Transferase

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Almost all DNA polymerases that have been characterized use DNA or RNA as templates to guide each integration event, specifically catalyzing the integration of mononucleotides into the growth primer. However, there is a unique DNA polymerase called terminal deoxynucleotidyl transferase (TdT), which uses only single-stranded DNA as a substrate. TdT has the unique ability to create genomic material de novo, making it one of the most attractive DNA polymerases for scientists. Although TdT was identified very early in mammals, it is still one of the least understood enzymes that catalyze DNA synthesis.

Fig 1. The catalytic process of TdT (Deshpande, S.; et al. 2019)

proteolysed In the past decades, different mRNA splicing variants of this enzyme have been identified and extensively studied in bovines, mice, and humans. So far, two TdT splice variants have been observed in mice, while bovines and humans have three respectively.

In mice, the two identified splice variants of TdT are TdTS (a short form consisting of 509 amino acids) and TdTL (a long form consisting of 529 residues). TDTS catalyzes the unbiased and non-templated addition of nucleotides to the antigen receptor gene, while TdTL has 3'→5' exonuclease activity. But others believe that TdTL does not have exonuclease enzyme activity, and shows almost the same enzymatic activity as TDTS in vitro. Although the mechanism of this regulation is not yet clear, these data indicate that the function of the two TdT murine isoforms is to balance the diversity of the antigen repertoire to maintain the integrity of the antigen receptor variable region. The human (h) and bovines (b) TdT isoforms are more complicated because each has three alternative splice variants named TdTS (short), TdTL1 (long) and TdTL2 (long).

Source and purification of TdT

TdT was first purified from calf thymus glands in 1971. Initially, the isolated protein was considered to be a proteolyzed form, which contained two peptides that were originally identified as a heterodimer of α and β subunits. However, subsequent studies confirmed that TdT is a monomeric protein with a molecular weight of 58,000 to 60,000 daltons. However, since it is easily hydrolyzed, it becomes very difficult to obtain an intact and highly active enzyme. Large amounts of TdT can be purified from cultured cell lines propagated from patients with acute lymphoblastic leukemia. However, this method is too expensive for mass production. At present, TdT is mainly purified to a homogeneous form by column chromatography, which is achieved by attaching a hexahistidine tag to the N-terminal that does not affect its enzymatic activity. Recombinant human TdT has been successfully overexpressed in the baculovirus expression system and purified to homogeneity in one step from Trichoplusia ni larvae using a monoclonal antibody affinity column. This advancement played an indispensable role in generating a large number of proteins required for TdT structural characterization.

Fig 2. Structures of mouse TdT (Loc’h, J.; et al. 2016)

Metal utilization

It is well known that all DNA polymerases require the presence of divalent metal ions to catalyze the phosphoryl transfer reaction associated with nucleotide incorporation. In this mechanism, an aspartate residue near the deoxyribose sugar of the incoming dNTP serves as the general base to abstract the proton from the 3′-OH to generate a more reactive nucleophile. Similarly, terminal transferases, like all DNA polymerases, also require divalent metal ions for catalysis. Unlike other enzymes, TdT is unique in its ability to use multiple divalent cations such as Co²⁺, Mn²⁺, Zn²⁺ and Mg²⁺. Generally, in the presence of divalent metal ions, the elongation rate of primer p(dA)n (where n is the chain length from 4 to 50) with dATP is arranged in the following order: Mg²⁺> Zn²⁺> Co²⁺> Mn²⁺. In addition, each metal ion has a different effect on the kinetics of nucleotide incorporation. For example, Mg²⁺ promotes the preferential utilization of dATP and dGTP, while Co²⁺ improves the catalytic polymerization efficiency of pyrimidine, dTTP, and dCTP. 

Biomedical applications of TdT

A large amount of evidence shows that changes in the activity/expression level of TdT play an important role in the occurrence and development of cancer and some chemical reactions. Most B- and T-cell acute lymphocytic leukemias (ALL) patients show different levels of TdT expression and multiple TdT subtypes in their blast cells. In addition, higher levels of TdT activity are closely related to poor prognosis and ultimately lead to shortened survival time. These findings have driven the development of TdT selective inhibitors, which can be used as chemotherapeutics for these related leukemias. For example, the nucleoside analog cordycepin (3'-deoxyadenosine) is cytotoxic to terminal transferase-positive leukemia cells, especially when used in combination with adenosine deaminase inhibitor deoxycoformycin. However, this analog has not been widely used due to its side effects. Recent studies have reported that certain aryldiketo hexenoic acids can inhibit the catalytic activity of TdT and polλ without acting as a chain terminator, and show strong cytotoxicity to TdT-positive leukemia cell lines (Molt4).

TdT can be used as a biocatalyst to label the 3'-termini of synthetic oligonucleotides with radionucleotides or different fluorescent probes, and then these labeled primers can be annealed to the complementary strand and used as a radioactive substrate for monitoring the activities of enzymes involved in nucleic acid metabolism include restriction endonucleases, template-dependent DNA polymerases and DNA glycosylases.