C4′ sugar oxidation of deoxyribonucleotide triphosphates by chromium(V) complexes

The Cr(V) complexes, bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) ([OCr^V(ehba)₂]⁻) and (2,2-bis(hydroxymethyl)-2-(bis(2-hydroxyethyl)amino)ethanolato)oxochromate(V) ([OCr^V(BT)]²⁻), were reacted with a series of deoxyribonucleotide triphosphates. Oxidation of deoxyribose at C4′ was observed by measuring the amount of thiobarbituric acid reactive species (TBARS) produced in these reactions. For both compounds, the TBARS obtained with purine nucleotides was between 2.25 and 3.5 times greater than what was observed with pyrimidine nucleotide. This result suggests that the identity of the nucleic acid base can influence the hydrogen atom abstraction at C4′. Overall, the amount of product obtained with [OCr^V(BT)]²⁻ was significantly less than what was observed with [OCr^V(ehba)₂]⁻, indicating that these two Cr(V) model complexes may oxidize DNA differently.     

Analysis of mutagenesis induced by a thermolabile T4 phage deoxycytidylate hydroxymethylase suggests localized deoxyribonucleotide pool imbalance

Summary To understand the molecular basis of mutation stimulated by deoxyribonucleotide pool imbalance, we studied a temperature-sensitive T4 phage gene 42 mutant (LB3), which specifies a thermolabile deoxycytidylate hydroxymethylase. Analysis of rII mutations, revertible to wild type along either GC-to-AT or AT-to-GC transition pathways, showed 8- to 80-fold stimulation of GC-to-AT mutations at a semi-permissive temperature (34° C). One such marker, rII SN103, which showed the highest stimulation at 34° C, was sequenced after amplification of the template by polymerase chain reaction. The mutant site in rII SN103 was identified at nucleotide position 265 from the rII B translational start as an AT-to-GC transition, which changes TCA to CCA. Sequence analysis of revertants and pseudorevertants generated at 34° C showed that both cytosines within this triplet can undergo change to either thymine or adenine, consistent with the hypothesis that hydroxymethyldeoxycytidine triphosphate pools are depleted at replication sites. However, dNTP pool measurements in extracts of 34° C cultures showed no significant deviations from values obtained at 30° C, suggesting that pool imbalances occur only locally, close to replication forks. Our studies support the hypothesis that the imitator phenotype displayed by ts LB3 at semi-permissive temperature is a consequence of perturbation of the flow of nucleotide precursors into the DNA replication machinery. A putative localized depletion of hm-dCTP presumably enlarges effective dTTP/hm-dCTP and dATP/hm-dCTP pool ratios, resulting in the observed C-to-T transition and C-to-A transversion mutations.     

Requirement for cell proliferation for the effects of 5-bromo-2′-deoxyuridine on cultures of chick chondrocytes

Replication was inhibited in cultures of chick chondrocytes either by growing the cells at high density in ‘pellets’, or by use of inhibitors of DNA synthesis. Exposure to BUdR in either condition did not result in the fibroblast-like morphology and marked inhibition of chondroitin sulfate synthesis which is obtained if replicating cells are exposed to the analog. Growth of chondrocytes for 3 days in media containing embryo extract resulted in a switch from predominantly chondroitin sulfate synthesis to the exclusive synthesis of a hyaluronic acid-like molecule.     

Changes in protein synthesis and in RNA poly A+ population after treatment of Dictyostelium amoebae by 5-bromo-2′-deoxyuridine

Incorporation of 5-bromodeoxyuridine (5-BUdR) into nuclear DNA severely interrupts the life cycle of Dictyostelium discoideum after the first generation of growth. Loose cellular aggregates are then formed, but no spore or stalk cells are detectable and no other morphological transformations are observed. The perturbation of gene expression in the life cycle has been studied at the protein level by two-dimensional gel electrophoresis after pulse labelling with 35S-methionine and also by changes in the patterns of polysomal messenger RNA population. The latter was monitored by hybridisation studies using specific cDNA probes for “vegetative” and “18 hr” messenger RNAs. In the presence of 5-BUdR major anomalies in polypeptide synthesis were observed after the loose aggregation stage. Some vegetative polypeptides, including actin, which are normally abundant only during growth to the aggregation stage, are oversynthesised during the period 12-24 hr after starvation. In this same interval the normal decline in the abundance of vegetative mRNA species was not observed. In marked contrast virtually half the normal “18 hr-specific polypeptides” were poorly synthesised. Likewise, the normal increase in abundance of the corresponding “18 hr-specific” poly A + RNA species in the polysomes did not occur. No major alteration in the timing of the appearance of new macromolecules during the cell cycle was observed in spite of extensive modification of gene expression by the incorporation of 5-BUdR into genomic DNA.     

In vivo determination of 5-bromo-2′-deoxyuridine incorporation into DNA tumor tissue by a new P-postlabelling thin-layer chromatographic method

The halopyrimidine 5-bromo-2′-deoxyuridine (BUDR) can serve as one of many indicators of tumor malignity, complementary to histologic grade. We have developed a thin-layer chromatographic (TLC) technique that can assess tumor DNA base composition and analogue (BUDR) incorporation which vies with immunochemistry for BUDR. This requires post-labeling DNA by nick-translation and radioactive 5′-phosphorylation of representative ³²P-α-dNMPs (deoxynucleotide monophosphates). Subsequent 3′-monophosphate digest exchanges a radioactive ³²PO₄ for the neighboring cold nucleotide. Separation in two dimensional PEI-cellulose TLC is carried out in acetic acid, (NH₄)₂SO₄, and (NH₄)HS0₄. TLC of dNMPs was applied to control HeLa DNA, and HeLa cells receiving BUDR. BUDR is detected in 10⁶ HeLa cells after 12–72 h incubations. Findings in HeLa DNA demonstrate normal TLC retention factors for all ³²P-dNMPs. Two dimensional R_F (x,y axes in cm) demonstrate: dAMP=1.4, 9.4; dCMP=10.0, 13.5; dGMP=4.6, 4.4; dTMP=9.0, 7.4; and BUDRMP 6.4, 6.6. This technique quantifies BUDR-which parallels tumor S phase, and serves as an indicator of labelling index (LI).     

Nucleosides and nucleotides. 126. Incorporation of a mutagenic nucleoside, 5-formyl-2′-deoxyuridine, into an oligodeoxyribonucleotide

An oligodeoxyribonucleotide containing 5-formyl-2′-deoxyuridine (1) was synthesized using a new protecting group of the formyl group.     

The role of 5′-methylthioadenosine phosphorylase in 5′-methylthioadenosine-mediated inhibition of lymphocyte transformation

To determine if increased 5′-methylthioadenosine phosphorylase activity in activated lymphocytes may be responsible for the decreased inhibitory effect noted when 5′-methylthioadenosine is added after stimulation, the activity of this enzyme was monitored during lymphocyte transformation. A direct correlation existed between the transformation process and 5′-methylthioadenosine phosphorylase activity; the longer the stimulation process progressed, the greater the enzyme activity. The 7-deaza analog of 5′-methylthioadenosine, 5′-methylthiotubercidin, was utilized to explore further the role that the phosphorylase may play in the reversal process. 5′-Methylthioadenosine acted as a potent inhibitor, but not a substrate, of the 5′-methylthioadenosine phosphorylase, and was an even more potent inhibitor of lymphocyte transformation than 5′-methylthioadenosine. However, in direct contrast to the 5′-methylthioadenosine effect, inhibition by 5′-methylthiotubercidin could not be completely reversed. These data suggest the 5′-methylthioadenosine phosphorylase plays an important role in reversing 5′-methylthioadenosine-mediated inhibition and that the potent, nonreversible inhibitory effects of 5′-methylthiotubercidin are due to its resistance to 5′-methylthioadenosine phosphorylase degradation.     

Parathyroid hormone inhibition of induction of alkaline phosphatase activity by hydrocortisone and 5-iodo-2′-deoxyuridine in HeLa cells

Parathyroid hormone blocked the induction by hydrocortisone or IUdR of alkaline phosphatase activity in HeLa S3 cells. Alkaline phosphatase activity was not affected by parathyroid hormone treatment alone. Parathyroid hormone added to cells preinduced with hydrocortisone prevented further induction of alkaline phosphatase activity and caused an eventual return of activity to the non-induced level.     

The role of adenosine 3′,5′-monophosphate in the transformation of cloudman mouse melanoma cells

The role of adenosine 3′,5′-monophosphate (cyclic AMP) in the regulation of mouse melanoma cell growth and differentiation was investigated. A variant melanoma (Cloudman S91-F) which displays a greater degree of transformation than the parental cell (Cloudman S91) was isolated. A correlation between cyclic AMP metabolism and transformation was made. Dibutyryl cyclic AMP depressed cell growth and increased pigmentation in both parental and variant cell lines. The parental cell line, however, was more responsive to melanocyte-stimulating hormone (MSH) which was found to affect cell growth and pigmentation by increasing cyclic AMP levels. The more transformed S91-F cell line contained lower levels of cyclic AMP than the parental cell line, and this fact correlated well with the higher degree of growth and lesser degree of pigmentation in the variant. Enzymatic analysis revealed that the hydrolysis of cyclic AMP in both cell lines was similar, while the adenylate cyclase activity of the variant cell line was lower than that of the parental cell line. Lineweaver-Burk plots demonstrated that the K_m′s for the enzymes in the two cell lines were the same but that the V_max of the S91-F cell line was significantly less than that of the S91 cell line. Thus, the lesion in the S91-F cell which is responsible for its more transformed characteristics seems to be one which affects adenylate cyclase at the level of the cell membrane.     

Studies on the penetration of mammalian cells by deoxyribonucleoside-5′-phosphates

We have tested the ability of [5′-³²P]-deoxyribonucleoside monophosphates (dNMPs) to penetrate living mouse fibroblast L cells and human HeLa cells. Under the conditions of our experiments, small numbers of apparently intact dNMP molecules appeared to penetrate into the interior of L cells and be incorporated into DNA. This incorporation was not due to mycoplasma contamination nor to extracellular hydrolysis of the dNMPs followed by resynthesis inside the cell. Under these same conditions, penetration of HeLa cells by intact dNMPs did not occur to a significant extent. However, HeLa cells were capable of hydrolyzing extracellular dNMPs to Pi and deoxyribonucleosides at a much faster rate than L cells. These experiments provide a starting point for attempts to specifically label the DNA in intact, living eukaryotic cells with [³²P]-dNMPs.     

Incorporation of 5-bromo-2′deoxyuridine (BUdR) in Dictyostelium discoideum DNA

The modality of incorporation of 5-bromo-2′deoxyuridine (BUdR) in the DNA of Dictyostelium discoideum was studied after one generation of growth of the amoebae in the presence of different concentrations of the drug. The analog was incorporated following the semiconservative pattern of DNA replication. BUdR incorporation in monosubstituted DNA has been measured both by CsCl isopycnic centrifugation or by base analysis chromatography; substitution of thymidine by its analog reaches a maximal value of 30% (60% in the substituted strand). Up to 20% substitution it is proportional to the drug concentration in the growth medium. In these conditions, thymidine substitution is higher in repetitive sequences of the DNA than in unique sequences; the percent of increase of thymidine substitution in repetitive fractions versus total DNA is inversely proportional to thymidine substitution in total DNA.     

Mechanisms of degradation of 2′-5′ oligoadenylates

We have studied the mechanisms of breakdown of 2'-5' oligoadenylates. We monitored the time-courses of degradation of ppp(A2'p5')nA (dimer to tetramer) and of 5'OH-(A2'p5')nA (dimer to pentamer) in unfractionated L1210 cell extract. The 5' triphosphorylated 2'-5' oligoadenylates are converted by a phosphatase activity. However, 2'-5' oligoadenylates are degraded mainly by phosphodiesterase activity which splits the 2'-5' phosphodiester bond sequentially at the 2' end to yield 5' AMP and one-unit-shorter oligomers. The nonlinear least-squares curve-fitting program CONSAM was used to fit these kinetics and to determine the degradation rate constant of each oligomer. Trimers and tetramers, whether 5' triphosphorylated or not, are degraded at the same rate, whereas 5' triphosphorylated dimer is rapidly hydrolyzed and 5'-OH dimer is the most stable oligomer. The interaction between degradation enzymes and the substrate strongly depends on the presence of a 5' phosphate group in the vicinity of the phosphodiester bond to be hydrolyzed; indeed, when this 5' phosphate group is present, as in pp/pA2'p5'A/or A2'/p5'A2'p5'A/, affinity is high and maximal velocity is low. Such a degradation pattern can control the concentration of 2'-5' oligoadenylates active on RNAse L either by limiting their synthesis (5' triphosphorylated dimer is the primer necessary for the formation of longer oligomers) and/or by converting them into inhibitory (e.g., monophosphorylated trimer) or inactive (e.g., nonphosphorylated oligomers) molecules.     

Nature of the SOS mutator activity: genetic characterization of untargeted mutagenesis in Escherichia coli

Summary In Escherichia coli, induction of the SOS functions by UV irradiation or by mutation in the recA gene promotes an SOS mutator activity which generates mutations in undamaged DNA. Activation of RecA protein by the recA730 mutation increases the level of spontaneous mutation in the bacterial DNA. The number of recA730-induced mutations is greatly increased in mismatch repair deficient strains in which replication errors are not corrected. This suggests that the majority of recA730-induced mutations (90%) arise through correctable, i.e. non-targeted, replication errors. This recA730 mutator effect is suppressed by a mutation in the umuC gene. We also found that dam recA730 double mutants are unstable, segregating clones that have lost the dam or the recA mutations or that have acquired a new mutation, probably in one of the genes involved in mismatch repair. We suggest that the genetic instability of the dam recA730 mutants is provoked by the high level of replication errors induced by the recA730 mutation, generating killing by coincident mismatch repair on the two unmethylated DNA strands. The recA730 mutation increases spontaneous mutagenesis of phage poorly. UV irradiation of recA730 host bacteria increases phage untargeted mutagenesis to the level observed in UV-irradiated recA ⁺ strains. This UV-induced mutator effect in recA730 mutants is not suppressed by a umuC mutation. Therefore UV and the recA730 mutation seem to induce different SOS mutator activities, both generating untargeted mutations.     

Culture of adult rat lung cells: Benzo(a)pyrene metabolism and mutagenesis

Summary A method is described for obtaining and culturing large numbers of lung cells from normal adult male rats. The lungs were perfused in situ to remove blood cells and then perfused via the trachea with a trypsin-collagenase solution to initiate tissue digestion. The tissue was further digested in the enzyme solution and approximately 2×10⁸ viable lung cells were obtained per animal. Primary cultures contained a mixed cell population. Through eight subcultures about 70% of the cell population possessed an epithelial-like morphology, whereas the remaining 30% was fibroblast-like. Three clones of epithelial-like cells were isolated at the fourth subculture. The mass culture lung cells and the epithelial-like clone that was studied retained a normal karyotype and did not grow in soft agar. Both the mass culture cells and the epithelial clone metabolized the lung carcinogen benzo(a)pyrene (BP) to water-soluble products. Furthermore, the mass culture lung cells metabolized BP to intermediate(s) which mutated Chinese hamster V79 cells from ouabain sensitivity to ouabain resistance. These lung cell cultures have potential use in cell transformation, mutation and carcinogen metabolism studies. Visiting scientist from Hungary. This research was supported by Grant 5 R01 CA20022 and Public Health Service Contract N01 CP33278 from the Division of Cancer Cause and Prevention, National Cancer Institute, National Institutes of Health.     

Enzymatic alcoholysis of 3′,5′-di-O-acetyl-2′-deoxynucleosides

Candida antarctica-B (CAL-B) lipase-catalysed alcoholysis of a set of 3′,5′-di-O-acetyl-2′-deoxynucleosides (1a–e) gave the corresponding 3′-O-acetyl-2′-deoxy-nucleosides (2a–e) in yields ranging from 50 to 96%. The alcohol employed in the biotransformation affected the rate of the enzymatic reaction and the yield of the 3′-O-acetylated product, but in all cases only this regioisomer was formed. The obtained results are in agreement with the regioselectivity displayed by CAL-B lipase in previously reported biotransformations of nucleosides. CAL-B catalysed alcoholysis of 2′,3′,5′-tri-O-acetyl-cytidine and 4-N-acetyl-2′,3′,5′-tri-O-acetylcytidine was also studied, affording with the same regioselectivity the corresponding free 5′-hydroxyl nucleosides.     

Regulation of DNA synthesis by guanosine-5′-diphosphate, cyclic guanosine-3′,5′-monophosphate, and cyclic adenosine-3′,5′-monophosphate in mouse lymphoid cells

The effect of various adenine and guanine nucleotides and nucleosides on DNA synthesis was studied in various types of mouse lymphoid cells. Two out of the ten compounds tested, namely guanosine-5′-diphosphate (GDP) and cyclic guanosine-3′,5′-monophosphate (cGMP) increased the thymidine incorporation into the DNA of the spleen cells and counteracted completely or partially the inhibitory action of cyclic adenosine-3′,5′-monophosphate (cAMP) on spleen cells stimulated by various B or T cell mitogens. GDP seems to act preferentially on thymus cells while cGMP acts better on bone marrow cells. The possible significance of the results for the mechanism of the mitogenic signal is discussed.     

5′ Phosphorylation of DNA in mammalian cells: Identification of a polymin P-precipitable polynucleotide kinase

Proteins that catayze 5′ phosphorylation of an oligodeozyribonucleotide substrate can be fractionated by polumin P treatment of whole cell extrats of calf thymus glands. Anion exchange chromatography on Q-Sepharose revealed three separable peaks of activity in the polymin P supernatant fraction, and one peak of activity in the Polymin P pellet fraction. The latter activity, polymin P-precipitable polynucleotide kinase (PP-PNK), was futher purified with a 1,500-fold increase of specific activity compared to the crude polymin fraction. Oligonucleotides, a dephosphorylated 2.9-kb EcoRI fragment, and poly(A) were phosphorylated by the enzyme preparation, but thymidine 3′monophosphate was not a substrate. PP-PNk preparations exhibited an apparent K_M of 52 μM for ATP and 8 μM for oligo dT₂₅. The enzyme preparation displayed no detectable 3′ phosphatase or cyclic 2′,3′ phosphohydrolase activities. The sedimentation coefficient of the PP-Pnk activity was 3.85 as determined by sucrose density gradient analysis; the stokes radius was 45 Å, leading to an estimated molecular mass of 72 kDa. The enzyme had a pH optimun in the neutral to alkaline range in several buffer systems and is distinct from the DNA Kinase with an acidic pH optimum previously described in calf thymus. © Wiley-Liss, Inc.     

2′,3′-Dideoxy-3′-aminonucleoside 5′-triphosphates inhibit the repair of DNA in rat liver chromatin

2′,3′-Dideoxy-3′-aminonucleoside 5′-triphosphates are shown to be strong inhibitors of repair DNA synthesis in γ-irradiated rat liver chromatin. The activity of these compounds is comparable with that of the most effective inhibitor of the DNA polymerase β-catalyzed repair DNA synthesis.