Recombinant hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) was reported to possess terminal transferase recombinants activity, the ability to add unmolded nucleotides to the 3′ end of viral RNAs. However, this TNTase was later purported to be a cellular enzyme that copurifies with the HCV RdRp. In this report, we present evidence that TNTase activity is an inherent function of highly purified HCV and bovine viral diarrhoea virus RdRps from prokaryotic and eukaryotic cells.
A change of the highly conserved GDD catalytic motif in HCV RdRp to GAA abolished both RNA synthesis and TNTase activity. Furthermore, the nucleotides added through this TNTase activity are strongly influenced by the sequence near the 3′ end of the viral template RNA, perhaps explaining the discrepant observations above between RdRp preparations. Finally, RdRp TNTase activity was shown to restore the ability to direct the initiation of RNA synthesis in vitro on an initiation-defective RNA substrate, implying that this activity maintains the integrity of the ends of the viral genome.
Recombinant terminal deoxynucleotidyl transferase (TDT) is a DNA polymerase that catalyzes the addition of deoxynucleotides to the 3fi hydroxyl terminus of DNA. A TdT Technical Bulletin is available.
Applications: Homopolymer of vector tails and insert for cloning. Labelling of oligonucleotides with biotin (1,2), 32P- or 35S-label (3), or in apoptosis (TUNEL) (4,5).
Source: Purified from TdT calf thymus E. coli clone.
Performance and quality tests: endonuclease, exodeoxyribonuclease 3´ and 5´ and levels of incorporation tested.
Unit definition: One unit incorporates 1 nmol of dATP into acid precipitable material in 1 hour at 37°C, using d(pA)50 as a primer.
Hazard Warning: Toxic; potassium cacodylate contained in the reaction buffer. It also contains cobalt chloride, a highly toxic chemical.
Unit reaction conditions: 0.2 M potassium cacodylate (pH 7.2), 10 mM MgO4 C4 H6, 1 mM 2-mercaptoethanol, 0.5 mg/mL BSA, 100 µM d(pA)50, 1 mM [3H]dATP and enzyme in 0.15 ml for 1 h at 37°C.
For research use only. It should not be used in diagnostic procedures.
Kind of product: DTT
Shipping condition: Approved for shipment on wet or dry ice
Enzyme: Terminal deoxynucleotidyl transferase
Compatible buffer: 5X buffer, reaction buffer
Content and storage
Recombinant terminal deoxynucleotidyl transferase (rTdT) is supplied with a vial of 5X buffer [500 mM potassium cacodylate (pH 7.2), 10 mM CoCl2, 1 mM DTT]. Store at -20°C.