DNA Ligase

CONTENT Introduction Mechanism of DNA ligase Unit of enzyme activity Types of DNA Ligase: 1)Bacteriophage T4 DNA ligase 2)E.coli DNA Ligase 3)Taq DNA Ligase 4)T4 DNA Ligase Applications Reference

Introduction DNA ligase catalyses the formation of phosphodiester bond between the 5’-phosphate of one strand of DNA or RNA and the 3’-hydroxyl of another. This enzyme is used to covalently link or ligate fragments of DNA together The DNA ligase used in molecular cloning differ in their abilities to ligate noncanonical substrate,such as blunt ended duplexeDNA:RNA hybrid or ssDNAs.

T7 DNA ligase

Mechanism of DNA Ligase The mechanism of DNA ligase is to form two covalent phosphodiester bonds between 3' hydroxyl ends of one nucleotide, ("acceptor") with the 5' phosphate end of another ("donor"). ATP is required for the ligase reaction, which proceeds in three steps: adenylation (addition of AMP) of a residue in the active center of the enzyme, pyrophosphate is released. ATP → AMP + Ppi PPi + H2O → 2 Pi (2) transfer of the AMP to the 5' phosphate so-called donor, formation of a pyrophosphate bond; (3) formation of a phosphodiester bond between the 5' phosphate of the donor and the 3' hydroxyl of the acceptor.

The reaction occurs in three stages in all DNA ligases: 1. Formation of a covalent enzyme-AMP intermediate linked to a lysine side-chain in the enzyme. 2. Transfer of the AMP nucleotide to the 5’ phosphate of the nicked DNA strand. 3. Attack on the AMP-DNA bond by the 3’-OH of the nicked DNA sealing the phosphate backbone and resealing AMP. The following figure illustrates the three reaction stages:

Depending up on the source,the enzyme requires either ATP or NAD+ as cofactors

Unit of Enzyme Activity For all ligase, one Weiss unit is the amount of Enzyme required to convert 1 n mol of radiolabeled phosphate from pyrophosphate into absorbable material in 20 min at 370 c under standard assay condition. one Weiss unit equals about 67 cohesive end ligation unit.

Bacteriophage T4 DNA Ligase (ATP) The most widely used DNA ligase is derived from the T4 bacteriophage. It is a monomeric polypeptide MW 68KDa is encoded by bacteriophage gene30. It has broader specificity and repairs single stranded duplex DNA, RNA or DNA:RNA hybrids. .

Application: 1 ligation of cohesive ends: 2 ligation of blunt ended termini: this reaction is much slower than ligation of sticky ends and the ligation is improved by addition of monovalent cation and low concentration of PEG .3 Ligation of synthetic linkers or adapter

E.Coli DNA ligase It is derived from E.coli cell and requires NAD+ as cofacter. It is a monomeric enzyme of MW 74KDa which catalyzes the formation of the phosphodiester bond in duplex DNA containing cohesive ends. This enzyme has narrower substrate specificity, making it a useful tool in specific application.

Application: 1) ligation of cohesive ends:E.coli DNA ligase is used to catalyze sticky end ligation. 2)cloning of full length cDNA: E.coli DNA ligase has been employed in a procedure for high efficiency cloning of full length cDNA

Taq DNA ligase [NAD+ ] The gene encoding thermostable ligases have been identified from several thermophilic bacteria. Several of this ligase have been cloned and expressed to high levels in E.coli It is uses in the detection of mutation as thermostable DNA ligase retain their activities after exposure to higher temp for multiple rounds it is uses in DNA amplificaton reaction to detect mutation in mammalian DNA.

T4 RNA Ligase T4 RNA ligase is the only phage RNA ligase that has been extensively characterized and used in genetic engineering. This enzyme catalyzed the phosphodiester bond formation of RNA molecule with hydrolysis of ATP to PPI It is monomeric enzyme with 373 deduced amino acid residues is a product of the T4 gene 63

. Mammalian ligases . DNA ligase I: ligates the nascent DNA of the lagging strand after the DNA polymerase I has removed the RNA primer from the Okazaki fragments. DNA ligase II: alternatively spliced form of DNA ligase III found in non-dividing cells. DNA ligase III: complexes with DNA repair protein XRCC1 to aid in sealing base excision mutations and recombinant fragments. DNA ligase IV:. It catalyzes the final step in the non-homologous end joining DNA double-strand break repair pathway. It is also required for V(D)J recombination, the process which generates diversity in immunoglobulin and T-cell receptor loci during immune system development. DNA repair protein XRCC1 also known as X-ray repair cross-complementing protein 1 is a protein that in humans is encoded by the XRCC1 gene. XRCC1 is involved in DNA repair where it complexes with DNA ligase III. base excision repair (BER) is a cellular mechanism that repairs damaged DNA throughout the cell cycle. It is responsible primarily for removing small, non-helix-distorting base lesions from the genome. The related nucleotide excision repair pathway repairs bulky helix-distorting lesions. BER is important for removing damaged bases that could otherwise cause mutations by mispairing or lead to breaks in DNA during replication. V(D)J recombination, less commonly known as somatic recombination, is the unique mechanism of genetic recombination that occurs only in developing lymphocytes during the early stages of T and B cell maturation. The process results in the highly diverse repertoire of antibodies/immunoglobulins (Igs) and T cell receptors (TCRs) found on B cells and T cells, respectively.

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