Incorporation Of Deoxyribonucleotides Research Articles

Elucidating sequence-function relationships in a template-independent polymerase to enable novel DNA recording applications.

Harnessing DNA as a high-density storage medium for information storage and molecular recording of signals has been of increasing interest in the biotechnology field. Recently, progress in enzymatic DNA synthesis, DNA digital data storage, and DNA-based molecular recording has been made by leveraging the activity of the template-independent DNA polymerase, terminal deoxynucleotidyl transferase (TdT). TdT adds deoxyribonucleotides to the 3' end of single-stranded DNA, generating random sequences of single-stranded DNA. TdT can use several divalent cations for its enzymatic activity and exhibits shifts in deoxyribonucleotide incorporation frequencies in response to changes in its reaction environment. However, there is limited understanding of sequence-structure-function relationships regarding these properties, which in turn limits our ability to modulate TdT to further advance TdT-based tools. Most TdT literature to-date explores the activity of murine, bovine or human TdTs; studies probing TdT sequence and structure largely focus on strictly conserved residues that are functionally critical to TdT activity. Here, we explore non-conserved TdT sequence space by surveying the natural diversity of TdT. We characterize a diverse set of TdT homologs from different organisms and identify several TdT residues/regions that confer differences in TdT behavior between homologs. The observations in this study can design rules for targeted TdT libraries, in tandem with a screening assay, to modulate TdT properties. Moreover, the data can be useful in guiding further studies of potential residues of interest. Overall, we characterize TdTs that have not been previously studied in the literature, and we provide new insights into TdT sequence-function relationships.

Microenvironmental Effect of 2'-O-(1-Pyrenylmethyl)uridine Modified Fluorescent Oligonucleotide Probes on Sensitive and Selective Detection of Target RNA.

2'-O-(1-Pyrenylmethyl)uridine modified oligoribonucleotides provide highly sensitive pyrene fluorescent probes for detecting specific nucleotide mutation of RNA targets. To develop more stable and cost-effective oligonucleotide probes, we investigated the local microenvironmental effects of nearby nucleobases on pyrene fluorescence in duplexes of RNAs and 2'-O-(1-pyrenylmethyl)uridine modified oligonucleotides. By incorporation of deoxyribonucleotides, ribonucleotides, 2'-MeO-nucleotides and 2'-F-nucleotides at both sides of 2'-O-(1-pyrenylmethyl)uridine (U(p)) in oligodeoxynucleotide probes, we synthesized a series of pyrene modified oligonucleotide probes. Their pyrene fluorescence emission spectra indicated that only two proximal nucleotides have a substantial effect on the pyrene fluorescence properties of these oligonucleotide probes hybridized with target RNA with an order of fluorescence sensitivity of 2'-F-nucleotides > 2'-MeO-nucleotides > ribonucleotides ≫ deoxyribonucleotides. While based on circular dichroism spectra, overall helix conformations (either A- or B-form) of the duplexes have marginal effects on the sensitivity of the probes. Instead, the local substitution reflected the propensity of the nucleotide sugar ring to adopt North type conformation and, accordingly, shifted their helix geometry toward a more A-type like conformation in local microenvironments. Thus, higher enhancement of pyrene fluorescence emission favored local A-type helix structures and more polar and hydrophobic environments (F > MeO > OH at 2' substitution) of duplex minor grooves of probes with the target RNA. Further dynamic simulation revealed that local microenvironmental effect of 2'-F-nucleotides or ribonucleotides was enough for pyrene moiety to move out of nucleobases to the minor groove of duplexes; in addition, 2'-F-nucleotide had less effect on π-stack of pyrene-modified uridine with upstream and downstream nucleobases. The present oligonucleotide probes successfully distinguished target RNA from single-mutated RNA analyte during an in vitro assay of RNA synthesis.

Strand Annealing and Terminal Transferase Activities of a B-family DNA Polymerase

DNA replication polymerases have the inherent ability to faithfully and rapidly copy a DNA template according to precise Watson-Crick base pairing. The primary B-family DNA replication polymerase (Dpo1) in the hyperthermophilic archaeon, Sulfolobus solfataricus, is shown here to possess a remarkable DNA stabilizing ability for maintaining weak base pairing interactions to facilitate primer extension. This thermal stabilization by Dpo1 allowed for template-directed synthesis at temperatures more than 30 °C above the melting temperature of naked DNA. Surprisingly, Dpo1 also displays a competing terminal deoxynucleotide transferase (TdT) activity unlike any other B-family DNA polymerase. Dpo1 is shown to elongate single-stranded DNA in template-dependent and template-independent manners. Experiments with different homopolymeric templates indicate that initial deoxyribonucleotide incorporation is complementary to the template. Rate-limiting steps that include looping back and annealing to the template allow for a unique template-dependent terminal transferase activity. The multiple activities of this unique B-family DNA polymerase make this enzyme an essential component for DNA replication and DNA repair for the maintenance of the archaeal genome at high temperatures.

Kinetic Basis of Sugar Selection by a Y-Family DNA Polymerase from Sulfolobus solfataricus P2

DNA polymerases use either a bulky active site residue or a backbone segment to select against ribonucleotides in order to faithfully replicate cellular genomes. Here, we demonstrated that an active site mutation (Y12A) within Sulfolobus solfataricus DNA polymerase IV (Dpo4) caused an average increase of 220-fold in matched ribonucleotide incorporation efficiency and an average decrease of 9-fold in correct deoxyribonucleotide incorporation efficiency, leading to an average reduction of 2000-fold in sugar selectivity. Thus, the bulky side chain of Tyr12 is important for both ribonucleotide discrimination and efficient deoxyribonucleotide incorporation. Other than synthesizing DNA as the wild-type Dpo4, the Y12A Dpo4 mutant incorporated more than 20 consecutive ribonucleotides into primer/template (DNA/DNA) duplexes, suggesting that this mutant protein possesses both a DNA-dependent DNA polymerase activity and a DNA-dependent RNA polymerase activity. Moreover, the binary and ternary crystal structures of Dpo4 have revealed that this DNA lesion bypass polymerase can bind up to eight base pairs of double-stranded DNA which is entirely in B-type. Thus, the DNA binding cleft of Dpo4 is flexible and can accommodate both A- and B-type oligodeoxyribonucleotide duplexes as well as damaged DNA.

Fidelity and Processivity of Reverse Transcription by the Human Mitochondrial DNA Polymerase

We have characterized, by transient-state kinetic methods, the polymerase and exonuclease activities of the human mitochondrial DNA polymerase (pol gamma) during reverse transcription, employing a synthetic oligonucleotide consisting of a DNA primer and an RNA template. In comparison with the kinetic parameters observed with a DNA template, the rate of correct deoxynucleotide incorporation was reduced 25-fold (5.5+/-0.2 s(-1)), whereas the dissociation constant (Kd) for nucleotide binding was increased 4-fold (12+/-1 microm). In addition, discrimination against mismatches was reduced approximately 20-fold to only 15,000 on average. The proofreading exonuclease favored the removal of an incorrect nucleotide (0.0021+/-0.0002 s(-1) for correct versus 0.034+/-0.004 s(-1) for incorrect), and the partitioning between incorporation beyond a mismatch (5.5x10(-5)+/-0.4x10(-5) s(-1)), and exonuclease removal of that mismatch favors removal of the mismatch. These data suggest that the "reverse transcriptase activity" of mitochondrial polymerase could be physiologically relevant. However, the enzyme stalls and is unable to efficiently incorporate beyond a single nucleotide with an RNA template. Additionally, we present a refined method for calculating net discrimination, which more accurately describes the contributions of correct and incorrect incorporation. The biological and biotechnological significance of these results are discussed.

Incorporation of deoxyribonucleotides and ribonucleotides by a dNTP-binding cleft mutated reverse transcriptase in hepatitis B virus core particles

Our recent observation that hepatitis B virus (HBV) DNA polymerase (P) might initiate minus-strand DNA synthesis without primer [Kim et al., (2004) Virology 322, 22–30], raised a possibility that HBV P protein may have the potential to function as an RNA polymerase. Thus, we mutated Phe 436, a bulky amino acid with aromatic side chain, at the putative dNTP-binding cleft in reverse transcriptase (RT) domain of P protein to smaller amino acids (Gly or Val), and examined RNA polymerase activity. HBV core particles containing RT dNTP-binding cleft mutant P protein were able to incorporate 32P-ribonucleotides, but not HBV core particles containing wild type (wt), priming-deficient mutant, or RT-deficient mutant P proteins. Since all the experiments were conducted with core particles isolated from transfected cells, our results indicate that the HBV RT mutant core particles containing RT dNTP-binding cleft mutant P protein could incorporate both deoxyribonucleotides and ribonucleotides in replicating systems.

The eureka enzyme: the discovery of DNA polymerase

The identification and partial purification by Arthur Kornberg and his colleagues in 1956 of an enzyme - DNA polymerase I of Escherichia coli - that catalysed the stable incorporation of deoxyribonucleotides into DNA in vitro came as a surprise. At the time, most scientists in the field believed that DNA synthesis was too complicated to be accurately reflected outside the living cell.

A sensitive new method for clinically monitoring cytarabine concentrations at the DNA level in leukemic cells

Cytarabine (ara-C), a major antileukemic agent, is phosphorylated in the cell to cytarabine triphosphate (ara-CTP), which is then partly incorporated into DNA. The drug incorporation into DNA poisons the extending primer against further incorporation of deoxyribonucleotides including dCTP, ultimately inhibiting DNA synthesis. While intracellular ara-CTP concentration has been found to predict clinical outcome, cytotoxicity in vitro is determined primarily by the extent of drug incorporation into DNA. However, clinically appropriate quantitation methods for ara-C at the DNA level have not been available. We developed a sensitive new method for monitoring ara-C incorporated into DNA in vivo. After DNA from leukemic cells was fractionated using the Schmidt-Thannhauser-Schneider method, it was degraded to constituent nucleosides to release ara-C, which was isolated from the nucleosides using HPLC and then measured by radioimmunoassay. Recovery for DNA fractionation, ara-C release by degradation, and ara-C isolation were 92.0 ± 6.4%, 90.7 ± 9.4%, and 98.5 ± 1.4%, respectively. The method was found to determine ara-C incorporation into DNA of ara-C-treated HL 60 cells in vitro with minimal interassay variation. The values determined were compatible with those determined by scintillation counting in parallel experiments using tritiated ara-C. Our method could be used to monitor DNA-incorporated ara-C concentrations during ara-C therapy, together with plasma ara-C and intracellular ara-CTP concentrations. ara-C incorporation into DNA appeared to be associated with intracellular retention of ara-CTP or persistence of plasma ara-C. Thus, the present method is sensitive, accurate, precise, and may permit therapeutic drug monitoring at the DNA level for better individualization of antileukemic regimens.

Amplification of Telomeric Arrays via Rolling-circle Mechanism

Alternative (telomerase-independent) lengthening of telomeres mediated through homologous recombination is often accompanied by a generation of extrachromosomal telomeric circles (t-circles), whose role in direct promotion of recombinational telomere elongation has been recently demonstrated. Here we present evidence that t-circles in a natural telomerase-deficient system of mitochondria of the yeast Candida parapsilosis replicate independently of the linear chromosome via a rolling-circle mechanism. This is supported by an observation of (i) single-stranded DNA consisting of concatameric arrays of telomeric sequence, (ii) lasso-shaped molecules representing rolling-circle intermediates, and (iii) preferential incorporation of deoxyribonucleotides into telomeric fragments and t-circles. Analysis of naturally occurring variant t-circles revealed conserved motifs with potential function in driving the rolling-circle replication. These data indicate that extrachromosomal t-circles observed in a wide variety of organisms, including yeasts, plants, Xenopus laevis, and certain human cell lines, may represent independent replicons generating telomeric sequences and, thus, actively participating in telomere dynamics. Moreover, because of the promiscuous occurrence of t-circles across phyla, the results from yeast mitochondria have implications related to the primordial system of telomere maintenance, providing a paradigm for evolution of telomeres in nuclei of early eukaryotes.

Terminal deoxynucleotidyl transferase is down-regulated by AP-1-like regulatory elements in human lymphoid cells.

Terminal deoxynucleotidyl transferase (TdT) is a template-independent DNA polymerase that catalyses the incorporation of deoxyribonucleotides into the 3'-hydroxyl end of DNA templates and is thought to increase junctional diversity of antigen receptor genes. TdT is expressed only on immature lymphocytes and acute lymphoblastic leukaemia cells and its transcriptional expression is tightly regulated. We had previously found that protein kinase C (PKC) activation down-regulates TdT expression. PKC-activation induces the synthesis of the Fos and Jun proteins, known as the major components of activation protein 1 (AP-1) transcriptional factor implicated in transcriptional control. Here we report the identification of several DNA-protein interactions within the TdT promoter region in non-TdT expressing human cells. Sequence analysis revealed the presence of a putative AP-1-like DNA-binding site, suggesting that AP-1 may play a relevant role in TdT transcriptional regulation. Using a different source of nuclear extracts and the AP-1-TdT motif as a probe we identified several DNA-protein retarded complexes in electrophoretic mobility shift assays. Super-band shifting analysis using an antibody against c-Jun protein confirmed that the main interaction is produced by a nuclear factor that belongs to the AP-1 family transcription factors. Our findings suggest that the TdT gene expression is down-regulated, at least in part, through AP-1-like transcription factors.

Terminal Deoxynucleotidyl Transferase Indiscriminately Incorporates Ribonucleotides and Deoxyribonucleotides

Terminal deoxynucleotidyl transferase (TdT) catalyzes the condensation of deoxyribonucleotides on 3'-hydroxyl ends of DNA strands in a template-independent manner and adds N-regions to gene segment junctions during V(D)J recombination. Although TdT is able to incorporate a few ribonucleotides in vitro, TdT discrimination between ribo- and deoxyribonucleotides has never been studied. We found that TdT shows only a minor preference for incorporation of deoxyribonucleotides over ribonucleotides on DNA strands. However, incorporation of ribonucleotides alone or in the presence of deoxyribonucleotides generally leads to premature chain termination, reflecting an impeded accommodation of ribo- or mixed ribo/deoxyribonucleic acid substrates by TdT. An essential catalytic aspartate in TdT was identified, which is a first step toward understanding the apparent lack of sugar discrimination by TdT.

Coupling Ribose Selection to Fidelity of DNA Synthesis: THE ROLE OF Tyr-115 OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 REVERSE TRANSCRIPTASE

The catalytic efficiency of incorporation of deoxyribonucleotides by wild-type human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) was around 100-fold higher than for dideoxyribonucleotides, in Mg(2+)-catalyzed reactions, and more than 10,000-fold higher than for nucleotides having a 2'-hydroxyl group in Mg(2+)- and Mn(2+)-catalyzed reactions. Mutant RTs with nonconservative substitutions affecting Tyr-115 (Y115V, Y115A, and Y115G) showed a dramatic reduction in their ability to discriminate against ribonucleotides in the presence of both cations. However, selectivity of deoxyribonucleotides versus ribonucleotides was not affected in mutants Y115W and F160W. The substitution of Tyr-115 with Val or Gly had no effect on discrimination against dideoxyribonucleotides, but these mutants were less efficient than the wild-type RT in discriminating against cordycepin 5'-triphosphate. We also show that Tyr-115 is involved in fidelity of DNA synthesis, but substitutions at this position have different effects depending on the metal cofactor used in the assays. In Mg(2+)-catalyzed reactions, removal of the side chain of Tyr-115 reduced misinsertion and mispair extension fidelity, while opposite effects were observed in Mn(2+)-catalyzed reactions. Our results indicate that the aromatic side chain of Tyr-115 plays a role as a "steric gate" preventing the incorporation of nucleotides with a 2'-hydroxyl group in a cation-independent manner, while its influence on fidelity could be modulated by Mg(2+) or Mn(2+).

Suppression of dexamethasone-stimulated DNA synthesis in an oncogene construct containing rat cell line by a DNA site-oriented ligand of poly-ADP-ribose polymerase: 6-Amino-1,2-benzopyrone

The cellular inhibitory effects of 6-amino-1,2-benzopyrone (6-ABP), a DNA site-specific ligand of adenosine diphosphoribosyl transferase (ADPRT), were determined in a dexamethasone-sensitive EJ-ras gene construct containing cell line (14C cells). Dexamethasone in vitro transforms these cells to a tumorigenic phenotype and also stimulates cell replication. At a nontoxic concentration (0.2 mM) 6-ABP treatment of intact cells for 4 days inhibits the dexamethasone-stimulated increment of cellular DNA content, depresses replicative DNA synthesis as assayed by thymidine incorporation to the level of cells that were not exposed to dexamethasone, and in permeabilized cells reduces the dexamethasone-stimulated increase of deoxyribonucleotide incorporation into DNA to the level of untreated cells. In situ pulse labeling of cells pretreated with 6-ABP indicated an inhibition of DNA synthesis at a stage prior to the formation of the 10-kb intermediate species. The drug had no direct effect on cellular DNA polymerases as tested in vitro, and the inhibition of DNA synthesis in permeabilized cells following drug treatment for 4 days was abolished by externally added DNA templates. Neither dexamethasone nor the drug influenced the cellular quantity of ADPRT molecules, tested immunochemically.

Evidence for the presence of DNA primase in mitochondria of Saccharomyces cerevisiae

Chromatography of a DNA polymerase preparation from mitochondria of Saccharomyces cerevisiae on DNA-cellulose column, using Tris-HCl (pH 7.5) buffer containing 0.6 M NaCl as eluent, was found to yield a fraction exhibiting DNA primase-like activity free of DNA polymerase. This fraction could support the synthesis of 12–15 residue-long oligoribonucleotides on single-stranded natural or synthetic DNA templates. The oligoribonucleotides could be further elongated by incorporation of deoxyribonucleotides in the presence of Klenow fragment.

Purification and characterization of the dnaA46 gene product.

The alleles of dnaA46, dnaA5, and dnaA204 have been cloned from the Escherichia coli chromosome into a protein-overproducing vector. Under conditions of induced expression, similar amounts of dnaA46 and dnaA5 gene products and less of the dnaA204 gene product were observed. Facilitated by elevated expression levels, an improved method for purification of dnaA+ protein and the purification of dnaA46 protein have been obtained. The replication activity of dnaA46 protein did not appear to be thermolabile upon prior incubation at various temperatures. Thermolabile replication activity was observed under conditions of coupled DNA synthesis. In contrast with the wild type protein, DNA synthesis dependent on dnaA46 protein showed a pronounced lag before incorporation of deoxyribonucleotides.

Impairment of nucleotide metabolism by iron-chelating deferoxamine

The effect of deferoxamine on nucleotide metabolism in HL-60 leukemic cells was studied to explore the mechanism of its antiproliferation activity. It was found that in intact cells deferoxamine markedly inhibited the ribonucleotide reduction and incorporation of bases (adenine, hypoxanthine), ribonucleosides (inosine, guanosine) and deoxyribonucleosides (thymidine, deoxyadenosine, deoxyguanosine) into nucleic acids. Although deferoxamine did not inhibit thymidine and uridine incorporation into free nucleotides, inhibition of hypoxanthine and adenine incorporation into nucleotides as well as inhibition of nucleotide biosynthesis de novo was found. Nucleotide catabolism, protein synthesis, and intracellular levels of ribonucleotides were not affected significantly by deferoxamine. These results showed that deferoxamine selectively affects several specific reactions of nucleotide metabolism. Inhibition of ribonucleotide reduction, inhibition of ribonucleotide and deoxyribonucleotide incorporation into nucleic acids, as well as inhibition of purine biosynthesis, may alter significantly cellular physiology and, therefore, contribute significantly to the antiproliferative activity of deferoxamine.

Identification and characterization of cauliflower mosaic virus replication complexes—Analogy to hepatitis B viruses

Putative replication complexes of cauliflower mosaic virus (CaMV) which resemble hepatitis B virus replication complexes in several respects have been identified. The CaMV replication complexes contain a heterogeneous population of minus-strand DNA ranging in size from less than 600 nucleotides to full genome length of 8000 nucleotides. The minus-strand DNA of these replication complexes is largely single stranded or in a DNA/RNA hybrid structure. All of the plus-strand DNA within the complexes is in a DNA duplex which contains regions of single-stranded minus-strand DNA. These DNAs appear to be replication intermediates arising from reverse transcription of the CaMV 35 S RNA and to be encapsidated into virions or virion-like particles. The same fractions from sucrose density gradients that contain the putative replication intermediates also contain an enzymatic activity that incorporates deoxyribonucleotide triphosphates in both minus- and plus-strand CaMV DNA. This replication activity is consistent with a reverse transcription model for CaMV replication based on results which demonstrate that incorporation of deoxyribonucleotides into minus-strand DNA is refractory to actinomycin D while incorporation into plus-strand DNA is inhibited by the drug.

Inhibitors of DNA polymerases: their selectivity and mode of action.

The first DNA polymerase in mammalian cells was discovered by Bollum (1958), but only in the last years the existence of multiple forms of DNA polymerases was recognized in these cells. In the recent years, there has been a great interest in the purification and characterization of different eukaryotic DNA polymerases, and to study the functional role(s) of each of these enzymes. The field of DNA polymerases gained an unexpected importance as in 1970, a novel DNA polymerase was discovered as a constituent of retrovirus (Temin and Mizutani, 1970; Baltimore 1970), now known as reverse transcriptase (RT). In addition, several new DNA-polymerizing activities are induced in eukaryotic cells infected with DNA viruses. Another unique enzyme which also exhibits a deoxyribonucleotide polymerizing activity is the terminal deoxyribonucleotidyl transferase (TdT). This enzyme, though a cellular enzyme, is unique in the sense that, so far, only thymus tissue has been shown to contain it. In the strictest sense TdT is not a DNA polymerase as it does not require a template to direct the DNA synthesis; nevertheless, it does catalyze the incorporation of deoxyribonucleotides into DNA and thus we consider it here as one of the DNA synthetic enzymes. Like RT the TdT also merits considerable attention as a biochemical marker’for certain types of leukemias, as will be discussed by Dr. Sarin and Dr. Mertelsmann is this proceeding.

Evidence for the absence of DNA proofreading in HeLa cell nuclei.

[3H]2-Aminopurine deoxyribonucleoside triphosphate and [32P]dATP were added exogenously at equimolar concentrations to washed HeLa cell nuclei both in the presence and absence of cell cytoplasm. The observed ratio of 2-aminopurine/adenine deoxyribonucleotide incorporation into DNA was about 12%, which is consistent with 2-aminopurine misinsertion frequencies measured in cell-free assays, for various DNA polymerases including alpha-polymerase from calf thymus, Escherichia coli polymerase I, and several mutant and wild type bacteriophage T4 polymerases. Based on the 12% 2-aminopurine/adenine misincorporation ratio, we propose that proofreading of replicating DNA is not occurring in HeLa nuclei, and that discrimination against 2-aminopurine incorporation is governed primarily by a 1.1 kcal/mol difference in free energy between 2-aminopurine.thymine and adenine.thymine base pairs rather than by properties attributable to either the mammalian DNA polymerase or HeLa cell nuclear replication apparatus.

The effect of phenethyl alcohol on in vitro DNA synthesis in Escherichia coli

The effect of phenethyl alcohol on DNA synthesis was examined using several in vitro systems of Escherichia coli H560; i.e., ether-treated cells, membrane fractions and folded chromosomes fortified with DNA polymerase. In all systems, the incorporation of deoxyribonucleotides was much reduced for the phenethyl alcohol-treated cells compared with the non-treated cells. The total activity of DNA polymerases in polA 1 cells (mostly DNA polymerase II) was not impaired for the phenethyl alcohol-treated cells and the reduction of the rate of DNA synthesis in vitro was ascribed to the reduction of the chromosomal template activity which was related to trypsin sensitive protein components. The analysis of chromosomes from the phenethyl alcohol-treated cells revealed the remarkable reduction of a protein component of molecular weight approx. 58 000 in contrast with a protein component of molecular weight approx. 30 000.