Role of carcinogen-modified deoxynucleotide precursors in mutagenesis

Agents which damage or modify cellular DNA will generally also modify the nucleotide precursor pools, sometimes preferentially (Topal and Baker, 1982). There are at least two different ways that incorporation of modified (possibly promutagenic) nucleotides could, theoretically, make a significant contribution to the mutations induced by these agents. Modified bases may exhibit ambiguous base pairing and produce mutations during normal replication or they may induce secondary mutations as a result of processing subsequent to incorporation. There are important precedents for such possibilities. Classical studies on mutagenesis with prototype mutagens like 2-aminopurine (2-AP) and 5-bromouracil clearly show that mutations can occur by incorporation of deoxynucleotides of tautomeric or ionized (Sowers et al., 1987) bases into newly synthesized DNA (Ronen, 1979; Lasken and Goodman, 1984, Coulondre and Miller, 1977). 5-Hydroxymethyl-2′-deoxyuridine (HMdU), a product of oxidative DNA damage, can also be (re)incorporated into cellular DNA with both toxic and mutagenic consequences (Kaufman, 1987; Shirname-More et al., 1987). Furthermore, modified nucleotides may alter the pool sizes of the normal nucleotides and indirectly produce toxic and mutagenic effects. However, these effects are generally see at high, nonphysiological, concentrations of the modified precursors and may not be relevant under physiological conditions. The relative importance of modified deoxynucleotide precursors in the production of mutations by alkylating and oxidative DNA-damaging agents is discussed.     

Deoxycytidine transport and pyrimidine deoxynucleotide metabolism in phytohaemagglutinin-stimulated pig lymphocytes.

Increased entry of deoxy[3H]cytidine begins at about 12h after addition of phytohaemagglutinin to peripheral pig lymphocyte cultures, and is accompanied by a parallel stimulation of deoxycytidine kinase up to the beginning of DNA synthesis at 24h. The increased deoxycytidine uptake is characterized by an increase in Vmax. without alteration of the apparent Km (0.7 +/- 0.11 muM). Although the entries of both nucleosides are promoted at the same time, the stimulation of deoxycytidine uptake is less than that of thymidine, and the two nucleosides are transported by separate systems. In addition to deoxycytidien kinase, the synthesis of deoxycytidylate deaminase and thymidylate synthetase are stimulated after addition of phytohaemagglutinin, but to a lesser extent than that of thymidine kinase. The importance of the latter enzyme in forming dTMP, and of thymidylate kinase in providing dTTP, is discussed.     

The role of ribosomes in stabilizing bacteriophage T4 deoxynucleotide kinase mRNA.

In the present investigation, an approach toward defining the role of ribosomes in stabilizing functional messenger RNA in cell-free extracts is described. The data presented show that initiation of protein synthesis is necessary for maximal functional stability of bacteriophage T4 deoxynucleotide kinase mRNA $\text{in vitro}$ and suggest that much of the stability is attained by interaction of the deoxynucleotide kinase mRNA initiation site with a 30S ribosomal subunit. Data is also presented which suggest that any of several $\text{E}$. $\text{coli}$ ribonucleases could serve as a messenger ribonuclease $\text{in vivo}$.     

Quantitative analysis of deoxynucleotide substitutions in the codon-anticodon helix

The role of 2' hydroxyl groups in the codon-anticodon helix was evaluated by introducing single deoxynucleotides into each of the six positions in the helix and measuring the affinity of tRNA to either the A site or the P site of Escherichia coli 70S ribosomes. In perfect agreement with the X-ray structure of the Thermus thermophilus 30S subunit, A site binding was weaker in five of the six positions but P site binding was unaffected. Since the addition of paromomycin restores A site binding, it appears that the deoxynucleotide substituted complexes are impaired in their ability to promote the ribosomal conformational change that accompanies tRNA binding.     

Site-specific deoxynucleotide substitutions in yeast U6 snRNA block splicing of pre-mRNA in vitro.

We have identified 2'-hydroxyl groups of the U6 phosphate-ribose backbone which are required for reconstitution of splicing activity in U6-depleted yeast extract. To screen the 2'-hydroxyls of yeast U6 at nucleotides 39-88, spanning the conserved central domain, synthetic U6 RNAs were constructed with deoxyribonucleotides incorporated site specifically. Only four individual deoxynucleotide substitutions blocked splicing activity: dA51 (in the ACAGAG sequence), dA62 (next to the AGC triad), and dU70 and dC72 (both in the loop of the 3' intramolecular stem-loop). Native gel analysis revealed that these deoxy-substituted U6 RNAs were competent for assembly of spliceosomes. Interestingly, a 2'-O-methyl substituent at A51, A62, U70 or C72 did not inhibit splicing activity, indicating that the essential 2'-OH groups at these positions in U6 act as hydrogen bond acceptors or neutral coordinated ligands. The requisite 2'-hydroxyls at A62, U70 and C72 show both similarities and differences relative to the positions of essential 2'-hydroxyls of catalytic domain V of group II ribozymes. The identification of the essential 2'-hydroxyls at positions 62, 70 and 72 corroborates that the 3' intramolecular stem-loop in U6 plays an important role in pre-mRNA splicing.     

Anatomy of bovine mammillitis DNA II. Size and arrangements of the deoxynucleotide sequences.

We previously reported that bovine mammillitis virus (BMV) DNA consists of two covalently linked components designated L and S and estimated to be 71.5 x 10(6) and 15.7 x 10(6) in molecular weight, respectively; the components invert relative to each other, giving rise to four equimolar populations differing soley in the relative orientation of the two components. We now report that (i) BMV DNA has a contour length corresponding to a molecular weight of 89 x 10(6). (ii) Component L consists of a unique sequence (Ul) bracketed by sequences ab and its inverted repeat b'a', estimated to be of molecular weights 66.1 x 10(6), 2.7 x 10(6), and 2.7 x 10(6), respectively. (iii) Component S consists of a unique sequence (Us) bracketed be sequence ca and its inverted repeat a'c', estimated to be of molecular weights 8.3 x 10(6), 3.7 x 10(6), and 3.7 x 10(6), respectively. (iv) The a sequences present at the termini of a complete linear molecule (abUlb'a'a'c'Usca) are arranged in tandem so that the DNA can circularize after limited digestion with arranged in tandem so that the DNA can circularize after limited digestion with lambda 5'-exonuclease. The size of the a sequences was estimated to be 0.7 x 10(6) in molecular weight. (v) At least portions of the a sequences are repeated in an inverted orientation immediately adjacent to or near the a sequence. Thus, BMV DNA mimics herpes simplex virus type 1 DNA with respect to the arrangement but not size of deoxynucleotide sequences. The evolutionary relationship of BMV DNA relative to other herpesvirus DNAs is discussed.     

Bacteriophage T4 deoxynucleotide kinase: gene cloning and enzyme purification.

Gene 1 of bacteriophage T4 has been cloned into a lambda pL expression vector, resulting in the overproduction of deoxynucleotide kinase. A procedure that includes affinity chromatography on Cibacron Blue F3GA-agarose has been used to purify milligram quantities of enzymes from a 2-liter culture. The enzyme has been partially characterized in vitro and in vivo, and it appears to be identical to the deoxynucleotide kinase isolated from T4-infected Escherichia coli. These results prove the earlier contention that the phosphorylation of three dissimilar deoxynucleotides (5-hydroxymethyldeoxycytidylate, dTMP, and dGMP), to the exclusion of most others, is catalyzed by a single protein.     

Studies in phytosterol biosynthesis. Mechanism of biosynthesis of cycloartenol

1. The mechanism of cycloartenol biosynthesis in leaves of Solanum tuberosum was investigated with the use of [2-¹⁴C,(4R)-4-³H₁]mevalonic acid. 2. The ³H/¹⁴C atomic ratio in cycloartenol was 6:6, the same as that in squalene; this eliminates lanosterol as a possible biosynthetic precursor of cycloartenol, and indicates that a hydrogen migration from C-9 to C-8 occurs. 3. Chemical isomerization of the cycloartenol to lanosterol (³H/¹⁴C ratio 5:6) and parkeol (³H/¹⁴C ratio 6:6) confirms the hydrogen migration from C-9 to C-8. 4. Possible mechanisms for the biosynthesis of cycloartenol and parkeol are discussed. 5. The ³H/¹⁴C ratio for 24-methylenecycloartanol was 6:6, demonstrating that the hydrogen atom at C-24 is retained during alkylation of the cycloartenol side chain.     

A new endonuclease recognizing the deoxynucleotide sequence CCNNGG from the cyanobacterium Synechocystis 6701.

A new sequence-specific endonuclease from the cyanobacterium Synechocystis species PCC 6701 has been purified and characterized. This enzyme, SecI, is unique in recognizing the nucleotide sequence: 5' -CCNNGG-3' 3' -GGNNCC-5' and cleaves it at the position indicated by the symbol. Two other restriction endonucleases, SecII and SecIII, found in this organism are isoschizomers of MspI and MstII, respectively.     

Crystal structure of bacteriophage T4 deoxynucleotide kinase with its substrates dGMP and ATP.

NMP kinases catalyse the phosphorylation of the canonical nucleotides to the corresponding diphosphates using ATP as a phosphate donor. Bacteriophage T4 deoxynucleotide kinase (DNK) is the only member of this family of enzymes that recognizes three structurally dissimilar nucleotides: dGMP, dTMP and 5-hydroxymethyl-dCMP while excluding dCMP and dAMP. The crystal structure of DNK with its substrate dGMP has been determined at 2.0 A resolution by single isomorphous replacement. The structure of the ternary complex with dGMP and ATP has been determined at 2.2 A resolution. The polypeptide chain of DNK is folded into two domains of equal size, one of which resembles the mononucleotide binding motif with the glycine-rich P-loop. The second domain, consisting of five alpha-helices, forms the NMP binding pocket. A hinge connection between the domains allows for large movements upon substrate binding which are not restricted by dimerization of the enzyme. The mechanism of active centre formation via domain closure is described. Comparison with other P-loop-containing proteins indicates an induced-fit mode of NTP binding. Protein-substrate interactions observed at the NMP and NTP sites provide the basis for understanding the principles of nucleotide discrimination.     

A computational model of mitochondrial deoxynucleotide metabolism and DNA replication

We present a computational model of mitochondrial deoxynucleotide metabolism and mitochondrial DNA (mtDNA) synthesis. The model includes the transport of deoxynucleosides and deoxynucleotides into the mitochondrial matrix space, as well as their phosphorylation and polymerization into mtDNA. Different simulated cell types (cancer, rapidly dividing, slowly dividing, and postmitotic cells) are represented in this model by different cytoplasmic deoxynucleotide concentrations. We calculated the changes in deoxynucleotide concentrations within the mitochondrion during the course of a mtDNA replication event and the time required for mtDNA replication in the different cell types. On the basis of the model, we define three steady states of mitochondrial deoxynucleotide metabolism: the phosphorylating state (the net import of deoxynucleosides and export of phosphorylated deoxynucleotides), the desphosphorylating state (the reverse of the phosphorylating state), and the efficient state (the net import of both deoxynucleosides and deoxynucleotides). We present five testable hypotheses based on this simulation. First, the deoxynucleotide pools within a mitochondrion are sufficient to support only a small fraction of even a single mtDNA replication event. Second, the mtDNA replication time in postmitotic cells is much longer than that in rapidly dividing cells. Third, mitochondria in dividing cells are net sinks of cytoplasmic deoxynucleotides, while mitochondria in postmitotic cells are net sources. Fourth, the deoxynucleotide carrier exerts the most control over the mtDNA replication rate in rapidly dividing cells, but in postmitotic cells, the NDPK and TK2 enzymes have the most control. Fifth, following from the previous hypothesis, rapidly dividing cells derive almost all of their mtDNA precursors from the cytoplasmic deoxynucleotides, not from phosphorylation within the mitochondrion. simulation; nucleotide phosphorylation; nucleoside transport; mitochondrial DNA     

dTMP biosynthesis in Archaea.

The biosynthesis of dTMP has been studied in cell extracts of two different members of the domain Archaea, Methanosarcina thermophila and Sulfolobus solfataricus. In M. thermophila, the dTMP was formed from dUMP and [methylene-2H2]-5,10-methylenetetrahydrosarcinapterin generated in situ from added [methylene-2H2] formaldehyde and the tetrahydrosarcinapterin present in the cell extract. In S. solfataricus, the 5,10-methyl-enetetrahydro derivative of a synthetic fragment of sulfopterin, the modified folate present in these cells, served as the C1 donor. These data indicate that the Archaea thymidylate synthases carry out the same basic reaction which occurs in other organisms but use the 5,10-methylenetetrahydro derivatives of modified folates as C1 donors.     

A novel artificial intelligence-based approach for identification of deoxynucleotide aptamers

The selection of a DNA aptamer through the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method involves multiple binding steps, in which a target and a library of randomized DNA sequences are mixed for selection of a single, nucleotide-specific molecule. Usually, 10 to 20 steps are required for SELEX to be completed. Throughout this process it is necessary to discriminate between true DNA aptamers and unspecified DNA-binding sequences. Thus, a novel machine learning-based approach was developed to support and simplify the early steps of the SELEX process, to help discriminate binding between DNA aptamers from those unspecified targets of DNA-binding sequences. An Artificial Intelligence (AI) approach to identify aptamers were implemented based on Natural Language Processing (NLP) and Machine Learning (ML). NLP method (CountVectorizer) was used to extract information from the nucleotide sequences. Four ML algorithms (Logistic Regression, Decision Tree, Gaussian Naïve Bayes, Support Vector Machines) were trained using data from the NLP method along with sequence information. The best performing model was Support Vector Machines because it had the best ability to discriminate between positive and negative classes. In our model, an Accuracy (A) of 0.995, the fraction of samples that the model correctly classified, and an Area Under the Receiving Operating Curve (AUROC) of 0.998, the degree by which a model is capable of distinguishing between classes, were observed. The developed AI approach is useful to identify potential DNA aptamers to reduce the amount of rounds in a SELEX selection. This new approach could be applied in the design of DNA libraries and result in a more efficient and faster process for DNA aptamers to be chosen during SELEX.