Evolving a Thermostable Terminal Deoxynucleotidyl Transferase.

Terminal deoxynucleotidyl transferase (TdT) catalyzes template free incorporation of arbitrary nucleotides onto single-stranded DNA. Due to this unique feature, TdT is widely used in biotechnology and clinical applications. One particularly tantalizing use is the synthesis of long de novo DNA molecules by TdT-mediated iterative incorporation of a 3' reversibly blocked nucleotide, followed by deblocking. However, wild-type (WT) TdT is not optimized for the incorporation of 3' modified nucleotides, and TdT engineering is hampered by the fact that TdT is marginally stable and only present in mesophilic organisms. We sought to first evolve a thermostable TdT variant to serve as backbone for subsequent evolution to enable efficient incorporation of 3'-modified nucleotides. A thermostable variant would be a good starting point for such an effort, as evolution to incorporate bulky modified nucleotides generally results in lowered stability. In addition, a thermostable TdT would also be useful when blunt dsDNA is a substrate as higher temperature could be used to melt dsDNA. Here, we developed an assay to identify thermostable TdT variants. After screening about 10 000 TdT mutants, we identified a variant, named TdT3-2, that is 10 °C more thermostable than WT TdT, while preserving the catalytic properties of the WT enzyme.

Mimivirus encodes a multifunctional primase with DNA/RNA polymerase, terminal transferase and translesion synthesis activities.

Acanthamoeba polyphaga mimivirus is an amoeba-infecting giant virus with over 1000 genes including several involved in DNA replication and repair. Here, we report the biochemical characterization of gene product 577 (gp577), a hypothetical protein (product of L537 gene) encoded by mimivirus. Sequence analysis and phylogeny suggested gp577 to be a primase-polymerase (PrimPol)-the first PrimPol to be identified in a nucleocytoplasmic large DNA virus (NCLDV). Recombinant gp577 protein purified as a homodimer and exhibited de novo RNA as well as DNA synthesis on circular and linear single-stranded DNA templates. Further, gp577 extends a DNA/RNA primer annealed to a DNA or RNA template using deoxyribonucleoties (dNTPs) or ribonucleotides (NTPs) demonstrating its DNA/RNA polymerase and reverse transcriptase activity. We also show that gp577 possesses terminal transferase activity and is capable of extending ssDNA and dsDNA with NTPs and dNTPs. Mutation of the conserved primase motif residues of gp577 resulted in the loss of primase, polymerase, reverse transcriptase and terminal transferase activities. Additionally, we show that gp577 possesses translesion synthesis (TLS) activity. Mimiviral gp577 represents the first protein from an NCLDV endowed with primase, polymerase, reverse transcriptase, terminal transferase and TLS activities.

In situ grown DNA nanotail-templated silver nanoclusters enabling label-free electrochemical sensing of terminal deoxynucleotidyl transferase activity.

A novel label-free electrochemical strategy was established based on the unique electro-catalytic activity of graphene oxide (GO)-supported terminal deoxynucleotidyl transferase (TdT)-generated C-rich DNA nanotail-templated silver nanoclusters (DNA-AgNCs). TdT can catalyze the deoxycytidine triphosphate (dCTP) to the 3'-OH terminus of single-stranded DNA (ssDNA) with no template; then, in the presence of Ag(I), TdT-generated C-rich DNA sequence was employed for the synthetic template of AgNCs because of the formed complexes of nitrogen atoms of cytosine based with silver atoms. We proved that in situ grown DNA nanotail-templated AgNCs can be adsorbed on GO-modified electrode and possess high electro-catalytic activity to H2O2 reduction, presenting a good electrochemical indicator for signal readout. Under optimal conditions, the proposed biosensor could be employed for quantitatively monitoring TdT activity and within a dynamic range from 0.4 to 90U/mL and a low limit of detection is 0.08U/mL. With high sensitivity and excellent selectivity, this strategy offers a facile, convenient and specific electrochemical method for TdT activity detection and its relevant inhibitors screening. It holds a promising potential in the practical application of TdT-based biochemical research, disease diagnosis and drug discovery.

Crystallization of the catalytic domain of murine terminal deoxynucleotidyl transferase.

The catalytic domain of murine terminal deoxynucleotidyl transferase (TdT) has been crystallized in the space group P2(1)2(1)2(1), with unit-cell parameters a = 47.1, b = 86.2, c = 111.7 A. The crystals diffract to a resolution of 2.4 A using synchrotron radiation and a full data set has been collected from the native crystals. The enzyme was shown to be active in the crystalline state.

High-level expression of murine terminal deoxynucleotidyl transferase in Escherichia coli grown at low temperature and overexpressing argU tRNA.

Terminal deoxynucleotidyl transferase (TdT) is a highly conserved vertebrate enzyme that possesses the unique ability to catalyze the random addition of deoxynucleoside 5'-triphosphates onto the 3'-hydroxyl group of a single-stranded DNA. It plays an important role in the generation of immunoglobin and T-cell receptor diversity. TdT is usually obtained from animal thymus gland or produced in a baculovirus system, but both procedures are rather tedious, and proteolysis occurs during purification. Attempts to overexpress TdT in bacteria have been unsuccessful or have yielded an enzyme with a lower specific activity. A dearth of TdT has thus hampered detailed structural and functional studies. In the present study, we report that by lowering growth temperature and overexpressing a rare arginyl tRNA, it is possible to boost the production in Escherichia coli of murine TdT with minimal proteolysis and high specific activity.

ATP-dependent processivity of a telomerase activity from Saccharomyces cerevisiae.

Extracts of Saccharomyces cerevisiae were shown to support the elongation of oligodeoxynucleotides with telomere-like sequences. The primer sequence specificity of this elongation activity, its incorporation of dG and dT but not dA or dC from the corresponding triphosphates, and its sensitivity to RNase A and RNase H are all consistent with it being a telomerase. In contrast to the reported properties of other telomerases, the presence of ATP enhances the efficiency of initiation of the yeast enzyme and improves its processivity. Hydrolysis of ATP appears to be unnecessary for the observed effects, as the beta,gamma-imido or the gamma-thio derivative of ATP is nearly as effective.

Gel shift and UV cross-linking analysis of Tetrahymena telomerase.

Telomerase is an unusual ribonucleoprotein that synthesizes new telomeres onto chromosome ends. The enzyme has been most extensively characterized in ciliates, where the RNA component has been cloned from several species, and its elongation properties have been characterized in detail. To understand the substrate specificity and protein composition of telomerase, we have used gel shift and UV cross-linking to characterize the enzyme from the ciliate Tetrahymena thermophila. In a mobility shift assay, a complex was identified that contained telomerase RNA, co-purified with telomerase activity, and was sensitive to nuclease treatment. The mobility shift complexes specifically formed using several different single-stranded, telomeric sequences but not non-telomeric primers. These results suggest that the specificity of telomerase for G-rich primer sequences occurs at least in part at the level of primer binding. UV cross-linking analysis identified a 100-kDa cross-linked protein that may be a telomerase component.

Isolation and characterization of the Xenopus terminal deoxynucleotidyl transferase.

A cDNA clone containing the sequence of the Xenopus homologue of terminal deoxynucleotidyl transferase (TdT) was isolated and analyzed. The derived amino acid sequence shares > 50% identity with the human, mouse, and bovine TdT sequences. By hybridization and PCR analyses Xenopus TdT mRNA was found in the thymus and not in the spleen, kidney, intestine, or liver. During ontogeny TdT appears in significant amounts in the thymus of tadpoles at metamorphic climax but little in the earlier midlarval stages. The emergence of TdT only late in development correlates with the paucity of N region addition in larval Ig heavy chain sequences. Past experiments on the ontogeny of the Xenopus immune system revealed a less efficacious tadpole immune response in skin graft rejection and Ab heterogeneity; the absence of TdT in the tadpole stages fits well with the notion of lower larval TCR and Ab diversity.

Identification of a nonprocessive telomerase activity from mouse cells.

Telomerase activity was identified in extracts from several different mouse cell lines. Addition of telomeric TTAGGG repeats was specific to telomeric oligonucleotide primers and sensitive to pretreatment with RNase A. In contrast to the hundreds of repeats synthesized by the human and Tetrahymena telomerase enzymes in vitro, mouse telomerase synthesized only one or two TTAGGG repeats onto telomeric primers. The products observed after elongation of primers with circularly permuted (TTAGGG)3 sequences and after chain termination with ddATP or ddTTP indicated that mouse telomerase pauses after the addition of the first dG residue in the sequence TTAGGG. The short length of the products synthesized by mouse telomerase was not due to a diffusible inhibitor in the mouse extract, because the human telomerase continued to synthesize long products when mixed with mouse fractions. Primer challenge experiments showed that the human enzyme synthesized long TTAGGG repeats processively in vitro, whereas the mouse telomerase appeared to be much less processive. The identification of short telomerase reaction products in mouse extracts suggests that extracts from other organisms may also generate only short products. This knowledge may aid in the identification of telomerase activity in organisms where activity has not yet been detected.

Use of immobilized triazine dyes in the purification of DNA topoisomerase I (Topo I) and terminal deoxynucleotidyl transferase (TdT) from calf thymus.

The interaction between TdT and Topo I, and twelve various triazine dyes immobilized on Sepharose CL-6B was studied. Yellow lightproof 2KT-Sepharose and Bordeaux 4ST-Sepharose were used to purify TdT and Topo I, respectively. The principal role of copper ions, complexed to the dye molecules, in the dye-protein interaction was evaluated.

Interaction of terminal transferase with single-stranded DNA.

A 58-kDa monomer of terminal transferase was isolated from calf thymus using a monoclonal antibody affinity column. The enzymatic activity was comparable to that of the 44-kDa alpha beta dimer isolated by conventional methods. Binding of the two enzyme forms to single-stranded DNA was monitored by fluorescence. The site size of both forms was approximately 11 +/- 2 nucleotides. Binding of the 44-kDa alpha beta dimer to polydeoxyadenosine was examined under several conditions. The cooperativity parameter increased from about 90 in the presence of Mg2+ to 300-400 in the absence of Mg2+. The observed dissociation constant of 3-5 microM was essentially independent of salt concentration, whereas the intrinsic dissociation constant decreased about 5-fold in the presence of Mg2+. The binding parameters of the 58-kDa monomer were independent of buffer composition and were similar to those of the 44-kDa alpha beta dimer in the presence of Mg2+. These results indicate that the additional 14-kDa peptide sequences present in the high molecular mass monomer form are not part of the DNA-binding site of terminal transferase.

Purification of high molecular mass species of calf thymus terminal deoxynucleotidyltransferase.

We have developed a simplified column chromatographic procedure for the simultaneous purification of two high molecular mass forms (58 kd and 45 kd) and a standard two subunit 44 kd from of terminal deoxynucleotidyltransferase (TdT) from calf thymus chromatin. The procedure involves high salt extraction of the chromatin fraction followed by successive chromatographies on phosphocellulose, DEAE sephadex, and hydroxylapatite matrices. While all 3 species of TdT comigrate throughout these steps, separation of individual species is achieved on a single stranded DNA agarose column. The combined yield of the 45 kd and 58 kd TdTs is quite high (approximately 8 mg/5000g tissue), 45 kd being the major species (approximately 60%) and the 58 kd constituting about 30%. The 44 kd species containing two subunits usually represents under 10% of the total. All the three forms of TdT showed similar specific activity and preference for purine deoxynucleoside triphosphates (dNTPs). The Km for individual dNTP with all three species of TdT is quite similar and decreases in the order dCTP greater than dTTP greater than dATP greater than dGTP. The Km for both synthetic primer and activated DNA with the 3 TdTs was, in increasing order, two subunit 44 kd less than 45 kd less than 58 kd TdT. Both 58 kd and 45 kd TdT displayed two optima for Mn++ (0.1 mM and 1 mM) and a single sharp optimum for Mg++ (2.5 mM). The two subunit 44 kd TdT exhibited a single but broad optimum for Mn++ (1 mM) and for Mg++ (10 mM).

Molecular biology of terminal transferase.

Terminal transferase is an unusual deoxynucleotide polymerizing enzyme found only in prelymphocytes. The protein was purified to homogeneity from calf thymus glands in 1971 as a 32 kDa protein with a two peptide structure. Subsequent biochemical and immunological analyses of terminal transferase protein in crude extracts from a number of animal species showed a single peptide with a molecular weight of about 58,000. The two peptide structure found earlier was caused by proteolysis. Homogeneous 58 kDa terminal transferase has now been produced from human lymphoblastoid cells and calf thymus glands by immunoaffinity chromatography. In vitro phosphorylation studies showed that the terminal transferase protein contains one phosphorylation site near one end of the polypeptide chain, and the phosphorylation of the enzyme has been confirmed by in vivo labeling experiments. Unambiguous demonstration of the molecular weight of the human terminal transferase was obtained by translation of the cloned human terminal transferase DNA sequence to a 58,308 Da protein. The translated amino acid sequence also provided a possible phosphorylation site near the amino-terminus of the protein. Preliminary analysis of the genomic structure shows a simple intron/exon pattern with the total human terminal transferase gene spanning at least 65 Kb.

Identification of a specific telomere terminal transferase activity in Tetrahymena extracts.

We have found a novel activity in Tetrahymena cell free extracts that adds tandem TTGGGG repeats onto synthetic telomere primers. The single-stranded DNA oligonucleotides (TTGGGG)4 and TGTGTGGGTGTGTGGGTGTGTGGG, consisting of the Tetrahymena and yeast telomeric sequences respectively, each functioned as primers for elongation, while (CCCCAA)4 and two nontelomeric sequence DNA oligomers did not. Efficient synthesis of the TTGGGG repeats depended only on addition of micromolar concentrations of oligomer primer, dGTP, and dTTP to the extract. The activity was sensitive to heat and proteinase K treatment. The repeat addition was independent of both endogenous Tetrahymena DNA and the endogenous alpha-type DNA polymerase; and a greater elongation activity was present during macronuclear development, when a large number of telomeres are formed and replicated, than during vegetative cell growth. We propose that the novel telomere terminal transferase is involved in the addition of telomeric repeats necessary for the replication of chromosome ends in eukaryotes.

Affinity purification and refined structural characterization of terminal deoxynucleotidyltransferase.

A total of 56 stable murine hybridoma monoclones that produce homogeneous antibodies against human or calf terminal deoxynucleotidyltransferase have been established. All of the antibodies exhibited specific binding to various Mr forms of terminal transferase and eight possessed neutralizing activity. Results are presented that permitted characterization of ten of these antibodies with respect to their immunoglobulin class, their recognition of calf or human terminal-transferase Mr species by immunoblotting techniques and their recognition of distinct antigenic sites. Terminal transferase was purified in a single step by using an immunoaffinity column constructed with a monoclonal antibody exhibiting a high binding affinity for the enzyme. Single monoclonal antibodies were also used to bind selectively to terminal-transferase antigen in tissue slices and individual cells.

A specific endonuclease which co-purifies with calf thymus terminal deoxynucleotidyl transferase.

An endonuclease which introduces single nicks in superhelical DNA co-purifies with the enzyme, terminal deoxynucleotidyl transferase. This activity is found in all homogeneous preparations of terminal transferase tested, and remains associated with the polymerase activity during additional fractionation methods. Kinetic data suggest that the nuclease and polymerase occupy distinct active sites, although multifunctionality has not been proven. However, the ability of the polymerase to synthesize oligodeoxynucleotide products on endonuclease-generated singly-nicked circular duplex DNA may represent an important biological function or signal in lymphoid cells.

Proteolytic degradation of calf thymus terminal deoxynucleotidyl transferase.

A high molecular weight preparation of terminal transferase containing 58,000- and 44,000-dalton peptides has been purified from calf thymus glands. The relationship of these terminal transferase peptides to the low molecular weight form was established with an immunoblot procedure using rabbit antibody directed against the homogeneous calf thymus low molecular weight terminal transferase (32,000 daltons). The 58,000- and 44,000-dalton enzyme species are each shown to be enzymatically active by renaturation in situ after electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate. These results suggest that the homogeneous terminal transferase previously described is derived from the higher molecular weight species by proteolysis during fractionation. Controlled degradation of the high molecular weight calf thymus terminal transferase with trypsin produces fully active enzyme containing alpha- and beta-peptides similar to those found in the 32,000-dalton species. Isoelectric focusing experiments show a decrease of isoelectric pH of the enzyme with proteolysis.