DNA ligases indicate the basic class of enzymes necessary for all entities to sustain structural integrity of the genome. This enzyme connects two strands of DNA together as a result of association between phosphate group of one strand and deoxyribose group on the other strand. The DNA ligase is functional in joining the Okazaki fragments that take shape on the lagging strand while the DNA replicates. The DNA ligase is able to join two of the DNA fragments as a result of formation of a phosphodiester bond between them with the help of a molecule of energy.
Cellular phenomena of recombination, DNA replication and repair generate breaks in the phosphate backbone DNA structure. It compromises genome’s stability posing a threat to the loss of genetic content in addition to the introduction of the deleterious chromosomal mutations.
The catalytic activity of DNA ligases eventually repairs DNA breaks by forming phosphodiester bonds between adjacent nucleotides in duplex DNA. The DNA ligase activity necessitates a nucleotide cofactor following a unique 3-step reaction mechanism involving covalent modification. The process includes covalent alteration of the DNA substrate and ligase enzyme.
The DNA ligase can either be used for the introduction of genes of interest into the plasmid vectors or for the creation of fusion genes by uniting one gene into the other. This phenomenon is referred to as ligation. Ligation can be carried out on lengths of DNA having sticky or blunt ends following restriction digests. The DNA fragments in the blunt end ligation are directly joined by the DNA ligase.
DNA Ligase – Function
The importance of DNA ligases to maintain genomic integrity is immense. It does so by joining the breaks in the DNA’s phosphodiester backbone occurring while recombination and replication takes place in addition to the result of DNA damage and its repairing.
The primary role of DNA ligase is to ligate two strands of DNA, which could be single or double strands despite the fact that ligases are differently used for various purposes in vitro and vivo processes.
Role of DNA ligase in replication
4 different daughter single-stranded DNA molecules are produced in the process of replication from a single DNA duplex. To bring about complete replication, various enzymes perform a range of activities. Replication of DNA is initiated with the introduction of the RNA primer via primase enzyme. Primer’s 3′ end is used as the initial point to add nucleotides by the DNA polymerase at the leading strand. The process terminates at the lagging strand through the synthesis of the Okazaki fragments. When the process is about to complete, the primer is extracted out and loaded with nucleotides in the gaps between the Okazaki fragments by the DNA polymerase. However, the strands so produced are yet to be joined.
The role of DNA ligase here is to fill gaps by producing phosphodiester links between the gaps once the primer between the Okazaki fragments is removed. Though ligation 5′ end of a strand and 3′ end of another end are joined through the elimination of the pyrophosphate from the triphosphate. On the contrary, the DNA replication uses ligation, the same is not applicable on blunt ended ds DNA or double stranded DNA.
Role of DNA ligase in recombination DNA technology
DNA ligase I, II or IV are used in cloning experiments, but eukaryotic DNA ligase is not used. The phage T4 DNA ligase instead, is used to perform various ligation methods. Two types of DNA ends are generated by restriction digestion, they are blunt or sticky ends. For various molecular biological techniques, different ends are generated.
DNA Ligase – Types
The following are the different types of DNA Ligase:
Eukaryotic DNA Ligase
DNA Ligase I – It ligates the nascent DNA on the lagging strand particularly the gaps between the Okazaki fragments.
DNA Ligase II – These are not deemed to be true ligases as it does not possess its own gene. The eukaryotic DNA ligase II is generated from gene encoding DNA ligase III. Primarily, it participates in the DNA repair pathway.
DNA Ligase III – Participates in the DNA repair, particularly the nucleotide excision repair. This ligase is the one found in the mitochondrial DNA also.
DNA Ligase IV – It joins double-stranded DNA and does take part in double-strand break repair pathway, specially, the non-homologous end-joining.
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