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ligase
LIGASE
JOINING OF NUCLEIC ACID EITHER DNA OR RNA- THROUGH PHOSPHODIESTER BOND. WIDESPREAD AND IDENTIFIED IN A RANGE OF ORGANISM. TWO TYPES
DNA LIGASE RNA LIGASE
DNA LIGASE
Dna ligase is an important cellular enzyme, as its function is to repair broken phosphodiester bonds that may occur at random or as a consequence of DNA replication or recombination or repairing. The first DNA ligase was purified and characterized in 1967
TWO
CLASSES
OF
DNA
THE FIRST USES NAD+ AS A COFACTOR AND ONLY FOUND IN BACTERIA. THE SECOND USES ATP AS A COFACTOR AND FOUND IN EUKARYOTES, VIRUSES AND BACTERIOPHAGES THE SMALLEST KNOWN ATP-DEPENDENT DNA LIGASE IS THE ONE FROM THE BACTERIOPHAGE T7 (MOLECULAR MASS 41 KDA).
AN ENZYME AMP COMPLEX BINDS TO A NICK BEARING 3 OH AND 5 P GROUPS. THE AMP REACTS WITH THE PHOSPHATE GROUP. ATTACK BY THE 3 OH GROUP ON THIS MOIETY GENERATES A NEW PHOSPHODIESTER BOND, WHICH SEALS THE NICK
b) Hydrogen bonding between complementary bases causes the molecules, transiently, to stick together. DNA ligase (indicated by gray shading) catalyzes the formation of a phosphodiester bond between the 5 phosphate on one molecule and the 3 hydroxyl on the other. c) The two molecules are now covalently linked by the top strand. The nick in the bottom strand may also be sealed by DNA ligase, or may be repaired by the host bacterium.
coRI, the bases making up the EcoRI restriction site are indicated in blue.
This function is crucial to the success of many experiments, and dna ligase is therefore a key enzyme in genetic engineering. The two most intensively studied and widely used dna ligases are E. coli dna ligase and T4 dna ligase. The enzyme used most often in experiments is t4 dna ligase, which is purified from e. Coli cells infected with bacteriophage t4.
T4 DNA LIGASE
Monomeric enzyme Aminoacids- 487 Obtained from t4 bacteriophage infected e coli. Encoded by gene 30 of t4 bacteriophage. Molecular weight77000(determined by sedimentation equilibrium)
SUBSTRATE
Cohesive termini Blunt ends DNA-RNA hybrids RNA-RNA hybrids ( 5 phosphate and 3 Hydroxyl) Rate of Blunt end ligation by T4 ligase not linearly depend upon enzyme concentration and works efficiently only in high concentration of DNA and enzyme
Condensing agent such as peg, ficoll and hexamminecobalt chloride accelerates the blunt end ligation by a factor of 1000 and permit ligation at lower enzyme, ATP and DNA concentration. Blunt end ligation is inhibited by high concentration of na(>50mm) and phosphate(>25mm)
COFACTORS
Required for forming a covalent amp-enzyme intermediate. T4 DNA ligase ATP It will also utilize dAtp(at 0.5% of the rate) which acts as a competitive inhibitor with ATP
TEMPERATURE
Very sensitive to the temp. Depend upon length of the joining fragment and its Tm. It has an optimum temp for ligating cohesive end of 4 degree C For sealing nick-37 degree c Blunt end ligation-25 degree c for 16 mers or longer Inactivated by heating at 65C for 10 minutes or 70C for 5 minutes
Higher level (0.2 M) of ammonium , sodium, potassium, cesium, and lithium ion inhibits completely. Unaffected by low concentration of ammonium ion Blunt end ligation is inhibited by 25 mM phosphate and 50 mM sodium ion. Polyamine such as spermine and spermidine also inhibit but can be overcome by increasing the DNA concentration.
Requires divalent cation for activity T4 ligase has a magnesium optimum of 10 mm whereas mn2+ is only 25% as efficient . In joining DNA:RNA hybrids Mn 2+ is twice as effective as Mg2+ Ph 40%- 6.9 65%-8.3 Optimum- 7.5 to 8.0
Temperature-
Cohesive end- 10 to 15
COMPARISON
APPLICATION
Cloning of restriction enzyme generated DNA fragments Cloning of PCR products Joining of double-stranded oligonucleotide linkers or adaptors to DNA
Site-directed mutagenesis Amplified fragment length polymorphism (AFLP) Ligase-mediated RNA detection Nick repair in duplex DNA, RNA or DNA/RNA hybrids Self-circularization of linear DNA.
Source-https://www.neb.com/products/dnamodifying-enzymes-and-cloningtechnologies/dna-ligases/dna-ligases
T3 DNA Ligase
Sticky ends, blunt ends, and nick sealing can all be efficiently catalyzed by T3 DNA Ligase . As with T4 DNA Ligase, blunt-end ligation is enhanced by the addition of PEG 6000 to the reaction. T3 DNA Ligase exhibits a higher tolerance (2-fold) for NaCl in the reaction compared to T4 DNA Ligase, making the enzyme a versatile choice for in vitro molecular biology protocols requiring DNA ligase activity.
ElectroLigase
ElectroLigase is specifically formulated for robust ligation of all types of DNA ends (blunt-, sticky-, T/A) and is directly compatible, without desalting or purification, with electrocompetent cells used for transformation by electroporation.
RNA LIGASE
T4 RNA Ligase catalyzes the ATPdependent intra- and intermolecular formation of phosphodiester bonds between 5'-phosphate and 3'hydroxyl termini of oligonucleotides, single-stranded RNA and DNA. This enzyme is found in E coli after infection with T- seven phage. In vivo role is unclear
Monomeric enzyme Mol. Mass- 48000 (determined by sedimentation equilibrium) Nucleic acid substrate
Cofactor
Single stranded RNA It can also act on variety of single or double stranded RNA or DNA molecule It acts on very small pieces of RNA (upper limit is 40 mers) ATP Magnesium ions are required
Application
RNA 3'-end labeling with cytidine 3',5'-bis [alpha-32P] phosphate Joining RNA to RNA Synthesis of oligoribonucleotides and oligodeoxyribonucleotides Specific modifications of tRNAs Oligodeoxyribonucleotide ligation to singlestranded cDNAs for 5' RACE (Rapid Amplification of cDNA Ends) Site-specific generation of composite primers for PCR
Inactivation
Inactivated by heating at 70C for 10min. Inhibitors: metal chelators, SH group-modifying reagents (8) 43.6kDa monomer
Quality Control
The absence of ribonucleases, exodeoxyribonucleases, endodeoxyribonucleases, and phosphatases confirmed by appropriate quality tests.
Source
RNA Ligases Thermostable 5 AppDNA/RNA Ligase 5 DNA Adenylation Kit T4 RNA Ligase 1 (ssRNA Ligase) T4 RNA Ligase 2 (dsRNA Ligase) T4 RNA Ligase 2, truncated T4 RNA Ligase 2, truncated K227Q T4 RNA Ligase 2, truncated KQ T4 RNA Ligase Reaction Buffer
END of PART 1
INTRODUCTION
TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE (TDT) IS A TEMPLATE INDEPENDENT DNA POLYMERASE Add dNTPS to the 3-OH of oligodeoxyribonucleotides and single and double strand of DNA.
STRUCTURE
Monomeric Mol. Wt.- 60000 Da AMINO ACIDS- 508 to529(depending UPON SOURCE) A high degree of sequence homology(>80%)in tdt between different species
REACTION BUFFER
ACTIVITY IS STRONGLY INHIBITED BY THE AMMONIUM ION AS WELL AS CHLORIDE, IODIDE AND PHOSPHATE ANIONS. POTASSIUM OR SODIUM CACODYLATE(dimethyl arsenic acid) BUFFERS ARE PREFERRED- SHOWN TO BE OPTIMAL FOR POLYPURINE AND POLYPYRIMIDINE SYNTHESIS
DIVALENT CATION
Polymerization requires presence of divalent cations. Order of efficiency for damp addition to oligonucleotide is as following Mg>Zn>Co>Mn (all are divalent cation) FOR DGTP- MAGMESIUM ION FOR PYRIMIDINE- COBALT ION
TdT binds its substrate with high Km value of 100 micro M FOR dATP ; dGTP , 500 micro M for dTTP and dCTP ; 1 micro M for oligonucleotide primer and upto 1 mM for homopolymer primer ends.
Substrate concentration should be higher for optimal activity of TdT.. Higher concentration can be achieved by working with small volumes. The no of nucleotide added is determined by the ratio of mol dNTPs : Mol 3- OH Termini in the mixture.
Inactivation
Inactivated by heating at 70C for 10 min or by addition of EDTA. Inhibitors: metal chelators, ammonium, chloride, iodide, phosphate ions
Inhibition
Quality Control
The absence of endo-, exodeoxyribonucleases, phosphatases and ribonucleases confirmed by appropriate quality tests.
Source
E.coli cells carrying a cloned gene encoding calf thymus terminal deoxynucleotidyl transferase.
APPLICATION
Addition of homopolymeric tails to plasmid DNA and to cDNA Double- or single-stranded DNA 3-termini labeling with radioactively labeled or nonradioactively labeled nucleotides Addition of single nucleotides to the 3 ends of DNA for in vitro mutagenesis Production of synthetic homoand heteropolymers RACE (Rapid Amplification of cDNA Ends) Addition of vNTPs, ddNTPs and cordycepin triphosphate for chain termination in controlled manner.
Contn.
Improved method for cDNA cloning. The first strand is tailed with oligo(dC) allowing the second strand to be initiated using an oligo(dG) primer
With RNA as template TDT shows variable performance which strongly depends upon the tertiary structure of acceptor RNA 3'-end and the nature of nucleotide. Generally, it is lower than using dna as a template.
(www.neb.com/products/m0315-terminal-transferase)
Terminal Transferase
Terminal transferase (TdT) is a template independent polymerase that catalyzes the addition of deoxynucleotides to the 3' hydroxyl terminus of DNA molecules. Protruding, recessed or blunt-ended double or single-stranded DNA molecules serve as a substrate for TdT. The 58.3 kDa enzyme does not have 5' or 3' exonuclease activity. The addition of Co2+ in the reacton makes tailing more efficient. Highlights
Isolated from a recombinant source Labeling of the 3' ends of DNA with modified nucleotides (e.g., ddNTP, DIG-dUTP) Supplied with 10X Reaction Buffer and 2.5mM CoCl2
Applications
Transferase gene from calf thymus. Reagents Supplied The following reagents are supplied with this product: CoCl2 ............10X Terminal Transferase Reaction Buffer................10X Addition of homopolymer tails to the 3' ends of DNA Labeling the 3' ends of DNA with modified nucleotides (e.g., ddNTP, DIG-dUTP) TUNEL assay (in situ localization of apoptosis) TdT dependent PCR
END OF PART- 2
INTRODUCTION
Ligation efficiency depends on the DNA ends in the reaction
Complementary sticky ends Ligation is efficient annealing of complementary overhangs brings 5P and 3OH into close proximity Blunt ends Ligation is inefficient need high concentrations of ligase and DNA molecular crowding reagents (like PEG 8000) improve intermolecular ligation, then dilute to promote intramolecular ligation
Cloning strategy
Terminal transferase to add polynucleotide tails to foreign DNA and vector DNA Cloning foreign DNA by adding linkers Cloning foreign DNA by adding adaptors
LINKER
Linkers are the chemically synthesized double stranded DNA oligonucleotides containing on it one or more restriction sites for cleavage by restriction enzymes, e.g. Eco RI, Hind III, Bam HI, etc. Linkers are ligated to blunt end DNA by using T4 DNA ligase. Both the vector and DNA are treated with restriction enzyme to develop sticky ends. The staggered cuts i.e. sticky ends are then ligated with T4 DNA ligase with very high efficiency to the termini of the vector and recombinant plasmid DNA molecules are produced.
EcoRI linker:
Add 2,4,6 "extra" base pairs in addition to the restriction site. This changes reading frame. ATG ATG ATG ATG if introduced between middle pair: (Met-Met-MetMet)
=ATG ATG CGG AAT TCC GAT GAT G 10-mer (Met-Met-Arg-AsnSer-Asp-Asp) =ATG ATG CCG GAA TTC CGG ATG ATG 12-mer (Met-Met-ProGlu-Phe-Arg-Met-Met).
end)
LIMITATIONS OF LINKER
It may be the case that the restriction enzyme used to generate the cohesive ends in the linker will also cut the foreign DNA at internal sites.
CHOOSE ANOTHER RESTRICTION ENZYME
But there may not be a suitable choice if the foreign DNA is large and has sites for several restriction enzymes.
Methylate internal restriction sites with the Appropriate modification methylase for example EcoRI methylase.
ADAPTER
Adapters are also the chemically synthesized partialy double stranded DNA oligonucleotides, with a blunt end at one side, and a cohesive end at the other side which contains a recognition site for a restriction enzyme.
Using T4 DNA ligase, adapters can be added to a blunt-ended DNA fragment
But unlike linkers, an adaptor is synthesized so that it already has one sticky end.
The idea is of course to ligate the blunt end of the adaptor to the blunt ends of the DNA fragment, to produce a new molecule with sticky ends.
This may appear to be a simple method but in practice a new problem arises; The sticky ends of individual adaptor molecules could base pair with each other to form dimers, so that the new DNA molecule is still blunt ended. The sticky ends could be recreated by digestion with a restriction endonuclease, but that would defeat the purpose of using adaptors in the first place. The answer to the problem lies in the precise chemical structure of the ends of the adaptor molecule
Adaptors and the potential problem with their use. (a) A typical adaptor. (b) Two adaptors could ligate to one another to produce a molecule similar to a linker (c) after ligation of adaptors a blunt-ended molecule is still bluntended and the restriction step is still needed
Normally the two ends of a polynucleotide strand are chemically distinct, a fact that is clear from a careful examination of the polymeric structure of DNA. One end, referred to as the 5' terminus, carries a phosphate group (5'-P); the other, the 3 terminus, has a hydroxyl group (3'-OH). In the double helix the two strands are antiparallel, so each end of a double-stranded molecule consists of one 5'-P terminus and one 3'-OH terminus.
Ligation normally takes place between the 5'-P and 3'-OH ends. Adaptor molecules are synthesized so that the blunt end is the same as natural DNA, but the sticky end is different. The 3'- OH terminus of the sticky end is the same as usual, but the 5'P terminus is modified; it lacks the phosphate group (removed by Alkaline Phosphates treatment), and is in fact a 5'-OH terminus. DNA ligase is unable to form a phosphodiester bridge between 5'-OH and 3'-OH ends.
The result is that, although base pairing is always occurring between the sticky ends of adaptor molecules, the association is never stabilized by ligation. Adaptors can therefore be ligated to a DNA molecule but not to themselves. After the adaptors have been attached, the abnormal 5'-OH terminus is converted to the natural 5'-P form by treatment with the enzyme polynucleotide kinase, producing a sticky-ended fragment that can be inserted into an appropriate vector.
REFERENCE
Primrose S.B. & Twyman R.M. 2006. Principles of gene manipulation and genomics. 7th ed. Blackwell publishing, Malden. Brown, T.A. (Terence A.).2010.Gene cloning and DNA analysis : an introduction / T.A. Brown.6th ed. John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex UK Nicholl,Desmond S. T. 2008. An Introduction to Genetic Engineering,Third Edition, CAMBRIDGE UNIVERSITY PRESS Cambridge, New York, Melbourne, Madrid, Cape Town, Singapore, So Paulo https://www.neb.com