Terminal deoxynucleotidyl transferase

DNA nucleotidylexotransferase

Amino acids 19-125 of human DNA nucleotidylexotransferase. PDB rendering based on 2coe.
Available structures
PDB Ortholog search: PDBe, RCSB
Identifiers
Symbols DNTT ; TDT
External IDs OMIM: 187410 MGI: 98659 HomoloGene: 3014 ChEMBL: 4810 GeneCards: DNTT Gene
EC number 2.7.7.31
Orthologs
Species Human Mouse
Entrez 1791 21673
Ensembl ENSG00000107447 ENSMUSG00000025014
UniProt P04053 P09838
RefSeq (mRNA) NM_001017520 NM_001043228
RefSeq (protein) NP_001017520 NP_001036693
Location (UCSC) Chr 10:
96.3 – 96.34 Mb		 Chr 19:
41.03 – 41.06 Mb
PubMed search

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.[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]

Function and regulation

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, that has exonuclease activity, 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, 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 radioactive isotopes, 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]

See also

References

  1. ↑ Isobe M, Huebner K, Erikson J, Peterson RC, Bollum FJ, Chang LM, Croce CM (Sep 1985). "Chromosome localization of the gene for human terminal deoxynucleotidyltransferase to region 10q23-q25". Proceedings of the National Academy of Sciences of the United States of America 82 (17): 5836–40. doi:10.1073/pnas.82.17.5836. PMC 390648. PMID 3862101.
  2. ↑ 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. PMID 3467897.
  3. 1 2 3 Motea EA, Berdis AJ (May 2010). "Terminal deoxynucleotidyl transferase: the story of a misguided DNA polymerase". Biochimica et Biophysica Acta 1804 (5): 1151–66. doi:10.1016/j.bbapap.2009.06.030. PMC 2846215. PMID 19596089.
  4. ↑ Haeryfar SM, Hickman HD, Irvine KR, Tscharke DC, Bennink JR, Yewdell JW (Jul 2008). "Terminal deoxynucleotidyl transferase establishes and broadens antiviral CD8+ T cell immunodominance hierarchies". Journal of Immunology 181 (1): 649–59. doi:10.4049/jimmunol.181.1.649. PMID 18566432.
  5. ↑ 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. ISBN 0-7817-6519-6.
  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. PMID 17854898.
  7. ↑ 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. PMID 17663723.
  8. ↑ 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. PMID 22464913.
  9. ↑ 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. PMID 15537631.
  10. ↑ 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. PMID 10629138.
  11. ↑ 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. ISBN 1-84110-100-1.

Further reading

External links

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