Ligases

In this enzyme, the association between the deoxyribose group on one strand of DNA and phosphate group on the other strand of DNA forms a link that joins the two DNA strands together. The Okazaki fragments that form on the lagging strand during DNA replication are joined together by the DNA ligase, which is functional in this process. Using the assistance of a molecule of energy, the DNA ligase is able to join two DNA fragments together, resulting in the formation of phosphodiester bonds between the two DNA fragments.

DNA Replication and Recombination

When the cellular processes of recombination, DNA replication, and repair occur, breaks in the phosphate backbone DNA structure are created. It jeopardizes the stability of the genome, posing a threat to the loss of genetic content as well as the introduction of deleterious chromosomal mutations, among other consequences.

DNA ligases are enzymes that catalyse the formation of phosphodiester bonds between adjacent nucleotides in duplex DNA. This catalytic activity eventually results in the repair of DNA breaks. When performing DNA ligase activity, a nucleotide cofactor is required, which is accomplished through a three-step reaction mechanism involving covalent modification. The process entails the covalent modification of the DNA substrate as well as the ligase enzyme.

The DNA ligase can be used for a variety of purposes, including the introduction of genes of interest into plasmid vectors and the creation of fusion genes by joining two genes together. Ligation is the term used to describe this phenomenon. Ligation can be performed on DNA lengths that have sticky or blunt ends after they have been subjected to restriction digests. The DNA ligase joins the DNA fragments together directly in the blunt end ligation method of ligation.

DNA Ligase – What it does

The importance of DNA ligases in the preservation of genomic integrity cannot be overstated. When DNA damage occurs and is repaired, it joins the breaks in the phosphodiester backbone that occur during recombination and replication. This is in addition to the result of DNA damage and the repair of that damage.

Despite the fact that ligases are used for a variety of different purposes in vitro and in vivo processes, the primary function of DNA ligase is to ligate two strands of DNA, which could be single or double strands.

The role of DNA ligase in the replication process

One DNA duplex can result in the production of four different single-stranded DNA molecules during the process of DNA replication. Several enzymes carry out a variety of functions in order to complete the replication process completely. Through the action of the primase enzyme, the introduction of the RNA primer into DNA causes replication to begin. The 3′ end of the primer is used as the starting point for the addition of nucleotides by the DNA polymerase at the leading strand. 

The process comes to a close at the lagging strand when the Okazaki fragments are synthesised from the lagging strand. DNA polymerase extracts the primer and inserts nucleotides into the gaps between the Okazaki fragments at the end of the process, just before the process is finished. The strands that have been produced so far have not yet been joined.

Once the primer between the Okazaki fragments has been removed, the role of DNA ligase is to fill in the gaps between the Okazaki fragments by forming phosphodiester links between the gaps. Ligation is the process of joining two strands together by eliminating the pyrophosphate from the triphosphate at the 5′ and 3′ ends of one strand, respectively. On the contrary, DNA replication is accomplished through ligation; however, this is not the case for blunt ended ds DNA or double stranded DNA.

The role of DNA ligase in recombinant DNA technology

In cloning experiments, DNA ligase I, II, or IV are used; however, eukaryotic DNA ligase is not used in these experiments. The phage T4 DNA ligase, on the other hand, is used to perform a variety of ligation procedures. When DNA ends are generated by restriction digestion, they can be classified as blunt ends or sticky ends. Ends are generated for a variety of molecular biological techniques, each with its own characteristics.

DNA Ligase – There Are Several Types

Among the various kinds of DNA Ligase are the following types:

A DNA ligase found in eukaryotes

Genetically modified yeasts produce DNA ligase I, which joins the nascent DNA on the lagging strand, particularly in the gaps between the Okazaki fragments.

DNA Ligase II – Because it does not have its own gene, DNA Ligase II is not considered to be true ligases. The eukaryotic DNA ligase II is derived from the DNA ligase III gene, which is found in bacteria. It primarily functions as a component of the DNA repair pathway.

DNA Ligase III – Plays a role in DNA repair, specifically nucleotide excision repair, and is found in the nucleus. This ligase can also be found in the mitochondrial DNA, which is interesting.

DNA Ligase IV – This enzyme joins double-stranded DNA together and is involved in the double-strand break repair pathway, specifically the non-homologous end-joining step.

Conclusion

DNA ligase activity necessitates the presence of a nucleotide cofactor and is characterised by a three-step reaction mechanism that involves covalent modification of both the ligase enzyme and the substrate DNA. In the course of conducting X-ray crystallographic analysis, a variety of DNA ligases at various stages of the ligation reaction were captured. These structures demonstrate the presence of unique covalent reaction intermediates, provide insights into the mechanism of the ligase reaction, and demonstrate the extensive conformational flexibility and dynamic nature of the modular structure.