, during replication, begins to add new base pairs that are complementary to the strand. In eukaryotic cells, the polymerases alpha, delta, and epsilon are the primary polymerases involved in DNA replication.

Step 3: Synthesis of Leading and Lagging Strands

One of the strands is oriented in the 3′ to 5′ direction (towards the replication fork), this is the leading strand. The other strand is oriented in the 5′ to 3’direction (away from the replication fork), this is the trailing strand. Because of their different orientation.

Leading Strand Synthesis

A short piece of RNA called a primer (made by an enzyme called a primase) comes by and attaches to the end of the leading strand. The primer serves as the starting point for DNA synthesis.

The DNA polymerase binds to the leading strand and then walks along with it, adding new complementary nucleotide bases (A, C, G, and T) in the 5′ to 3′ direction to the DNA strand.

This type of replication is known as continuous.

Legging strand Synthesis

The DNA polymerase always runs in the 5 ‘to 3’ direction. So how can both strands be synthesized at the same time?

When both strands have been synthesized continuously while the replication fork is moving. A one strand would therefore have to be subjected to a 3 to 5 synthesis. This problem was solved by Reiji Okazaki and colleagues in the 1960s.

Okazaki found that one of the new strands of DNA was synthesized in short pieces (now these fragments are known as Okazaki fragments). This work ultimately led to the conclusion that one strand is synthesized continuously and others discontinuously.

The synthesis of any Okazaki fragment seems straightforward. However, the reality is quite complex. Because the DNA polymerase moves with the replication fork (5’ to 3’ direction). So how can you replicate two antiparallel DNA strands at the same time and in a coordinated manner?

To solve this problem, DNA polymerase III uses one set of its core subunits (the core polymerase) to continuously synthesize the leading strand. While the other two sets of the core subunit lie from one Okazaki fragment to the next on the looped duct.

Refer this article for more about DNA polymerase

In vitro, there are only two sets of core subunits containing DNA polymerase III holoenzymes that can synthesize both the leading and lagging strand. However, the third set of core subunits increases the efficiency of delayed strand synthesis as well as the processivity of the overall replisome.

in thi image we are discusing about how the okazaki fregment formation takes place, and how DNA polymerase III core subunite work in the formation of this short fregment.
Enzymetic mechanism of Okazaki frgment formation

As shown in the figure, when DnaB helicase binds before DNA polymerase III. It begins by unwinding the DNA on the replication fork as it moves along with the trailing strand template in the 5 ‘to 3’ direction. Primase occasionally associates with DnaB helicase and synthesizes a short RNA primer.

A new slide clamp is now positioned on the primer through the clamp loading complex of DNA polymerase III. When the synthesis of the Okazaki fragment is complete.

Replication halts and the core subunits of DNA polymerase III dissociate from their slide clamps (and form the complete Okazaki fragment) and associate with the new clamp. This initiates the synthesis of a new Okazaki fragment. Two sets of core subunits can be involved in the synthesis of two different Okazaki fragments at the same time.

Once an Okazaki fragment is complete, its RNA primers are removed by DNA polymerase I or RNase H1. And that space is replaced by DNA by the polymerase. The remaining nick has now been sealed by the DNA ligase.

Step 4: Remove Primer and Gap Fill

Once both the continuous and discontinuous strands are formed, an enzyme called an exonuclease (DNA polymerase I or RNase H1) removes all RNA primers from the original strands. These primers were replaced by suitable DNA bases. The remaining nick was sealed by the DNA ligase.

DNA ligase catalyzes the formation of a phosphodiester bond between a 3′-hydroxyl group on the end of one strand of DNA and a 5′-phosphate on the end of another strand

Step 5: Proofreading

The DNA replication takes place with very high fidelity. It contains the wrong nucleotide once for every 104–105 polymerized nucleotides. The accuracy of replication depends on the ability of replicative DNA polymerases to select the correct nucleotide for the polymerization reaction.

This high fidelity is not achieved in a single step but rather is generated through the operation of several successive error avoidance and processing steps. These steps include the selection of the correct DNA base by the DNA polymerase (i.e. the nucleotide insertion step), the editing (i.e. the removal) of polymerase miss insertion errors by exonucleolytic proofreading, and finally, the post-replicative DNA mismatch repair, which detects DNA mismatches and corrects newly replicated DNA.

Termination of DNA replication:

DNA replication ceases when two forks of replication meet on the same stretch of DNA, and the following events occur, but not necessarily in this order: forks converge until all of the intervening DNA is unwound; remaining gaps are filled and tied; Catenans are removed, and replication proteins are discharged.

Step 6: End Of the Replication

DNA replication finishes when the two-replication fork of the circular E. Coli chromosome meets again at a termination site (ter).

This ter sequence is placed on the chromosome to create a trap that a replication fork can enter but not leave. On the other hand, the Ter sequence acts as a binding site for the “Tus” protein. Which are acts as a terminology substance.

Now the Tus-Ter complex can only stop a replication fork in one direction. Because there is only one Tus-Ter complex function per replication cycle – the complex that one of the replication forks first encounters.

This point across from the replication fork is generally stopped if they collide (clash or meet). So when the first (arrested) fork hits the Tus-Ter complex, the other fork will stop. Next, the last few hundred base pairs of DNA are replicated between these large protein complexes (this mechanism is still unknown).

After completion, the parent strand and its complementary DNA strand wrap themselves in the familiar double helix shape. In the end, replication produces two DNA molecules, each with a strand from the parent molecule and a new strand.

FAQs

What is DNA Replication?

In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all living organisms acting as the most essential part for biological inheritance.

What is the Process of Replication in DNA?

DNA replication is the process by which a double-stranded DNA molecule is copied to produce two identical DNA molecules. Once the DNA in a cell is replicated, the cell can divide into two cells, each of which has an identical copy of the original DNA.

Why is DNA replication important?

The purpose of DNA replication is to produce two identical copies of a DNA molecule. This is essential for cell division during growth or repair of damaged tissues. DNA replication ensures that each new cell receives its own copy of the DNA.

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