A DNA sequence can be copied or broken separately from the original site, circularized, inserted into another site, and regulates subsequent genes. This process is called transposition. This sequence is called a jumping gene or transposon. Transposons are basic units that exist on chromosomal DNA and can replicate and move independently.
Enzymes that perform transposition functions, usually encoded by transposons, recognize specific sequences at both ends of the transposon, and can detach the transposon from adjacent sequences and then insert it into a new DNA target. Site, no homology requirement.
Transposons include inverted repeat sequences at both ends and a transposable gene in the middle. The inverted repeat sequence is where the transposase binds, and the transposable gene in the middle is the gene segment that jumps back and forth between genes.
Tn5 has a full length of about 5.8kb and consists of a core sequence encoding three antibiotics (neomycin, bleomycin, and streptomycin) and two inverted IS50 sequences, of which IS50R and IS50L The sequence is highly homologous, with only one base of IS50L mutated. IS50 has a 19 bp inverted end (outside end, OE and inside end, IE). The two inverted ends are different by 7 bp. This inverted end is the action site of transposase (Tnp). Both IS50L and IS50R contain genes encoding transposase (TnP) and transposition repressor protein (lnh). However, due to the base mutation in IS50L, translation is terminated prematurely, so only IS50R can produce normal active TnP and lnh.
When transposition occurs, two transposase molecules bind to the OE ends of the Tn5 transposon to form two Tnp-OE complexes. Subsequently, the two complexes associate and the ends interact and cleave. Polymerizes to form a synaptonemal complex composed of a dimeric protein and two molecules of DNA. Only after the formation of the synaptonemal complex does Tnp have the activity of cutting DNA. The formation of this structure is conducive to coordinated cleavage and transfer of the Tn5 DNA chain, and helps prevent Tnp from cutting only one end of the transposon DNA chain. The Tnp bound to the left end is responsible for catalyzing the hydrolysis of the phosphodiester bond at the right end, while the Tnp bound to the right end is responsible for catalyzing the hydrolysis of the phosphodiester bond at the left end. Activating Tn p water molecules, this activated water molecule hydrolyzes the DNA chain, forming two 3’-OH nucleophilic groups at both ends of Tn5. The nucleophilic groups then attack the complementary chain to form a hairpin structure. Then another activated water molecule hydrolyzes the hairpin structure to form blunt-ended Tn5, and the entire synaptonemal complex leaves the donor strand and binds to the target DNA. The 3'-OH of Tn5 nucleophilically attacks the target sequence, forming a 9 bp sticky end between the transposon insertion site, and a positive valence is formed between the 3'-OH of the transposon and the 5'-P of the target DNA. key, the transposon is inserted into the target sequence. The gap is filled under the action of DNA polymerase, and a 9 bp direct repeat sequence is formed at both ends of the transposon. The entire transposition process completes the process of genes being cut from the original DNA and then pasted into the DNA of another recipient, achieving gene "jumping" (pictured).
ATAC-seq, also known as Assay for Transposase-Accessible Chromatin with high throughput sequencing, is a high-throughput sequencing technology that uses transposase to explore accessible chromatin.