Terminal Deoxynucleotidyltransferase

Views:
 
Category: Education
     
 

Presentation Description

No description available.

Comments

Presentation Transcript

Terminal Deoxynucleotidyltransferase:

Terminal Deoxynucleotidyltransferase By Arvind Kumar Mudgal

Significance of Polymerases:

Significance of Polymerases Maintenance vs variation An enzyme that is unique even in the context of low-fidelity polymerases, is responsible for the generation of the random genetic information that is essential for the efficacious function of the vertebrate.

Introduction:

Introduction It was noted that a major activity recoverable from calf thymus glands was not due to a polymerase that was able to copy a template, but to a polymerase that formed DNA polymers whose composition was determined by the type and composition of deoxynucleoside triphosphates provided in the reaction mixture

PowerPoint Presentation:

Among normal tissues terminal transferase is generally found only in mammalian bone marrow and thymus Very high levels of terminal transferase activity have been detected in lymphoblastic leukemic cells from patients with chronic myelogenous leukemia in blastic crisis and in lymphoid cells transformed by Abelson murine leukemic virus

PowerPoint Presentation:

Thus the enzyme represents a useful marker of neoplastic cells in certain human diseases

PowerPoint Presentation:

Terminal transferase has proven to be a valuable tool for the synthesis of model polydeoxynucleotides , and more recently for the construction of recombinant DNAs .

Activity 1:

Activity 1 Is terminal deoxynucleotidyl transferase a somatic mutagen in lymphocytes ?

Family X:

Family X The X family of DNA polymerases is a subdivision of a larger superfamily of nucleotidyl transferases . Members of this family can be found in Achaea, Bacteria, Eukaryota , and viruses

PowerPoint Presentation:

TdT is known to catalyze nontemplated , random nucleotide addition at the V(D)J junctions thereby increasing antigen receptor diversity.

Purification and Properties:

Purification and Properties Terminal transferase from calf thymus was first purified to homogeneity by Chang and Bollum Has a molecular weight of 32360 and can be dissociated into two polypeptide chains with molecular weights of 26,500 and 8,000

PowerPoint Presentation:

The amino acid composition has been determined for the homogeneous enzyme and each of its subunits ; a significant excess of acidic over basic amino acids was found . This finding is surprising since the enzyme has an isoelectric pH of 8.6 and behaves as a basic protein on electrophoresis and chromatography

PowerPoint Presentation:

The pH optimum is approximately 7.2 and maximum activity is demonstrated in cacodylate buffer . The enzyme has a turnover number of about

PowerPoint Presentation:

The enzyme is stable at low temperatures and at pH 4.5, but is not stable at temperatures above 40" or to protein denaturants such as urea, sodium dodecyl sulfate (SDS) and organic solvents

PowerPoint Presentation:

A new terminal transferase has been isolated from calf thymus tissue by Johnson and Morgan that is a single polypeptide with a molecular weight of 79,000. Diebel and Coleman purified terminal transferase that has a molecular weight of 63,000 and consists of a single polypeptide Methods could be the reason

The Reactions Catalyzed :

The Reactions Catalyzed Oligodeoxynucleotide initiator d( pX ) is bound by the enzyme ( E); this is followed by repetitive grafting of mononucleotide units from a deoxynucleoside triphosphate to the terminal 3'-hydroxyl of the initiator and the release of inorganic pyrophosphate The reaction is irreversible,

dNTPs:

dNTPs Any of the common deoxynucleoside triphosphates can be polymerized by terminal transferase although the values differ for each

PowerPoint Presentation:

To catalyze template-independent polymerase activity, TdT requires a primer at least as large as a trinucleotide , a free 3-OH moiety for extension of that primer, and a free primer 5-phosphate. pdApdApdA is an active primer but not dApdApdA

PowerPoint Presentation:

Deoxynucleoside triphosphates with substituted amino groups are substrates for the terminal transferase , but the rate of polymer synthesis is much lower and the ultimate polymer length less than with the nonsubstituted bases.

INITIATOR:

INITIATOR At 35” the enzyme has an absolute requirement for an oligodeoxynucleotide containing at least three phosphate groups, d( pXpXpX ), and a free 3 ’- hydroxyl The kinetic analysis of polymerization of dATP using a variety of oligodeoxynucleotides of different base composition and length as initiators have been reported

PowerPoint Presentation:

The initiator chain length is increased to 5-7 nucleotides the polymerization rate also increases. Conditions that enhance initiator incorporation are lower temperature , high buffer concentration, and high enzyme specific activity

METAL IONS:

METAL IONS Several divalent metals can substitute for magnesium in activating the enzymatic polymerization of deoxynucleotides The extension of chains with dCTP or dTTP low concentrations of cobalt are best. Human terminal transferase from leukemic cells show maximum activity when manganese is used

PowerPoint Presentation:

In addition to divalent metal ions, rates of polymerization of deoxynucleoside triphosphate and initiator utilization are affected by specific buffer ions and ionic strength.

PowerPoint Presentation:

In 40 mM potassium phosphate buffer, dATP polymerization begins immediately. Increasing the phosphate buffer concentration above 40 mM decreases polymerization, whereas increasing cacodylate to 200 mM doubles the polymerization rate measured at 40 mM cacodylate

KINETICS:

KINETICS V max and K m have been reported for the polymerization of the four deoxynucleoside triphosphates and the kinetic constants for dATP polymerization using a series of oligodeoxynucleotide initiators differing in base composition and chain length. Affinity of the terminal transferase for initiator is in the order of I > G > A > T > C

PowerPoint Presentation:

A maximum in for dATP polymerization with oligodeoxynucleotides of different chain length is observed when the length of the initiator is five to seven nucleotides. For each mole of deoxynucleoside triphosphate incorporated into the polymer one mole of pyrophosphate is formed

PowerPoint Presentation:

The equilibrium constant for the forward reaction has been estimated to be polymerization on an oligonucleotide initiator. If m is the mean number of added nucleotide units that equals the average mole ratio of deoxynucleoside triphosphate reacting with initiator molecules, and x is the number of units added to an individual chain, the mole fraction of polymer molecules with x units added should fit the Poisson expression

THE MECHANISM OF THE REACTION:

THE MECHANISM OF THE REACTION Experiments by Chang and Bollum on the inhibition of terminal transferase by metal ligands and length of initiator required for synthesis suggest that at least two sites are involved in binding initiator molecules to terminal transferase .

PowerPoint Presentation:

In their model the enzyme-metal ion complex binds to the 3’-hydroxyl and the phosphoryl group of the third nucleotide from the 3’ terminus. After binding and the addition of another nucleotide, the enzyme-bound metal dissociates from the initiator and again binds to the 3’-hydroxyl and phosphoryl group of the third nucleotide.

Practical Applications:

Practical Applications Terminal transferase has been used to synthesize a variety of model poiydeoxynucleotides , and to modify the ends of plasmids and genes preparatory to their union into recombinant molecules that can be replicated by a bacterium

PowerPoint Presentation:

In studies of the role of DNA structure in genetic regulations, Wells and his co-workers synthesized a number of duplex block polymers, such as the synthesis utilized a series of enzymes including pancreatic DNase , terminal transferase , and T4 DNA polymerase

PowerPoint Presentation:

Polymers with distinctively labeled termini are readily prepared with terminal transferase . Rougeon et al. used terminal transferase to add poly( dT ) to the 3‘ end of cDNA , and oligo ( dA ) primer was used to prime the second-strand synthesis with DNA polymerase

Immune System and Terminal Deoxynucleotidyl Transferase:

Immune System and Terminal Deoxynucleotidyl Transferase The main goal of the vertebrate adaptive immune system is to defend the organism from harmful foreign agents or“antigens ” These proteins are divided into two major classes: the immunoglobulins ( Ig’s and the T cell antigen receptors (TCRs,

PowerPoint Presentation:

There are approximately 10 14 unique Ig’s and around 10 18 unique TCRs. Given that there are somewhere between 25000 genes in the entire human genome, it would seem impossible that each of these antigen-binding proteins could be encoded by a unique gene

PowerPoint Presentation:

Recombination signal sequences

PowerPoint Presentation:

Recombinase activating gene

VDJ gene recombination:

VDJ gene recombination

authorStream Live Help