DNA Ligase.pptx biotechnology notes for students

Ligase reactions

1. DNA LIGASE

2. ‘ Ligase ‘ came from the Latin word ‘ ligare ‘, which means  “to bind” . Ligase is the class of enzyme that brings about the binding or joining of two molecules. It is the class of enzyme that catalyzes the binding or joining of two macromolecules   by forming new bonds like C-O, C-N, and C-S. Ligase enzyme catalyzes the ligase reaction. About 50 ligase enzymes are known and are often referred to as  “molecular glue” . DNA ligase is an example. It catalyzes the DNA fragments’ binding by forming a phosphodiester bond between complementary ends of the DNA fragments. Thus, DNA ligase plays a critical role in repairing, replicating, and recombination of DNA. DNA ligase joins the DNA Molecule covalently by catalyzing the formation of phosphodiester bonds between adjacent nucleotides. Introduction

3. DNA ligases are the ligases that join or bind the two DNA fragments (also known as  Okazaki fragments ) by forming a phosphodiester bond. Enzymes that join the nucleic acids molecules together. They can join both DNA and RNA. Catalyse the formation of a phosphodiester bond between 5’ phosphate of one strand and 3’ hydroxyl of another. They are Mg2+ dependent. Enzymes that join the nucleic acids molecules together. They can join both DNA and RNA. Catalyse the formation of a phosphodiester bond between 5’ phosphate of one strand and 3’ hydroxyl of another. They are Mg2+ dependent.

4. Mechanism of Action DNA ligase binds the 3′ hydroxyl ends of one nucleotide (or acceptor) with the 5′ phosphate end of another (donor) by two covalent phosphodiester bonds in the following steps: In the first step, the DNA fragments or DNA segments or Okazaki fragments are reorganized with their active sites. The lysine residue of the active site of the Ligase gets adenylated with the addition of AMP. The release of pyrophosphate accompanies adenylation of Ligase. 3. Ligase transfer the AMP to the 5′ phosphate end of the donor with the formation of the pyrophosphate bond. 4. The 5′ phosphate end of the donor is linked to the 3′ hydroxyl end of the receptor with phosphodiester bond formation.

5. Types of DNA Ligases- Bacteriophage T4 E.Coli - encoded by the lig gene. uses energy gained by cleaving nicotinamide adenine dinucleotide (NAD) to create the phosphodiester bond. It does not ligate blunt-ended DNA except under conditions of molecular crowding with polyethylene glycol Taq - Its half-life is 48 hours at 65 °C and greater than 1 hour at 95 °C. This exceptional thermostability permits extremely high hybridization stringency and ligation specificity. T4 - most-commonly used in laboratory research. It can ligate either cohesive or blunt ends of DNA, oligonucleotides, as well as RNA and RNA-DNA hybrids, but not single-stranded nucleic acids. It can also ligate blunt-ended DNA with much greater efficiency than E. coli DNA ligase. DNA ligase I: ligates the nascent DNA of the lagging strand DNA ligase III: complexes with DNA repair protein, has been found to be present in mitochondria. DNA ligase II: appears to be used in repair. DNA ligase IV: It catalyzes the final step in the non-homologous end joining DNA double-strand break repair pathway .

6. Molecular bond involved Ligase subclass Example Carbon-Oxygen bonds (C-O) EC 6.1 Aminoacyl tRNA synthetase Carbon- Sulfur bonds (C-S) EC 6.2 Succinyl coenzyme A synthetase, Thiokinase Carbon-Nitrogen bonds (C-N) EC 6.3 Ubiquitin ligase, argininosuccinate synthetase Carbon-Carbon bonds (C-C) EC 6.4 Gamma-glutamyl carboxylase, Polyketide synthase Phosphoric ester bonds EC 6.5 DNA ligase Nitrogen-metal bonds EC 6.6 Chelatases Subclasses of Ligases

7. Ligase Examples Ubiquitin Ligases (C-N bond) Glutamate–cysteine ligase (C-N bond) Aminoacyl tRNA synthetase (C-O bond) Succinyl coenzyme A synthetase (C-S bond) Acetyl—CoA synthetase (C-S bond) Pyruvate carboxylase (C-C bond) Acetyl-CoA carboxylase (C-C bond) Propionyl-CoA carboxylase (C-C bond) Methylcrotonyl -CoA carboxylase (C-C bond) DNA ligase (Phosphoric ester bonds) Magnesium chelatase (Nitrogen-metal bonds) Cobalt chelatase (Nitrogen-metal bonds) DNA synthetase

8. Ligase Functions Joining two complementary macromolecules like nucleic acid. DNA ligase enzymes carry out the repairing, replication, and recombination of DNA. Ligases are one of the most widely used enzymes in the molecular biology laboratory. Ligases are used in recombinant DNA cloning to bind annealed fragments of restriction endonuclease. E. coli  DNA ligases are used for high-efficiency cloning of full-length DNA.  Commercially available thermostable Taq ligases are used in amplification reactions because of their thermostable properties. Human DNA ligase IV is required for V(D) J recombination, the process that generates diversity in immunoglobulin and T-cell receptor loci during immune system development.