LIGASE

 Ligases’ popular names frequently incorporate the word “ligase,” as in DNA ligase, an enzyme extensively employed in molecular biology laboratories to link together DNA fragments. Ligases are also known as “synthetases” since they are utilised to generate new molecules.  A DNA ligase is a type of enzyme that is a part of the topoisomerase family of enzymes. It is a DNA-binding enzyme that catalyses the ligation of duplex DNA, thereby joining two DNA strands. 

In some organisms, ligases are encoded by separate genes, while in others, they are encoded by genes that are part of the topoisomerase family.  Ligase can catalyse the joining of two single-stranded DNA molecules or DNA fragments to circularise them. This can be used to prepare circular DNA templates from linear ones, such as plasmids, phage and other vectors. In the case of circularising a phage DNA, this process can be used to generate phage genomes for direct analysis (e.g. single-molecule real-time, SMRT. For example, it is used in the cloning of plasmids or bacteriophages  

Functions and Applications of DNA ligase

While the DNA ligase function is quite straightforward, the mechanism by which it achieves this function has puzzled scientists for a long time. However, it seems that there are a lot of similarities between the enzyme and the type of enzyme that breaks a DNA chain to separate a DNA strand. 

Ligase is a DNA repair enzyme that can seal single-strand breaks, double-strand breaks, and gaps in DNA molecules, and has the ability to remove 3′-OH termini from DNA ends and to join a 3′-terminus of a single-strand break, double-strand break, or gap to a 5′-terminus of the break. The process is called ligation. The protein is known as ligase. 

DNA Ligase applications for molecular biology include: DNA ligase cloning, DNA ligase PCR, DNA ligase gel assay , DNA ligase gel electrophoresis. 

DNA ligases are the enzymes that catalyse the joining of DNA fragments to each other, using phosphodiester bonds.The DNA ligase catalyses the joining of the 3′ hydroxyl group of one DNA with the 5′ phosphate group of another DNA. The DNA ligase enzyme joins the phosphates of two DNA strands together by inserting the phosphate of one DNA strand into the 3′ hydroxyl group of the other strand, and then a second insertion of a phosphate in the 5′ hydroxyl group of the other strand. 

 Ligase is widely used in molecular biology and molecular genetics. They are involved in a number of important physiological functions and cellular processes. Ligases are essential for DNA replication, repair, recombination, and transposition. DNA ligase also finds its application in the process of DNA polymerisation. DNA ligase can restore the denatured DNA fragments together after relieving the constraints of the temperature. 

This makes DNA ligase an enzyme that is similar to topo IV, and its function is also known as DNA breakage/reunion. There are some other researchers who have also studied this topic.  For example, a group at Purdue University studied DNA ligase in a bacterial pathogen called Mycobacterium tuberculosis, and they found that an unusual ligase is required for the survival of the bacterium because it helps the bacteria maintain a genome in which genes have been deleted. 

DNA Ligase mechanism

There are three well-known mechanisms for DNA ligase to act on double-strand breaks and single-strand breaks. The topology of double-strand breaks, single-strand breaks, and the ligation products change depending on the ligase mechanism the ligase uses. DNA ligase can only operate in the presence of ATP. This makes sense because phosphates are the major energy providers in biological processes, thus they provide the energy to make the ligation happen. The ligase forms a covalent bond between the 3′ and 5′ termini and breaks the phosphodiester bond of the DNA strand. The ligase has two active sites. 

The ligase creates a covalent bond between the 5′-OH terminus and the 3′-P terminus of a duplex DNA.  As the ligase adds nucleotides, it inserts the terminal phosphate from the 5′ OH strand into the scissile bond of the 3′-OH terminus of the duplex DNA. The ligase uses high energy phosphoanhydride bonds to catalyse the reaction.  In the absence of ligase, the broken strands would be joined by means of a random annealing process that results in double-strand breaks and deletions. 

Conclusion- 

The  enzyme ligase, also known as molecular glue in some publications, is involved in DNA repair by joining the ends of broken strands of DNA. It is a ubiquitous enzyme, found in both prokaryotes and eukaryotes. There are many different ligases in the cell, and they are involved in many different processes, such as DNA replication, recombination, and DNA packaging.