28-419: Terminal deoxynucleotidyl transferase dUTP nick end labeling ( TUNEL ) is a method for detecting DNA fragmentation by labeling the 3′- hydroxyl termini in the double-strand DNA breaks generated during apoptosis . TUNEL is a method for detecting apoptotic DNA fragmentation, widely used to identify and quantify apoptotic cells, or to detect excessive DNA breakage in individual cells. The assay relies on
56-512: A terminal transferase, it is known to also work in a more general template-dependent fashion. The similarities between TdT and polymerase μ suggest they are closely evolutionarily related. 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
84-614: 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
112-522: 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
140-682: Is facilitated by a subsection of TdT called Loop1 which selectively probes for short breaks in double-stranded DNA. Further, the discovery of this template dependant activity has led to more convincing mechanistic hypotheses as to how the distribution of lengths of the additions of the N regions arise in V(D)J recombination. Polymerase μ and polymerase λ exhibit similar in trans templated dependant synthetic activity to TdT, but without similar dependence on downstream double-stranded DNA. Further, Polymerase λ has also been found to exhibit similar template-independent synthetic activity. Along with activity as
168-517: The TUNEL assay ( T erminal deoxynucleotidyl transferase d U TP N ick E nd L abeling) 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 . In immunohistochemistry and flow cytometry, antibodies to TdT can be used to demonstrate the presence of immature T and B cells and pluripotent hematopoietic stem cells, which possess
196-647: The cell cycle -phase position, was originally developed by Gorczyca et al. Concurrently, the avidin-peroxidase labeling assay applicable for light absorption microscope was described by Gavrieli et al. Since 1992 the TUNEL has become one of the main methods for detecting apoptotic programmed cell death. However, for years there has been a debate about its accuracy, due to problems in the original assay which caused necrotic cells to be inappropriately labeled as apoptotic. The method has subsequently been improved dramatically and if performed correctly should only identify cells in
224-520: The De Novo synthesis of oligonucleotides, with TdT-dNTP tethered analogs capable of primer extension by 1 nt at a time. 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. Junctional diversity Junctional diversity describes the DNA sequence variations introduced by the improper joining of gene segments during
252-429: 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 leukemia 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 tumors of childhood. TdT has also seen recent application in
280-556: 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
308-511: 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. Human TdT isoforms have three variants TdTL1, TdTL2, and TdTS. TdTL1 is broadly expressed in lymphoid cell lines while TdTL2 is predominantly expressed in normal small lymphocytes. Both localize in the nucleus when expressed and both possess 3'->5' exonuclease activity. In contrast, TdTS isoforms do not possess exonuclease activity and perform
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#1732775401289336-524: 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,
364-399: The different variable gene segments (those segments involved in antigen recognition) of TCRs and immunoglobulins are rearranged and unused segments removed. This introduces double-strand breaks between the required segments. These ends form hairpin loops and must be joined together to form a single strand (summarised in diagram, right). This joining is a very inaccurate process that results in
392-423: The enzyme, terminal deoxynucleotidyl transferase (TdT), adds further random 'N' nucleotides. The newly synthesised strands anneal to one another, but mismatches are common. Exonucleases remove these unpaired nucleotides and the gaps are filled by DNA synthesis and repair machinery. Exonucleases may also cause shortening of this junction, however this process is still poorly understood. Junctional diversity
420-483: The fetal period. 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
448-703: The first sources of pure TdT and lead to the discovery that differences in activity exist between human and bovine isoforms. 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 pK a of the 3'-OH group and ultimately facilitate the departure of the resultant pyrophosphate by-product. 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,
476-508: The four base pairs when adding them to the N-nucleotide segments, it has shown a bias for guanine and cytosine base pairs. In a template-dependant manner, TdT can incorporate nucleotides across strand breaks in double-stranded DNA in a manner referred to as in trans in contrast to the in cis mechanism found in most polymerases. This occurs optimally with a one base-pair break between strands and less so with an increasing gap. This
504-436: 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 Mg , Mn , Zn and Co . The rate of enzymatic activity depends on
532-887: The last phase of apoptosis . New methods incorporate the dUTPs modified by fluorophores or haptens, including biotin or bromine , which can be detected directly in the case of a fluorescently-modified nucleotide (i.e., fluorescein-dUTP), or indirectly with streptavidin or antibodies , if biotin-dUTP or BrdUTP are used, respectively. The most sensitive of them is the method utilizing incorporation of BrdUTP by TdT followed by immunocytochemical detection of BrdU . Terminal deoxynucleotidyl transferase 2COE 1791 21673 ENSG00000107447 ENSMUSG00000025014 P04053 P09838 NM_001017520 NM_004088 NM_001043228 NM_009345 NP_001017520 NP_004079 NP_001036693 NP_033371 Terminal deoxynucleotidyl transferase ( TdT ), also known as DNA nucleotidylexotransferase ( DNTT ) or terminal transferase ,
560-414: 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
588-463: The necessary elongation during V(D)J recombination. Since a similar exonuclease activity hypothesized in murine TdTL is found in human and bovine TdTL, some postulate that bovine and human TdTL isoforms regulate TdTS isoforms in a similar manner as proposed in mice. Further, some hypothesize that TdTL1 may be involved in the regulation of TdTL2 and/or TdTS activity. Upon the action of the RAG 1/2 enzymes,
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#1732775401289616-472: 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 can fill the gaps, finally creating the new coding joint with the action of ligase to combine the segments. Although TdT does not discriminate among
644-431: The process of V(D)J recombination . This process of V(D)J recombination has vital roles for the vertebrate immune system , as it is able to generate a huge repertoire of different T-cell receptor (TCR) and immunoglobulin molecules required for pathogen antigen recognition by T-cells and B cells, respectively. Junctional diversity includes the process of somatic recombination or V(D)J recombination , during which
672-519: 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
700-458: 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 microhomology alignment during non-homologous end joining according to
728-525: 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
756-441: The use of terminal deoxynucleotidyl transferase (TdT), an enzyme that catalyzes attachment of deoxynucleotides, tagged with a fluorochrome or another marker , to 3'-hydroxyl termini of DNA double strand breaks. It may also label cells having DNA damage by other means than in the course of apoptosis. The fluorochrome-based TUNEL assay applicable for flow cytometry , combining the detection of DNA strand breaks with respect to
784-414: The variable addition or subtraction of nucleotides and, thus, generates junctional diversity. Generation of junctional diversity starts as the proteins, recombination activating gene -1 and -2 (RAG1 and RAG2), along with DNA repair proteins, such as Artemis , are responsible for single-stranded cleavage of the hairpin loops and addition of a series of palindromic , 'P' nucleotides. Subsequent to this,
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