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 Jexons of the TCR and BCR genes during antibody gene recombination, enabling the phenomenon of junctional diversity. In humans, terminal transferase is encoded by the DNTTgene.[1][2] 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.[3][4]
TdT is absent in fetal liver HSCs, significantly impairing junctional diversity in B-cells during the fetal period.[5]
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. Cobalt is a necessary cofactor, however the enzyme catalyzes reaction upon Mg and Mn administration in vitro.
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.[3][6][7] 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.[8]
Mechanism
This image shows the mechanism explained in the article.This image provides a visual representation of how TdT works in the process of antibody gene rearrangement. Know that although the image uses D and J segments, the same type of rearrangements happen with other segment pairs as well.
Upon the action of the RAG 1/2 enzymes, the cleaved double stranded DNA is left with hairpin structures at the end of each DNA segment created by the cleavage event. The hairpins are both opened by the Artemis complex, which has endonuclease activity when phosphorylated, providing the free 3' OH ends for TdT to act upon. Once the Artemis complex has done its job and added palindromic nucleotides (P-nucleotides) to the newly opened DNA hairpins, the stage is set for TdT to do its job. TdT is now able to come in and add N-nucleotides to the existing P-nucleotides in a 5' to 3' direction that polymerases are known to function. On average 2-5 random base pairs are added to each 3' end generated after the action of the Artemis complex. The number of bases added is enough for the two newly synthesized ssDNA segments to undergo micro homology alignment during non-homologous end joining according to the normal Watson-Crick base pairing patterns (A-T, C-G). From there unpaired nucleotides are excised by an exonuclease, like the Artemis Complex (which has exonuclease activity in addition to endonuclease activity), and then template dependent polymerases are able to fill the gaps, finally creating the new coding joint with the action of ligase to combine the segments. Although TdT does not discriminate among the four base pairs when adding them to the N-nucleotide segments, it has shown a bias for guanine and cytosine base pairs.[3]
Polymerase λ has also been found to exhibit similar template independent synthetic activity. Along with activity as a terminal transferase, it is known to also work in a template dependent fashion. This is interesting because TdT works exclusively via a template independent pathway for the addition of nucleotides.[9]
Uses
Terminal transferase has applications in molecular biology. It can be used in RACE to add nucleotides that can then be used as a template for a primer in subsequent PCR. It can also be used to add nucleotides labeled with radioactiveisotopes, for example in the TUNEL assay (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) for the demonstration of apoptosis (which is marked, in part, by fragmented DNA). It is also used in the immunofluorescence assay for the diagnosis of acute lymphoblastic leukemia.[10]
In immunohistochemistry, antibodies to TdT can be used to demonstrate the presence of immature T and B cells and pluripotent hematopoietic stem cells, which possess the antigen, while mature lymphoid cells are always TdT-negative. While TdT-positive cells are found in small numbers in healthy lymph nodes and tonsils, the malignant cells of acute lymphoblastic leukaemia are also TdT-positive, and the antibody can, therefore, be used as part of a panel to diagnose this disease and to distinguish it from, for example, small cell tumours of childhood.[11]
TdT has also seen recent application in the De Novo synthesis of oligonucleotides, with TdT-dNTP tethered analogues capable of primer extension by 1 nt at a time. [12] In other words, the enzyme TdT has demonstrated the capability of making synthetic DNA by adding one letter at a time to a primer sequence.
↑Yang-Feng TL, Landau NR, Baltimore D, Francke U (1986). "The terminal deoxynucleotidyltransferase gene is located on human chromosome 10 (10q23----q24) and on mouse chromosome 19". Cytogenetics and Cell Genetics. 43 (3–4): 121–6. doi:10.1159/000132309. PMID3467897.
↑Hardy R (2008). "Chapter 7: B Lymphocyte Development and Biology". In Paul W. Fundamental Immunology (Book) (6th ed.). Philadelphia: Lippincott Williams & Wilkins. pp. 237–269. ISBN0-7817-6519-6.
↑Cherrier M, D'Andon MF, Rougeon F, Doyen N (Feb 2008). "Identification of a new cis-regulatory element of the terminal deoxynucleotidyl transferase gene in the 5' region of the murine locus". Molecular Immunology. 45 (4): 1009–17. doi:10.1016/j.molimm.2007.07.027. PMID17854898.
↑Kubota T, Maezawa S, Koiwai K, Hayano T, Koiwai O (Aug 2007). "Identification of functional domains in TdIF1 and its inhibitory mechanism for TdT activity". Genes to Cells. 12 (8): 941–59. doi:10.1111/j.1365-2443.2007.01105.x. PMID17663723.
↑Zhang Y, Shi M, Wen Q, Luo W, Yang Z, Zhou M, Ma L (2012-01-01). "Antigenic stimulation induces recombination activating gene 1 and terminal deoxynucleotidyl transferase expression in a murine T-cell hybridoma". Cellular Immunology. 274 (1–2): 19–25. doi:10.1016/j.cellimm.2012.02.008. PMID22464913.
↑Maga G, Ramadan K, Locatelli GA, Shevelev I, Spadari S, Hübscher U (Jan 2005). "DNA elongation by the human DNA polymerase lambda polymerase and terminal transferase activities are differentially coordinated by proliferating cell nuclear antigen and replication protein A". The Journal of Biological Chemistry. 280 (3): 1971–81. doi:10.1074/jbc.M411650200. PMID15537631.
↑Faber J, Kantarjian H, Roberts MW, Keating M, Freireich E, Albitar M (Jan 2000). "Terminal deoxynucleotidyl transferase-negative acute lymphoblastic leukemia". Archives of Pathology & Laboratory Medicine. 124 (1): 92–7. doi:10.1043/0003-9985(2000)124<0092:TDTNAL>2.0.CO;2. PMID10629138.
↑Leong, Anthony S-Y; Cooper, Kumarason; Leong, F Joel W-M (2003). Manual of Diagnostic Cytology (2 ed.). Greenwich Medical Media, Ltd. pp. 413–414. ISBN1-84110-100-1.
↑Palluk, S. et al. Nature Biotechnology 2018, 36, 645-650.
Further reading
O'Malley DP, Orazi A (Aug 2006). "Terminal deoxynucleotidyl transferase-positive cells in spleen, appendix and branchial cleft cysts in pediatric patients". Haematologica. 91 (8): 1139–40. PMID16885057.
Yamashita N, Shimazaki N, Ibe S, Kaneko R, Tanabe A, Toyomoto T, Fujita K, Hasegawa T, Toji S, Tamai K, Yamamoto H, Koiwai O (Jul 2001). "Terminal deoxynucleotidyltransferase directly interacts with a novel nuclear protein that is homologous to p65". Genes to Cells. 6 (7): 641–52. doi:10.1046/j.1365-2443.2001.00449.x. PMID11473582.
Chang LM, Bollum FJ (1986). "Molecular biology of terminal transferase". CRC Critical Reviews in Biochemistry. 21 (1): 27–52. doi:10.3109/10409238609113608. PMID3524991.
Maezawa S, Hayano T, Koiwai K, Fukushima R, Kouda K, Kubota T, Koiwai O (May 2008). "Bood POZ containing gene type 2 is a human counterpart of yeast Btb3p and promotes the degradation of terminal deoxynucleotidyltransferase". Genes to Cells. 13 (5): 439–57. doi:10.1111/j.1365-2443.2008.01179.x. PMID18429817.
Dworzak MN, Fritsch G, Fröschl G, Printz D, Gadner H (Nov 1998). "Four-color flow cytometric investigation of terminal deoxynucleotidyl transferase-positive lymphoid precursors in pediatric bone marrow: CD79a expression precedes CD19 in early B-cell ontogeny". Blood. 92 (9): 3203–9. PMID9787156.
Fujita K, Shimazaki N, Ohta Y, Kubota T, Ibe S, Toji S, Tamai K, Fujisaki S, Hayano T, Koiwai O (Jun 2003). "Terminal deoxynucleotidyltransferase forms a ternary complex with a novel chromatin remodeling protein with 82 kDa and core histone". Genes to Cells. 8 (6): 559–71. doi:10.1046/j.1365-2443.2003.00656.x. PMID12786946.
Kubota T, Maezawa S, Koiwai K, Hayano T, Koiwai O (Aug 2007). "Identification of functional domains in TdIF1 and its inhibitory mechanism for TdT activity". Genes to Cells. 12 (8): 941–59. doi:10.1111/j.1365-2443.2007.01105.x. PMID17663723.
Suzuki Y, Yoshitomo-Nakagawa K, Maruyama K, Suyama A, Sugano S (Oct 1997). "Construction and characterization of a full length-enriched and a 5'-end-enriched cDNA library". Gene. 200 (1–2): 149–56. doi:10.1016/S0378-1119(97)00411-3. PMID9373149.
Liu L, McGavran L, Lovell MA, Wei Q, Jamieson BA, Williams SA, Dirks NN, Danielson MS, Dubie LM, Liang X (Jun 2004). "Nonpositive terminal deoxynucleotidyl transferase in pediatric precursor B-lymphoblastic leukemia". American Journal of Clinical Pathology. 121 (6): 810–5. doi:10.1309/QD18-PPV1-NH3T-EUTF. PMID15198352.
Yang B, Gathy KN, Coleman MS (Apr 1994). "Mutational analysis of residues in the nucleotide binding domain of human terminal deoxynucleotidyl transferase". The Journal of Biological Chemistry. 269 (16): 11859–68. PMID8163485.
Thai TH, Kearney JF (Sep 2004). "Distinct and opposite activities of human terminal deoxynucleotidyltransferase splice variants". Journal of Immunology. 173 (6): 4009–19. doi:10.4049/jimmunol.173.6.4009. PMID15356150.
Shimazaki N, Fujita K, Koiwai O (Mar 2002). "[Expression and function of terminal deoxynucleotidyl-transferase and discovery of novel DNA polymerase mu]". Seikagaku. The Journal of Japanese Biochemical Society. 74 (3): 227–32. PMID11974916.