“The excision of a transposon from a native location followed by its integration at another location in a host genome is called non-replicative mechanism of transposition.”
The non-replicative mechanism of transposition, a cut and paste mechanism also known as the conservative mode of the transposition that helps transposon to jump from one location to another without leaving a copy of it at the original location.
And this is the reason this type of transposition is also called cut and paste mechanism of transposition.
The transposon is cut from one place and integrated into another location on a non-homologous locus, during this process the DNA sequence of the TE remain conserved, therefore, it is also known as conservative transposition.
The cut and paste mechanism of transposition is practically applicable to the gene therapy via artificial transposons. A gene can be introduced into the genome by using transposons.
A gene of interest is excised from one vector and introduced into the host genome by transposase-mediated transposition.
By the conservative transposition, any gene can be introduced into vertebrates as well as invertebrates.
Sleeping beauty transposon system is the best example of non-replicative transposon-mediated gene therapy.
We had covered almost all the information about the transposons, in the present series of articles. Some of the previous articles are:
- Transposons: A Jumping Entity and a Foe with Benefits
- Transposase, Transposons and Antibiotic Resistance in Bacteria
- Transposons in eukaryotes
- Role of transposons in evolution
- Replicative Transposition of DNA transposons and Retrotransposons
Let me brief you the topic.
The transposons are the mobile genetic elements present into prokaryote as well as eukaryotes that can move from one location to another location.
Structurally, it contains a gene body that can encode a couple of genes, terminal repeats either inverted or direct and a piece of the previous host DNA.
See the structure below,
They are categorized into retrotransposons that can move via the RNA intermediate and the DNA transposons that can transfer via the DNA intermediates.
The transposition mechanism varies among different transposons.
Some of the transposons follow the replicative mechanism of transposition in which After excision from the native place a copy of it remains there.
We had covered an entire article on replicative transposition. Please read it first: Replicative transposons.
While in the non-replicative transposition, the TE moves unidirectional, leave a gap behind.
In the present article, we will discuss the mechanism of the non-replicative transposition.
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What is non-replicative transposition?
Without leaving a copy behind, the excision of a transposon from one location followed by its integration to another location in the genome is a non-replicative transposition.
It cuts the transposon from one location and pastes it at another location hence it is also named cut and paste mechanism of transposition.
Mechanism of transposition:
The non-replicative transposition initiated with the activity of a transposase protein.
The transposase protein encoded from the transposon, helps it to migrate within a genome.
“It is believed that transposons can move between different organisms too.”
The transposase creates a synaptic complex initially.
The synaptic complex is formed by binding of transposase proteins to its terminal repeats.
The terminal repeat sequences present on both the ends of a transposon help to move it, recognized by the transposase protein (two or sometimes 4 transposase).
The structure of the synaptic complex is given below,
Once it binds to TRs, it brings the two ends of it together and creates a stable DNA protein complex called a synaptic complex.
Sometimes this complex is even called a transpsosome. This is a very first step in the non-replicative transposition.
The synaptic complex actually decides the fate of the transposon, it ensures DNA cleaving and joining which are required to move the transposon.
The next step is excision.
In this step the DNA-protein complex, a transpososome cut the transposon in a sequential manner.
First, it cleaves one DNA strand on each side exactly at the junction between the TE and the flanking (previous) host DNA.
(Remember! the transposon contains some of the DNA sequences at their ends from their previous host DNA).
Here, the purpose of it is to generate a free 3’ OH end needed to fill the gap.
In the second part of this reaction, the transposon cleaves another strand of it at both the end however it is not necessary that the same transposon catalyze both reactions.
Our transposon is now completely excised from the original position and ready to move.
In the very next step, The transposase creates two single-stranded cuts on the host DNA.
Immediate after that, the free 3’ OH end of the transposon DNA attacks the 5’ and of the host DNA.
At a molecular level, the reaction occurred as mentioned below,
- The 3′ end attaches to the 5′ end of the host DNA.
- It cleaves the bonds and released two diphosphates by joining with the 5’ end of the target DNA.
- The reaction occurred on both ends at the same time.
The transposons fill the gap between the junction of 3’ end (of transposon) and 5’ end (of the target DNA).
A very crucial even occurred at this point.
Once the gaps are filled the transposases are separated from both the end but another end of the target DNA remains unfilled.
The entire process of non-replicative transposition is shown in the figure below,
The transposase cannot fill the gap between the 5’ and 3’ ends of the transposon and target DNA, respectively.
5 to 9 nucleotide gap remains unfilled on both ends.
The DNA repair mechanism of the host cell can do so.
The polymerase fills the gaps by adding nucleotides between the gap. The same gap is filled by the polymerase on the other side which generates two duplicate sites on both the flanking regions of a transposon.
The entire process governed by the cellular repair protein is called target site duplication.
See the figure below,
Target site duplication is another important characteristic of a transposon.
“The target site duplication length divulge a distance between the transposase action site on the host DNA.”
In the final step, the DNA ligase seals the gaps between the newly synthesized DNA.
Apart from their role in the transposition, the transposase also performs one important function needed to complete the reaction.
It protects the transposon DNA ends attacked by cellular enzymes during the process of strand transfer.
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- Site-Directed Mutagenesis: Methods and Applications
- Effect of PCR inhibitors on PCR amplification
- PCR reaction: Ten secrets that nobody tells you
After completion of the entire process of conservative transposition, a gap, more preciously we can say a double-stranded break remains as such, at the native or old place.
Later on, that gap is filled by the repair mechanism of a cell. It might be joined directly or filled by homologous recombination.
See the figure below,
The bacterial Tn10 transposon follows the non-replicative modes of transposition. Our sleeping beauty transposons also does the same.
A gap in the previous place of the transposon are sealed directly or with some other nucleotides, therefore new variations can be produced by non-replicative transposition.
Also, at the new insertion site, the transposon can ON/OFF gene expression, alter gene function or creates new allelic variation.
Therefore it is believed that transposon played a significant role in the process of evolution by creating new allelic variations in the population.