Terminal deoxynucleotidyl transferase (TdT), also known as DNA nucleotidylexotransferase (DNTT) or terminal transferase, is a specialized DNA polymerase expressed in immature, pre-B, pre-T lymphoid cells, and acute lymphoblastic leukemia/lymphoma cells. TdT adds N-nucleotides to the V, D, and J exons of the TCR and BCR genes during antibody gene recombination, enabling the phenomenon of junctional diversity. In humans, terminal transferase is encoded by the DNTT gene. As a member of the X family of DNA polymerase enzymes, it works in conjunction with polymerase λ and polymerase μ, both of which belong to the same X family of polymerase enzymes. The diversity introduced by TdT has played an important role in the evolution of the vertebrate immune system, significantly increasing the variety of antigen receptors that a cell is equipped with to fight pathogens. Studies using TdT knockout mice have found drastic reductions (10-fold) in T-cell receptor (TCR) diversity compared with that of normal, or wild-type, systems. The greater diversity of TCRs that an organism is equipped with leads to greater resistance to infection. Although TdT was one of the first DNA polymerases identified in mammals in 1960, it remains one of the least understood of all DNA polymerases. In 2016–18, TdT was discovered to demonstrate in trans template dependant behaviour in addition to its more broadly known template independent behaviour
TdT is absent in fetal liver HSCs, significantly impairing junctional diversity in B-cells during the fetal period.
Function and regulation
Generally, TdT catalyses the addition of nucleotides to the 3' terminus of a DNA molecule. Unlike most DNA polymerases, it does not require a template. The preferred substrate of this enzyme is a 3'-overhang, but it can also add nucleotides to blunt or recessed 3' ends. Further, TdT is the only polymerase that is known to catalyze the synthesis of 2-15nt DNA polymers from free nucleotides in solution in vivo. In vitro, this behaviour catalyzes the general formation of DNA polymers without specific length. The 2-15nt DNA fragments produced in vivo are hypothesized to act in signaling pathways related to DNA repair and/or recombination machinery. Like many polymerases, TdT requires a divalent cation cofactor, however, TdT is unique in its ability to use a broader range of cations such as , , and . The rate of enzymatic activity depends on the available divalent cations and the nucleotide being added.
TdT is expressed mostly in the primary lymphoid organs, like the thymus and bone marrow. Regulation of its expression occurs via multiple pathways. These include protein-protein interactions, like those with TdIF1. TdIF1 is another protein that interacts with TdT to inhibit its function by masking the DNA binding region of the TdT polymerase. The regulation of TdT expression also exists at the transcriptional level, with regulation influenced by stage-specific factors, and occurs in a developmentally restrictive manner. Although expression is typically found to be in the primary lymphoid organs, recent work has suggested that stimulation via antigen can result in secondary TdT expression along with other enzymes needed for gene rearrangement outside of the thymus for T-cells. Patients with acute lymphoblastic leukemia greatly over-produce TdT. Cell lines derived from these patients served as one of the first sources of pure TdT and lead to the discovery that differences in activity exist between human and bovine isoforms.
Mechanism
Similar to many polymerases, the catalytic site of TdT has two divalent cations in its palm domain that assist in nucleotide binding, help lower the pKa of the 3'-OH group and ultimately facilitate the departure of the resultant pyrophosphate by-product.
Isoform Variation
Several isoforms of TdT have been observed in mice, bovines, and humans. To date, two variants have been identified in mice while three have been identified in humans.
The two splice variants identified in mice are named according to their respective lengths: TdTS consists of 509 amino acids while TdTL, the longer variant, consists of 529 amino acids. The differences between TdTS and TdTL occur outside regions that bind DNA and nucleotides. That the 20 amino acid difference affects enzymatic activity is controversial, with some arguing that TdTL's modifications bestow exonuclease activity while others argue that TdTL and TdTS have nearly identical in vitro activity. Additionally, TdTL reportedly can modulate the catalytic activity of TdTS in vivo through an unknown mechanism. It is suggested that this aids in the regulation of TdT's role in V(D)J recombination.