Human Terminal Deoxynucleotidyl Transferase Research Articles

Computational Modeling Study of the Molecular Basis of dNTP Selectivity in Human Terminal Deoxynucleotidyltransferase.

Human terminal deoxynucleotidyl transferase (TdT) can catalyze template-independent DNA synthesis during the V(D)J recombination and DNA repair through nonhomologous end joining. The capacity for template-independent random addition of nucleotides to single-stranded DNA makes this polymerase useful in various molecular biological applications involving sequential stepwise synthesis of oligonucleotides using modified dNTP. Nonetheless, a serious limitation to the applications of this enzyme is strong selectivity of human TdT toward dNTPs in the order dGTP > dTTP ≈ dATP > dCTP. This study involved molecular dynamics to simulate a potential impact of amino acid substitutions on the enzyme's selectivity toward dNTPs. It was found that the formation of stable hydrogen bonds between a nitrogenous base and amino acid residues at positions 395 and 456 is crucial for the preferences for dNTPs. A set of single-substitution and double-substitution mutants at these positions was analyzed by molecular dynamics simulations. The data revealed two TdT mutants-containing either substitution D395N or substitutions D395N+E456N-that possess substantially equalized selectivity toward various dNTPs as compared to the wild-type enzyme. These results will enable rational design of TdT-like enzymes with equalized dNTP selectivity for biotechnological applications.

Substrate Specificity Diversity of Human Terminal Deoxynucleotidyltransferase May Be a Naturally Programmed Feature Facilitating Its Biological Function.

Terminal 2'-deoxynucleotidyl transferase (TdT) is a unique enzyme capable of catalysing template-independent elongation of DNA 3' ends during V(D)J recombination. The mechanism controlling the enzyme's substrate specificity, which is necessary for its biological function, remains unknown. Accordingly, in this work, kinetic and mutational analyses of human TdT were performed and allowed to determine quantitative characteristics of individual stages of the enzyme-substrate interaction, which overall may ensure the enzyme's operation either in the distributive or processive mode of primer extension. It was found that conformational dynamics of TdT play an important role in the formation of the catalytic complex. Meanwhile, the nature of the nitrogenous base significantly affected both the dNTP-binding and catalytic-reaction efficiency. The results indicated that neutralisation of the charge and an increase in the internal volume of the active site caused a substantial increase in the activity of the enzyme and induced a transition to the processive mode in the presence of Mg2+ ions. Surrogate metal ions Co2+ or Mn2+ also may regulate the switching of the enzymatic process to the processive mode. Thus, the totality of individual factors affecting the activity of TdT ensures effective execution of its biological function.

Insight into the mechanism of DNA synthesis by human terminal deoxynucleotidyltransferase.

Terminal deoxynucleotidyltransferase (TdT) is a member of the DNA polymerase X family that is responsible for random addition of nucleotides to single-stranded DNA. We present investigation into the role of metal ions and specific interactions of dNTP with active-site amino acid residues in the mechanisms underlying the recognition of nucleoside triphosphates by human TdT under pre-steady-state conditions. In the elongation mode, the ratios of translocation and dissociation rate constants, as well as the catalytic rate constant were dependent on the nature of the nucleobase. Preferences of TdT in dNTP incorporation were researched by molecular dynamics simulations of complexes of TdT with a primer and dNTP or with the elongated primer. Purine nucleotides lost the "summarised" H-bonding network after the attachment of the nucleotide to the primer, whereas pyrimidine nucleotides increased the number and relative lifetime of H-bonds in the post-catalytic complex. The effect of divalent metal ions on the primer elongation revealed that Me<sup>2+</sup> cofactor can significantly change parameters of the primer elongation by strongly affecting the rate of nucleotide attachment and the polymerisation mode.

Synthesis of the new nucleoside 5′-alpha-iminophosphates using Staudinger reaction

Efficient protocols were developed for the synthesis of a new compounds - nucleoside 5′-α-iminophosphates using the Staudinger reaction. These substances are alpha-phosphate mimetic nucleotide in which an oxygen atom is replaced by a corresponding imino (=N-R)-group. Various 5′-iminomonophosphates of nucleosides were obtained. A chemical method for the synthesis of triphosphate derivatives based on the iminomonophosphates has been designed. Thymidine 5′-(1,3-dimethylimidazolidin-2-ylidene)-triphosphate (ppp(DMI)T) was synthesized, its hydrolytic stability and substrate properties in relation to some DNA polymerases was firstly studied. It was shown that ppp(DMI)T can serve as substrate for enzyme catalyzed template-independent DNA synthesis by human terminal deoxynucleotidyltransferase TdT.

Insight into the Mechanism of DNA Synthesis by Human Terminal Deoxynucleotidyltransferase

Insight into the Mechanism of DNA Synthesis by Human Terminal Deoxynucleotidyltransferase

The antitumor effect of lobaplatin against Ishikawa endometrial cancer cells in vitro and in vivo

The new effective chemotherapeutic drugs are required urgently for advanced and recurrent endometrial carcinoma (EC), which is one of the most common gynaecological tumors among women worldwide. Preclinical studies have shown that lobaplatin, one of the third-generation platinum compounds, has possessed powerful anti-cancer efficacy on a series of tumors. The purpose of this study is to investigate its effect and molecular mechanism on the growth of endometrial cancer cell line Ishikawa in vitro and in vivo. The results of cell counting kit-8 (CCK-8) shown that lobaplatin concentration-dependent inhibited cell proliferations in human endometrial carcinoma ishikawa cells. Flow cytometry (FCM) assay demonstrated that lobaplatin affected the survival of endometrial carcinoma cell by arresting cell cycle at S phase and G2/M phase and inducing apoptosis in dose-dependent manner. Moreover, Western blot analysis also showed that the apoptosis-inducing effects of lobaplatin was associated with the reduction of Bcl-2 expression while upregulation of cleaved-caspase-3, cleaved-caspase-8, cleaved-caspase-9 and Bax. Meanwhile, lobaplatin significantly suppressed tumor growth of human Ishikawa xenograft models and terminal deoxynucleotidyl trans-ferase dUTP nick end labeling confirmed the significant occurrence of lobaplatin-treated tumor tissues of apoptosis. Therefore, lobaplatin could be an effective chemotherapeutic agent for human endometrial carcinoma and warrants further clinical investigation.

Functional analyses of polymorphic variants of human terminal deoxynucleotidyl transferase

Human terminal deoxynucleotidyl transferase (hTdT) is a DNA polymerase that functions to generate diversity in the adaptive immune system. Here, we focus on the function of naturally occurring single-nucleotide polymorphisms (SNPs) of hTdT to evaluate their role in genetic-generated immune variation. The data demonstrate that the genetic variations generated by the hTdT SNPs will vary the human immune repertoire and thus its responses. Human TdT catalyzes template-independent addition of nucleotides (N-additions) during coding joint formation in V(D)J recombination. Its activity is crucial to the diversity of the antigen receptors of B and T lymphocytes. We used in vitro polymerase assays and in vivo human cell V(D)J recombination assays to evaluate the activity and the N-addition levels of six natural (SNP) variants of hTdT. In vitro, the variants differed from wild-type hTdT in polymerization ability with four having significantly lower activity. In vivo, the presence of TdT varied both the efficiency of recombination and N-addition, with two variants generating coding joints with significantly fewer N-additions. Although likely heterozygous, individuals possessing these genetic changes may have less diverse B- and T-cell receptors that would particularly effect individuals prone to adaptive immune disorders, including autoimmunity.

Synthetic analogues of the manzamenones and plakoridines which inhibit DNA polymerase

An array of novel analogues of the marine oxylipins, the manzamenones and plakoridines, have been prepared in divergent fashion using an approach modelled on a biogenetic theory. Many of the target compounds show potent inhibition of DNA polymerases α and β and human terminal deoxynucleotidyl transferase (TdT).

Structure—Activity Relationships of Untenone A and Its Derivatives for Inhibition of DNA Polymerases.

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Structure–activity relationships of untenone A and its derivatives for inhibition of DNA polymerases

We found that untenone A and mannzamenone A inhibit mammalian DNA polymerases α and β, and human terminal deoxynucleotidyl transferase (TdT). The syntheses of both compounds and the structure–activity relationships of untenone A derivatives are described.

Vertebrate TBP-like protein (TLP/TRF2/TLF) stimulates TATA-less terminal deoxynucleotidyl transferase promoters in a transient reporter assay, and TFIIA-binding capacity of TLP is required for this function.

The TBP-like protein (TLP/TRF2/TLF), which belongs to the TBP family of proteins, is present in all metazoan organisms. Although the human TLP has been reported to interfere with transcription from TATA-containing promoters, the transcription activation potential of TLP in higher animals is obscure. We previously demonstrated that artificially promoter-recruited TLP behaves like an unconventional transcriptional activator. In this study, we investigated the effects of TLP on TATA-less promoters of mouse and human terminal deoxynucleotidyl transferase (TdT) genes by transient reporter assays. As expected, TLP repressed both basal and activator-augmented transcription from the TATA-containing adenovirus major late promoter (MLP) and E1B promoter. On the other hand, however, TLP significantly stimulated both basal and activated transcription from TdT promoters. We investigated the strength of the promoters in chicken DT40 cells that lack the TLP gene. The MLP showed higher activity but the TdT promoter showed lower activity in TLP-null cells than in the wild-type cells. Moreover, ectopic expression of mouse TLP in the TLP-null cells considerably stimulated the TdT promoter. Insertion of a TATA element upstream from the TdT core promoter resulted in a loss of TLP-mediated activation. The mouse TLP was demonstrated to bind specifically to TFIIA with greater strength than TBP. We constructed mutated TLPs having amino acid substitutions that impair TFIIA binding. A representative TLP mutant lacking TFIIA-binding ability could not stimulate transcription from the TdT promoter, whereas that mutation suppressed TLP-mediated transcription repression of TATA promoters. The results of the present study suggest that the vertebrate TLP potentiates exogenous TATA-less promoters and that TFIIA plays an important role in the TLP function.

Association of terminal deoxynucleotidyl transferase with Ku.

Terminal deoxynucleotidyl transferase (TdT) catalyzes the addition of nucleotides at the junctions of rearranging Ig and T cell receptor gene segments, thereby generating antigen receptor diversity. Ku is a heterodimeric protein composed of 70- and 86-kDa subunits that binds DNA ends and is required for V(D)J recombination and DNA double-strand break (DSB) repair. We provide evidence for a direct interaction between TdT and Ku proteins. Studies with a baculovirus expression system show that TdT can interact specifically with each of the Ku subunits and with the heterodimer. The interaction between Ku and TdT is also observed in pre-T cells with endogenously expressed proteins. The protein-protein interaction is DNA independent and occurs at physiological salt concentrations. Deletion mutagenesis experiments reveal that the N-terminal region of TdT (131 amino acids) is essential for interaction with the Ku heterodimer. This region, although not important for TdT polymerization activity, contains a BRCA1 C-terminal domain that has been shown to mediate interactions of proteins involved in DNA repair. The induction of DSBs in Cos-7 cells transfected with a human TdT expression construct resulted in the appearance of discrete nuclear foci in which TdT and Ku colocalize. The physical association of TdT with Ku suggests a possible mechanism by which TdT is recruited to the sites of DSBs such as V(D)J recombination intermediates.

Construction and expression of a chimeric gene encoding human terminal deoxynucleotidyltransferase and DNA polymerase β

A domain substitution experiment was carried out between the structurally related DNA-polymerizing enzymes Polβ and TdT to investigate the region of Polβ required for template utilization. Site-directed mutagenesis and recombinant DNA procedures were used for construction of a gene encoding a chimeric form of the two enzymes and termed TDT::POLB, in which the DNA region encoding amino acids (aa) 154–212 of TdT was replaced by the corresponding region encoding aa 1–60 of Polβ. The construction was confirmed by restriction analysis and DNA sequencing. Since this region of Polβ represents most of the N-terminal domain of the enzyme possessing single-stranded DNA (ssDNA)-binding activity, it was hypothesized that the chimeric protein, unlike TdT, might possess template-dependent DNA polymerase activity. The chimeric gene product was produced in Escherichia coli, purified and subjected to preliminary enzymological characterization. The finding that the chimeric TdT::Polβ protein possessed significant template-dependent polymerase activity suggests that aa 1–60 of Polβ are involved in template utilization during the polymerization reaction, as suggested by the previous finding that the 8-kDa N-terminal domain of Polβ possesses ssDNA-binding activity [Kumar ., J. Biol. Chem. 265 (1990a) 2124-2131; Kumar ., Biochemistry 29 (1990b) 7156-7159; Prasad ., J. Biol. Chem. 268 (1993) 22746-22755].

Initiation of transcription at the human terminal deoxynucleotidyl transferase gene promoter: a novel role for the TATA binding protein.

Control of initiation of transcription of the human terminal deoxynucleotidyl transferase (TdT) gene was investigated by using an in vitro transcription assay. The precise contribution of discrete basal promoter elements to transcription initiation was determined by testing deletion and substitution mutations. The primary element, contained within the region spanning -34 to -14 bp relative to the transcription start site, accounted for 80% of basal promoter activity. TdT promoter activity required the sequence ACCCT at -24 to -20 bp since a dramatic decrease in transcription initiation was observed after mutation of this sequence, whereas mutation of the adjacent sequence from -32 to -25 bp did not alter promoter activity. The secondary element contained sequences surrounding the transcription start site and had 20% of promoter activity. Deletion of both elements completely abolished transcription initiation. Initiator characteristics of the secondary element were revealed by using the in vitro assay: promoter sequences at the transcription start site were sufficient to direct accurate initiation at a single site. Mutation of the sequence GGGTG spanning the transcription start site resulted in loss of transcription initiation. Both the primary and secondary elements were nonhomologous to corresponding regions from the mouse TdT gene promoter. While the human basal promoter functioned in the absence of TATA consensus sequences or GC-rich SP1 binding sites, it was dependent on active TFIID. In contrast to other TATA-less promoters, purified TATA binding protein substituted for the TFIID complex and restored promoter activity to TFIID-inactivated nuclear extracts.

Identification of a tripartite basal promoter which regulates human terminal deoxynucleotidyl transferase gene expression.

In order to locate the promoter region of the human terminal deoxynucleotidyl transferase gene, serially truncated segments of the 5'-flanking region of the gene were cloned into a chloramphenicol acetyltransferase reporter vector. Transient transfection analyses of the terminal transferase-reporter gene constructs identified the basal promoter region within -34 to +40 base pairs relative to the transcription start site. Three promoter elements were defined in this region. The primary element is within 34 base pairs upstream of the transcription start site. The CAP site is 62 base pairs upstream of the translation start site. The secondary element involves sequences around the transcription start site. The third is located 25 base pairs downstream from the initiation site (+25 to +40). This tripartite basal promoter was not tissue specific; similar patterns of promoter activity were observed in terminal transferase expressing and non-expressing cells. Transfection analyses also indicated the presence of negative regulatory elements upstream of the basal promoter region, and these elements were preferentially active in cells expressing terminal transferase.

Mutational analysis of residues in the nucleotide binding domain of human terminal deoxynucleotidyl transferase.

Human terminal deoxynucleotidyl transferase (TdT) was overexpressed in a baculovirus system. The pure recombinant enzyme was identical in size, activity, kinetic constants, and metal effects to native enzyme. Three amino acids, within either the putative nucleotide binding domain and part of a DNA polymerase consensus sequence, YGDTDSLF, or a TdT consensus sequence, GGFRRGK, were altered by site-directed mutagenesis. The four mutant forms of terminal transferase were also overexpressed in the baculovirus expression system and purified from Trichoplusia ni larvae by a monoclonal antibody affinity column and compared with wild-type enzyme with respect to thermostabilities, secondary structure, metal effects, and kinetic parameters. Three of the four mutants retained 3-16% of wild-type activity under varying metal conditions, and one of the mutants, D343E, consistently exhibited less than 0.2% of wild-type TdT activity with dATP and no activity with dGTP. All mutants had alterations in the Km for dATP. Variations in Km for dGTP were not as consistent. The Km for the other substrate, DNA initiator (dA)50) in the presence of dATP remained essentially the same as that of wild-type TdT for all mutants except D343E. The enzyme activity of all mutants was stimulated by Zn2+ at low concentrations, and this effect was diminished and reversed at higher concentrations of ZnSO4. All mutants still retained significant amounts of the secondary structure as measured by circular dichroism. These results indicated that the aspartic acid residue at position 343 is located at or near the active site and is critical for the nucleotide binding and catalytic activity.

Mutational analysis of residues in the nucleotide binding domain of human terminal deoxynucleotidyl transferase

Mutational analysis of residues in the nucleotide binding domain of human terminal deoxynucleotidyl transferase

Identification of a tripartite basal promoter which regulates human terminal deoxynucleotidyl transferase gene expression.

Identification of a tripartite basal promoter which regulates human terminal deoxynucleotidyl transferase gene expression.

Is terminal deoxynucleotidyl transferase an intracellular mutagen?

We have established stably transformed mammalian cell lines expressing recombinant human terminal deoxynucleotidyl transferase. A 58 kDa, enzymatically active protein is produced by these cell lines. Using the lac I gene of pJYMib shuttle vector as mutagenic target, we found no increase in mutation retes in cells expressing terminal deoxynucleotidyl transferase compared to controls. Our results suggest that the presence of terminal deoxynucleotidyl transferase alone in mammalian cells does not increase mutation rates.

Tissue-specific expression of human terminal deoxynucleotidyl transferase is regulated at the transcriptional level

Transcriptional regulation of expression of the terminal deoxynucleotidyl transferase gene in normal thymus and in differentiation arrested cells was demonstrated by analyzing steady-state levels of TdT RNA as well as the relative transcription rate of the gene. Terminal transferase transcripts were detected only in those cells and tissues that contained antigen and enzyme activity. The relative rates of transcription correlated with levels of mRNA as well as with levels of the protein. These data suggest that expression of this gene in normal and leukemic cells is modulated at the level of transcription.