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DNA LIGASEEdit

DNA Ligase reaction

Ligase reaction

DNA ligase is an enzyme that catalyzes the repair of single-strand breaks by the formation of covalent phosphodiester bonds between adjacent 3’-hydroxyl (acceptor) and 5’-phosphoryl (donor)  in double-stranded DNA. It is an ATP dependent reaction in which adenylation (addition of AMP) of the 5' phosphate mediates the formation of a pyrophosphate bond which reacts to the 3' hydroxyl to create a phosphodiester bond. 

The enzyme system was first purified from T4 bacteriophage synthesized in Escherichia coli media.  

Discovery of DNA-LigaseEdit

Isolation

Weiss and Richardson were the first to characterize and purify DNA ligase from E. coli extract infected with T4 phage (Weiss and Richardson, 1967). It was first isolated in a six-steps chromatographic-fractionation process starting with the elimination of cell debris and addition of streptomycin, followed by several Diethylaminoethyl (DEAE)-cellulose column washes and a final phosphocellulose fractionation. The final extract contained 10% of the activity initially recorded in the E. coli media; along the process it was discovered that ATP and Mg++ were necessary to optimize the reaction. 

AssayEdit

Phosphomonoesters groups from T7 DNA were removed and replaced by p32-phosphomonoesters by incubation with ATP32 and polynucleotide kinase. The purpose of the assay was to measured the conversion of 5'  p32-phosphomonoesters in nicked DNA into a form which that remained acid-insoluble after transformation with ligase and subsecuent incubation with phosphatase. The incubation precipitate was tested through zonal sedimentation.

After incubation with Ligase and Phosphate, 95 percent of phosphomonoesters initially present in the DNA had become resistant to phosphatase, hence suggesting a loss of phosphomonoesters to p-diesters in the presence of ligase.  Further confirmation was achieve by attempting to hydrolyze the DNA product with E. Coli exononuclease I, which  cannot break double-stranded DNA. The experiment showed that 90 percent of the product was still intact after hydrolization. Finally sedimentation analysis showed that under Alkaline conditions single polynucleotide strands treated with ligase had higher molecular weight that non-treated DNA strands.

Ligase structural GeneEdit

  Right after Weiss and Richardson's paper, Fareed and Richardson published the characterization of Gene 30 as the structural gene for T4 phage-induced polynucleotide ligase. By using T4 conditional lethal mutants they were able to pinpoint Gene 30 by using single-defective-gene strains (am H39, am H39X, and am am E605) incapable of replicating in E. coli media due to a lack of ligase synthesis. In contrast, they also discovered two ligases more heat-labile than that induced by the wild-type phage.

Furthermore this experiment was a keystone to establish that T4 phage could synthesize its own DNA, suggesting that ligase is essential for replication since its absence results in phosphodiester interruptions, “abortively replicated” or sensitive nucleotide lysis.

Association of Ligase to Recombinant Molecules 

In 1964 Tomizawa and Anraku described the process of recombination in T4 phage by studying the formation of joint DNA molecules derived from different parental chromosomes. After the discovery of DNA ligase 1967, Anraku and Lehman (1969) were able to explain recombinant DNA by means of a ligase-mediated reaction that allowed the synthesis of phosphodiester bonds between joint DNA molecules into a single recombinant DNA strand.   

MethodsEdit

The experiment used three T4 phage conditional lethal mutants: am X5 and double mutant am EB6, defective in T4-induced DNA polymerase; and triple mutant am EB6-605, defective in 

Ligase

Joining enzyme activity following infection of E. coli with (a) T4 EB6 and (b) EB6-605. --.--.--, + ATP; -O--O-, +NAD. (Anraku and Lehman, 1969)

the T4-induced DNA polymerase and polynucleotide ligase. Cultures of E. coli were infected with p32 and BU labeled mutants. After incubation the cells were harvested, DNA extracted, and mixed with 3H-labeled T4 DNA (internal control). Samples were analyzed with alkaline CsCl density-gradient centrifugation and radioactivity was read by liquid-scintillation counting.    

ResultsEdit

  The experiment showed that infected E. coli with mutant defective in the T4 DNA polymerase lead to the generation of joint and recombinant DNA molecules; however, infection with mutants defective in both polymerase and ligase (am EB6-605) produced only joint-non-covalent molecules. This showed that active T4 ligase was necessary for the completition of recombinant DNA molecules from their precursors as well as the inability of the host enzyme to substitute for the phage-induced ligase. 

ReferencesEdit

Anraku, N and Lehman, I.R. 1969. Enzymatic Joining of Polynucleotides VII. Rof of the T4-induced Ligase in the formation of Recombinant Molecules. J.Mol.Bio. 46,467-479. 

Fareed, G.C. and Richardson, C.C. 1967. Enzymatic Breakage and Joining of Deoxyribonucleic Acid, II. The Structural Gene for Polynucleotic Ligase in Bacteriophage T4. Proc. N.A.S. 58, 665-672.

Tomizawa, J and Anraku, N. 1964. Molecular Mechanisms of Genetic Recombination in Bacteriophage II. Joining of Parental DNA Molecules of Phage T4. J.Mol.Bio. 8, 516.

Weiss, B and Richardson, CC. 1967. Enzymatic Breakage and Joining of Deoxyribonucleic Acid, I. Repair of single-strand breaks in DNA by an Enzyme System for Escherichia Coli infected with T4 Bacteriophage. Proc.N.A.S. 57, 1021-1028. 

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