Substrate For DNA Synthesis Research Articles

INFLUENCE OF X-RAY AND MICROWAVE RADIATION ON DEAMINATION OF PURINE NUCLEOTIDES IN YEAST CELLS CANDIDA GUILLIERMONDII NP-4

In metabolism of living cells a key role play purine nucleotides which cells can be supplied either by de novo synthesis from lower molecular weight precursors, or by alternate ways of nucleotide synthesis or so-called "nucleotide salvage pathways", which allow reusing of intermediate products of nucleotide metabolism in nucleotide synthesis. This way is important in the post-stress repair period, saving energy and substrates in the repairing cells. Purine nucleotides are allosteric inhibitors of enzymes of nucleotide salvage pathways, therefore the increase in their catabolism leads to a decrease of their amount in the cells, which contributes to the intensive work of the nucleotide salvage pathways and provides substrates for DNA synthesis. Investigation of deamination of purine nucleotides in yeasts Candida guilliermondii NP-4 irradiated with X-rays, millimeter and decimeter electromagnetic waves, as well as after post-radiation incubation of cells has been realized. It has been shown that under the influence of X-ray and microwave irradiation in yeasts, the intensity of deamination of purine nucleotide-polyphosphates - ADP, ATP, GDF and GTP, has changed, which in all probability is an adaptive mechanism in the repair of yeasts after irradiation, provides the work of nucleotide salvage pathways, and can be associated with the metabolism of these compounds.

Evaluation of 4′-[Methyl-11C]Thiothymidine in a Rodent Tumor and Inflammation Model

4'-[methyl-(11)C]thiothymidine ((11)C-4DST) is a novel radiopharmaceutical that can be used for tumor imaging because of its rapid incorporation into DNA as a substrate for DNA synthesis. The in vivo stability of (11)C-4DST is much greater than that of natural thymidine, because of the presence of a sulfur atom in the 4'-position. Here, we evaluated the tissue kinetics and biodistribution of (11)C-4DST in a rodent tumor and acute sterile inflammation model in comparison with the previously published biodistribution data of 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT), (18)F-FDG, (11)C-choline, (11)C-methionine, and 2 σ-receptor ligands in the same animal model. C6 tumor cells were implanted subcutaneously into the right shoulder and turpentine (0.1 mL) was injected intramuscularly into the left hind leg of male Wistar rats 11 d and 24 h, respectively, before the scanning day. The animals were anesthetized with isoflurane, and (11)C-4DST (20-50 MBq) was injected intravenously. A dynamic PET scan was performed for 60 min with either the shoulder or hind leg region in the field of view. The animals were sacrificed, and a biodistribution study was performed. (11)C-4DST showed the highest tumor uptake (standardized uptake value, 4.93) of all radiopharmaceuticals tested. Its tumor-to-muscle concentration ratio (12.7) was similar to that of (18)F-FDG (13.2). The selectivity of (11)C-4DST for tumor as compared with acute inflammation was high (37.7), comparable to that of the σ-ligand (18)F-FE-SA5845 and much higher than that of (18)F-FDG (3.5). Rapidly proliferating tissues (tumor and bone marrow) showed a steadily increasing uptake. In inflamed muscle, (11)C-4DST showed relatively rapid washout, and tracer concentrations in inflamed and noninflamed muscle were not significantly different at intervals greater than 40 min. Competition of endogenous thymidine for (11)C-4DST uptake in target tissues was negligible, in contrast to competition for (18)F-FLT uptake. Thus, pretreatment of animals with thymidine phosphorylase was not required before PET with (11)C-4DST. In our rodent model, (11)C-4DST showed high tumor uptake (sensitivity) and high tumor selectivity. The different kinetics of (11)C-4DST in rapidly proliferating and inflammatory tissue may allow distinction between tumor and acute inflammation in a clinical setting. These promising results for (11)C-4DST warrant further investigation in PET studies in patients with various types of tumors.

Analysis of the Ex Vivo and In Vivo Antiretroviral Activity of Gemcitabine

Replication of retroviral and host genomes requires ribonucleotide reductase to convert rNTPs to dNTPs, which are then used as substrates for DNA synthesis. Inhibition of ribonucleotide reductase by hydroxyurea (HU) has been previously used to treat cancers as well as HIV. However, the use of HU as an antiretroviral is limited by its associated toxicities such as myelosuppression and hepatotoxicity. In this study, we examined the ribonucleotide reductase inhibitor, gemcitabine, both in cell culture and in C57Bl/6 mice infected with LP-BM5 murine leukemia virus (LP-BM5 MuLV, a murine AIDS model). Gemcitabine decreased infectivity of MuLV in cell culture with an EC50 in the low nanomolar range with no detectable cytotoxicity. Similarly, gemcitabine significantly decreased disease progression in mice infected with LP-BM5. Specifically, gemcitabine treatment decreased spleen size, plasma IgM, and provirus levels compared to LP-BM5 MuLV infected, untreated mice. Gemcitabine efficacy was observed at doses as low as 1 mg/kg/day in the absence of toxicity. Higher doses of gemcitabine (3 mg/kg/day and higher) were associated with toxicity as determined by a loss in body mass. In summary, our findings demonstrate that gemcitabine has antiretroviral activity ex vivo and in vivo in the LP-BM5 MuLV model. These observations together with a recent ex vivo study with HIV-1[1], suggest that gemcitabine has broad antiretroviral activity and could be particularly useful in vivo when used in combination drug therapy.

PTP1B deficiency enhances liver growth during suckling by increasing the expression of insulin‐like growth factor‐I

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin and tyrosine kinase growth factor signaling. We have recently demonstrated that PTP1B deficiency increases GLUT2/insulin receptor (IR) A complexes and glucose uptake in suckling, but not adult, primary hepatocytes. Herein we have investigated intrahepatic glucose utilization in 3-5 days old wild-type and PTP1B(-/-) mice. PTP1B deficiency decreased glycogen, lactate, and pyruvate content in the livers from suckling mice. Conversely, the activity of glucose 6-phosphate dehydrogenase (G6PD), the rate limiting enzyme of the pentose phosphate cycle (PPC) which provides substrates for DNA synthesis, was enhanced in the liver of PTP1B(-/-) animals. Liver weight, liver-to-body mass ratio, DNA content, and PCNA expression were increased in PTP1B(-/-) suckling mice compared to the wild-type controls. At the molecular level, STAT 5B phosphorylation, IGF-I mRNA, and protein levels as well as IGF-IR tyrosine phosphorylation were increased in the livers of PTP1B-deficient neonates. Unexpectedly, hepatic and serum triglycerides (TG) were increased by PTP1B deficiency, although the expression of lipogenic enzymes remained as in the wild-type controls. However, the analysis of milk composition revealed higher TG content in lactating females lacking PTP1B. The effects of PTP1B deficiency on G6PD activity, STAT 5B/IGF-I/IGF-IR axis, PCNA expression and liver growth during suckling were maintained by transferring PTP1B(-/-) embryos (PTP1B(-/-T)) to a wild-type female. Conversely, PTP1B(-/-T) mice did not show hepatic fat accumulation. In conclusion, the present study suggests that PTP1B plays a unique role in the control of the physiological liver development after birth.

Phase III Multicenter Randomized Clinical Trial to Evaluate the Safety and Efficacy of CoFactor®/5-Fluorouracil/Bevacizumab Versus Leucovorin/5-Fluorouracil/Bevacizumab as Initial Treatment for Metastatic Colorectal Carcinoma

Folate Metabolism Intracellular reduced folates exists as a “pool” of 6 interconvertable forms. One of these forms, 5,10 methylenetetrahydrofolate (CH2THF), is the essential 1-carbon donor and reduced folate substrate for thymidylate synthase (TS), the enzyme that catalyzes the methylation of deoxyuridine-5 -monophosphate (dUMP) to deoxythymidine-5 -monophosphate (dTMP). This enzymatic pathway is critical because it provides the necessary nucleotide substrates for DNA synthesis, and for this reason, TS has been an important target for cancer chemotherapy (Figure 1).1 The fluoropyrimidine 5-fluorouracil (5-FU) exerts its cytotoxic activity, at least in part, through inhibition of TS. Specifically, it does so through the 5-FU metabolite fluorodeoxyuridine monopohosphate (FdUMP) with formation of a ternary complex made up of FdUMP, TS, and the reduced folate CH2THF, which then results in TS enzyme inhibition. Historically, leucovorin (LV; 5-formyltetrahydrofolate) has been used as a strategy to increase the intracellular reduced folate pools and increase TS enzyme inhibition. However, LV must be metabolized within the cell through multiple intracellular enzymatic steps to form CH2THF. In general, CH2THF levels are relatively low, in the range of approximately 0.1-0.5 mol/L in normal cells and even lower in human cancer biopsy tissues. Although treatment with LV results in an approximately 2-fold increase in CH2THF, maximum ternary complex formation is generally achieved at CH2THF concentrations approaching 12 mol/L.4 Individual tumor metabolism of LV to tetrahydrofolate and, ultimately, to CH2THF is unpredictable, and CH2THF levels are typically among the lowest of the activated intracellular reduced folate forms.5 However, it is clear that high intratumoral levels of CH2THF allow for maximal inhibition of TS.6-10 Preclinical and clinical investigations have consistently shown that resistance of tumors to 5-FU is associated, at least in part, with decreased intratumoral reduced folate levels, typically through decreased folylpolyglutamylation.11,12 Thus, direct administration of the essential reduced folate CH2THF in place of LV might offer significant advantages with respect to enhancing clinical activity.

Exogenous 8-oxo-dG is not utilized for nucleotide synthesis but enhances the accumulation of 8-oxo-Gua in DNA through error-prone DNA synthesis

7,8-Dihydro-8-oxoguanine (8-oxo-Gua) and its nucleoside in cytosol are derived from the repair of oxidative DNA and the cleanup of oxidatively damaged DNA precursors, respectively. While the harmful effects of 8-oxo-Gua present in DNA have been studied extensively, few have reported its effects on cytosolic function. Our previous study showed that the addition of 8-oxo-dG to culture media caused an accumulation of 8-oxo-Gua in nuclear DNA in several leukemic cells including KG-1, which lack 8-oxoguanine glycosylase 1 (OGG1) activity due to mutational loss. However, the mechanism underlying 8-oxo-Gua level increases in DNA has not been addressed. In this study, we elucidated the metabolic fate of 8-oxo-Gua-containing nucleotide and the effect of exogenous 8-oxo-dG on DNA synthesis in KG-1 cells. We found that 8-oxo-dGMP was rapidly dephosphorylated to 8-oxo-dG rather than phosphorylated to 8-oxo-dGDP, thus indicating that 8-oxo-Gua-cotaining molecule is not used as a substrate for DNA synthesis in KG-1 cells. In fact, radiolabled 8-oxo-dG was incubated but radioactivity was not detected in nuclear DNA of KG-1 cells, showing that 8-oxo-dG is not directly incorporated into DNA. Interestingly, the activity of DNA polymerase β, which synthesize DNA with low fidelity increased in KG-1 cells treated with 8-oxo-dG, whereas the expression of DNA polymerase α decreased. In addition, the accumulation of 8-oxo-Gua in KG-1 DNA was completely inhibited by a specific inhibitor of DNA polymerase β. Thus, our findings address that the insertion of 8-oxo-dG into KG-1 DNA is not due to the direct incorporation of exogenous 8-oxo-dG, but rather to the inaccurate incorporation of endogenous 8-oxo-dGTP by DNA polymerase β. It further suggests that 8-oxo-dG in the cytosol may function as an active molecule itself and perturb the well-defined DNA synthesis.

Saccharomyces cerevisiae Mre11 is a high-affinity G4 DNA-binding protein and a G-rich DNA-specific endonuclease: implications for replication of telomeric DNA

In Saccharomyces cerevisiae, Mre11p/Rad50p/Xrs2p (MRX) complex plays a vital role in several nuclear processes including cellular response to DNA damage, telomere length maintenance, cell cycle checkpoint control and meiotic recombination. Telomeres are comprised of tandem repeats of G-rich DNA and are incorporated into non-nucleosomal chromatin. Although the structure of the yeast telomeric DNA is poorly understood, it has been suggested that the G-rich sequences can fold into G4 DNA, which has been shown to inhibit DNA synthesis by telomerase. However, little is known about the factors and mechanistic aspects of the generation of appropriate termini for DNA synthesis by telomerase. Here, we show that S.cerevisiae Mre11 protein (ScMre11p) possesses substantially higher binding affinity for G4 DNA, over single- or double-stranded DNA, and binding was inhibited by poly(dG) or porphyrin. Binding of ScMre11p to G4 DNA was most robust, compared with G2′ DNA and the resulting protein–DNA complexes were strikingly very resistant to dissociation by NaCl. Remarkably, binding of ScMre11p to G4 DNA and G-rich single-stranded DNA was accompanied by the endonucleolytic cleavage at sites flanking the array of G residues and G-quartets in Mn2+-dependent manner. Collectively, these results suggest that ScMre11p is likely to play a major role in generating appropriate substrates for DNA synthesis by telomerase and telomere-binding proteins. We discuss the implications of these findings with regard to telomere length maintenance by telomerase-dependent and independent mechanisms.

Dehydroepiandrosterone-sulfate inhibits pancreatic carcinoma cell proliferation in vitro and in vivo

Background . Dehydroepiandrosterone-sulfate (DHEA-S) is a potent inhibitor of glucose-6 phosphate dehydrogenase, the rate limiting enzyme of the hexose monophosphate shunt, a biochemical pathway that provides substrate for DNA synthesis in neoplastic tissue. DHEA-S has been shown to inhibit the growth of neoplasms arriving from human skin, lung, colon, and mammary tissue. This study evaluates the effect of DHEA-S on human pancreatic cancer cell lines in vitro and in vivo. Methods . In vitro, the human pancreatic adenocarcinoma cell lines MiaPaCa-2, Capan-1, Capan-2, CAV and Panc-1 were treated with concentrations of 1.9 μmol/L to 1000 μmol/L DHEA-S in 1% dimethylsulfoxide (DMSO) for 5 consecutive days. Cell proliferation was determined by a nonradioactive cell proliferation assay and compared with DMSO treated controls. In vivo testing was performed by inoculating two cell lines, MiaPaCa-2 and Panc-1, into the flank of 40 male nude athymic mice in four study groups. After 1 week of growth, 667 mg/kg DHEA-S in 1% DMSO or 0.2 ml 1% DMSO alone in the control group was administered by daily intraperitoneal injection. Body weight and tumor size was recorded weekly, and tumor weight was measured after 3 weeks of treatment. Results . In vitro cell proliferation was decreased in the five cell lines by 36% to 62% of controls (p < 0.001) at 500 μmol/L DHEA-S. In vivo, after 2 weeks, tumor size was only 76% (p < 0.008) and 67% (p < 0.005) of the controls. After 3 weeks of treatment, tumor size was 73% (p < 0.001) and 53% (p < 0.001) of controls, and tumor weight was decreased by 73% in MiaPaCa-2 (p < 0.001) and 66% in Panc-1 (p < 0.001). Radioimmunoassay measurements of DHEA-S and testosterone from DHEA-S treated mouse plasma showed a significant increase in circulating levels of these hormones. Conclusions . DHEA-S achieves high serum levels after intraperitoneal injection without elevation of serum testosterone levels and produces no significant toxicity. Treatment with DHEA-S results in a significant reduction of proliferation of human pancreatic cancer cells in culture and when grown as subcutaneous tumors in athymic nude mice.

Gemcitabine and radiosensitization in human tumor cells.

Gemcitabine is a nucleoside analogue with excellent clinical activity against solid tumors. Within the cell, gemcitabine is rapidly phosphorylated to its active di- and triphosphate metabolites. Cytotoxicity with gemcitabine appears to be related to multiple effects on DNA replication, where gemcitabine triphosphate can serve as both an inhibitor and substrate for DNA synthesis. Gemcitabine diphosphate inhibits ribonucleotide reductase, producing decreases in cellular dNTP pool levels in a cell-specific manner. These two major characteristics of gemcitabine, reduction in cellular dNTP pools and incorporation into DNA, are features of other antimetabolites antitumor agents which also exhibit radiosensitizing properties. Based on these favorable metabolic characteristics and the clinical activity of gemcitabine in tumor types which are commonly treated with radiation, the ability of gemcitabine to enhance X-radiation induced cytotoxicity was evaluated. Gemcitabine has been shown to be a potent radiosensitizer in a variety of tumor cell lines, including HT-29 colorectal carcinoma, pancreatic cancer, breast, non-small cell lung and head and neck cancer cell lines. Gemcitabine was most effective as a radiosensitizer when administered at least 2 hours prior to irradiation. For most cell lines, radiosensitization was evident at non-cytotoxic concentrations. The extent of radiosensitization increased with both increasing gemcitabine concentration and duration of exposure. Radiosensitization did not require redistribution of cells into a more radiosensitive phase of the cell cycle. The major metabolic effects observed under radiosensitizing conditions were the accumulation of high levels of gemcitabine triphosphate, and a selective decrease in the cellular dATP pool. The pattern of dATP decrease paralleled the increase in radiosensitization, whereas the level of gemcitabine triphosphate was not associated with the enhanced sensitivity to radiation. Compared to other radiosensitizers, the advantage of gemcitabine is that is can induce radiosensitization at concentrations that are 1000 times lower than typical plasma levels obtained with this drug. These studies will be used as guidelines for developing clinical trials of gemcitabine with radiation.

Metabolism and metabolic actions of 4′-thiothymidine in L1210 cells

4′-Thiothymidine (S-dThd) is a potent inhibitor of L1210 cell growth and is active against P388 leukemia in mice. Because of these activities and its novel structure, we have begun studies of its metabolism and metabolic actions in L1210 cells in order to understand its mechanism of cytotoxicity. S-dThd inhibited the incorporation of radiolabeled precursors into DNA, but did not inhibit the incorporation of either uridine or leucine into RNA or protein, respectively, which indicated that the mechanism oi its toxicity was due to its inhibition of DNA synthesis. S-dThd did not decrease the concentration of any of the natural deoxynucleoside triphosphates, which indicated that its cytotoxicity was not due to the inhibition of ribonucleotide reductase. S-dThd was readily phosphorylated and used as a substrate for DNA synthesis. Because the rate of incorporation of S-dThd into DNA was 20% that of thymidine, it is likely that the mechanism of action of S-dThd is not due to inhibition of DNA polymerases by the 5′-triphosphate of S-dThd, but instead to its incorporation into the DNA and its subsequent disruption of some function of DNA.

Therapy related disturbances in nucleotides in cancer cells.

Ribonucleotides (NTP) and deoxyribonucleotides (dNTP) form the end products of the purine and pyrimidine synthetic pathways, both the de novo and the salvage pathways. They form the direct substrates for RNA and DNA synthesis. Nucleotide pools in the cell are the result of a dynamic equilibrium of synthesis and consumption. Besides their essential role in nucleic acid synthesis, ribonucleotides are essential for a number of other functions in the cell such as a source for energy supply (ATP), nucleotide sugar synthesis (UTP, CTP and GTP), lipid biosynthesis (CTP), signal transduction (ATP, GTP as part of the GTP binding protein, cyclic nucleotides such as cAMP and cGMP), protein elongation, tubulin-binding and ras-oncogene (all GTP), phosphate donors for kinase catalyzed reactions (mostly ATP, but other nucleotides can also act as a phosphate donor), allosteric activators and feedback regulators of several synthetic pathways, co-enzyme in several synthetic reactions such folyl-polyglutamate synthetase and phosphoribosylpyrophosphate (PRPP) synthetase. Besides being a substrate for DNA synthesis, dNTP play an essential role in DNA repair and as feedback regulators of a number of enzymes. E.g. dTTP is an inhibitor of dCMP deaminase [1], and of thymidine kinase [2]; dATP is a potent feedback inhibitor of ribonucleotide reductase [3, 4], inhibiting the synthesis of all deoxyribonucleoside diphosphates, dGTP that of dGDP, dUDP and dCDP, dTTP that of dUDP and dCDP, while ATP is an activator of this enzyme, and dTTP an activator of the reduction of GDP to dGDP and dGTP of that of ADP to dADP. Thus, any perturbation of the synthesis or use of both NTP and dNTP can have major effects on cellular functioning. Thus measurement of nucleotide pools in cells during exposure to any drug and especially to drugs directed against synthesis of (deoxy)ribonucleotides can give essential information on the mechanism of action of these drugs.KeywordsRibonucleotide ReductaseNucleotide PoolDeoxycytidine KinaseSolid Tumor Cell LinedNTP PoolThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Cell type-specific expression of human 8-oxo-7,8-dihydroguanosine triphosphatase in normal breast and skin tissues in vivo.

8-Oxo-7,8-dihydroguanosine triphosphate (8-oxo-dGTP) is formed from the oxidation of GTP in the nucleotide pools of cells during normal cellular metabolism and from exogenous sources. 8-Oxo-dGTP is a potent mutagenic substrate for DNA synthesis causing transversion mutations. In human cells this oxidized base is hydrolyzed to 8-oxo-7,8-dihydroguanosine monophosphate by 8-oxo-7,8-dihydroguanosine triphosphatase (8-oxo-dGTPase) to prevent the misincorporation of 8-oxo-dGTP into cellular DNA. In order to better understand specific human tissue and cell type responses to oxidative stress, we used colorimetric in situ hybridization, with an 8-oxo-dGTPase-specific antisense oligomer probe, to map, for the first time, the cellular distribution of 8-oxo-dGTPase mRNA in tissue sections of normal neonatal foreskin and adult human breast tissues. Paraffin embedded tissue sections were hybridized with a digoxigenin-labeled 39 base oligomer, antisense to 8-oxo-dGTPase cDNA. Hybridization of the probe to cells expressing the 8-oxo-dGTPase gene was visualized following immunodetection with an alkaline phosphatase-conjugated anti-digoxigenin antibody. Following color development, we were able to simultaneously identify tissue architecture and cell types with expression of the 8-oxo-dGTPase gene. There was no hybridization-specific color when sections were 'mock' hybridized, hybridized with a sense probe or treated with RNase. In skin dermis, fibroblasts express high levels of 8-oxo-dGTPAse mRNA. Within the epidermis, a gradient of expression was observed, from high to moderate levels in the replicating basal epithelial cells to undetectable in the non-mitotic suprabasal and granular epithelial cells. In the breast tissue, fibroblasts in the loosely connective tissue and myoepithelial cells expressed high levels of 8-oxo-dGTPase mRNA, while expression in the luminal epithelial cells was not detectable. Our data suggest that expression of 8-oxo-dGTP is heterogenous between cell types within an organ and may help to explain cell type-specific responses to oxidative stress, especially in replicating and potentially replicating cells with low levels of this protective protein.

Primary structure of human deoxycytidylate deaminase and overexpression of its functional protein in Escherichia coli

The cDNA encoding human dCMP deaminase was isolated from a lambda ZAPII expression library using an antibody generated against highly purified HeLa cell dCMP deaminase. The cloned cDNA consists of 1856 base pairs and encodes a protein of 178 amino acids with a calculated molecular mass of 19,985 daltons. The sequence of several cyanogen bromide-cleaved peptides derived from HeLa cell dCMP deaminase are all contained within the deduced amino acid sequence. A zinc binding region is present in the enzyme, similar to that reported for cytidine deaminase (Yang, E. C., Carlow, D., Wolfenden, R., and Short, S. A. (1992) Biochemistry 31, 4168-4174). Northern blot analysis revealed a predominant messenger RNA species of 1.9 kilobases. Expression of the active protein to about 10% of Escherichia coli's total protein was achieved by subcloning the open reading frame into a high expression system using the polymerase chain reaction. Polyacrylamide gel electrophoresis revealed a prominent protein band which comigrated with affinity purified HeLa dCMP deaminase, while Western blot analysis yielded an immunoreactive band which comigrated with the single immunoreactive affinity column purified dCMP deaminase band. The enzyme which possesses a kcat of 1.02 x 10(3) s-1 was purified to homogeneity in over 60% yield. The overexpression of dCMP deaminase should permit more exacting studies on the regulation of this important allosteric enzyme which provides substrate for DNA synthesis.

Probing the energetics of oligo(dT).poly(dA) by laser cross-linking.

Experimentally determined changes in free energy (delta G(o)) for thymine-thymine interactions occurring in oligo(dT).poly(dA) are dependent on the method used for preparation of the double-stranded template. A rapid laser cross-linking technique was used to examine the equilibrium between oligomers of (dT) bound to either poly(dA) or poly(rA). The single-pulse (4-6 nsec) ultraviolet laser excitation of these polynucleotides causes pyrimidine dimer formation between contiguous oligo(dT) molecules, resulting in a "ligation" of the oligomers. Analysis of the resulting data using standard binding isotherms allowed determination of the degree of cooperativity existing between oligomers. Using the cooperativity, delta G(o), delta H(o), and delta S(o) are calculated, thereby providing thermodynamic parameters for this interaction. The measured cooperativity of oligo(dT) molecule interactions allows direct calculation of the number of 3' ends available as nicked structures or the number of 3' ends associated with gaps for oligo(dT).poly(dA) when used as a substrate for DNA synthesis.

Monofunctional DNA-platinum(II) adducts block frequently DNA polymerases.

The question of whether monofunctional DNA platinum(II) adducts block synthesis of DNA by purified DNA polymerases of different types and origin has been investigated by comparing the time dependence of synthesis arrest and of DNA adduct formation. Activated salmon testis DNA is used as a suitable substrate for DNA synthesis allowing to probe inhibition by platinum(II) monoadducts for the variety of inherent template-primers. Reaction amplitudes are related to defined mixtures of dichloro and chloroaqua platinum(II) complexes. It is found that (i) all investigated DNA polymerases seem arrested (100% efficiency) at bifunctional DNA adducts. (ii) human DNA polymerase beta bypasses most of the monofunctional lesions of the three platinum(II) complexes investigated. (iii) Klenow fragment is blocked by monoadducts with increasing efficiency in the order cis-diamminechloroaquaplatinum(II) (0%) less than meso-[1,2-bis(2,6- dichloro-4-hydroxyphenyl)ethylenediamine] chloroaquaplatinum(II) (50%) less than trans-diamminechloro-aquaplatinum(II) (75%). (iv) Escherichia coli DNA polymerase I, Thermus aquaticus DNA polymerase, Physarum polycephalum DNA polymerase alpha, and calf thymus DNA polymerase alpha appear to be arrested by monoadducts. According to these examples, blocking efficiencies depend on the cis/trans-stereogeometry of fixation of the carrier ligands at platinum(II) residues, on the size/chemical nature of the platin(II) carrier ligand and on the type/origin of DNA polymerase.

Effects of 2-chloro-2'-deoxyadenosine 5'-triphosphate on DNA synthesis in vitro by purified bacterial and viral DNA polymerase

2-Chloro-2'-deoxyadenosine 5'-triphosphate (CldATP) was compared with dATP as a substrate for DNA synthesis by bacterial and viral DNA polymerases in vitro. Lengths of chain extension and DNA synthesis pause sites were determined by comparison with products generated by dideoxynucleotide sequencing methods on the same end-labeled primer/template duplex after high-resolution polyacrylamide gel electrophoresis. Reverse transcriptase (RT) from human immunodeficiency virus (HIV-1) and avian myeloblastosis virus (AMV) incorporated CldATP efficiently. DNA strand elongation continued past most chloroadenine (ClA) insertion sites but resulted in shorter chains than when dATP was inserted. Phage T4 DNA polymerase incorporated CldATP least efficiently; Klenow fragment of Escherichia coli DNA polymerase I and modified T7 DNA polymerase (Sequenase) showed intermediate ability to utilize the analogue. Incorporation of several consecutive ClA residues into the replicating strand dramatically reduced the ability of Sequenase, Klenow fragment, and T4 DNA polymerases to continue strand elongation. In the absence of the corresponding normal deoxyribonucleoside triphosphate during DNA synthesis, ClA was frequently misincorporated as thymine, cytosine, or guanine by both AMV RT and HIV-1 RT but rarely, if at all, by Klenow fragment, Sequenase, and T4 DNA polymerase. Except T4, for most DNA polymerases, CldATP at 10-20-fold molar excess over dATP was not a strong competitive inhibitor of dATP, as judged by the amount of strand extension and polymerase pause sites during DNA synthetic reactions. Our results indicate that the degree of strand extension in the presence of CldATP, the number and location of polymerase pause sites, and the amount of misincorporation of the analogue are both polymerase- and sequence-dependent.

Potent inhibitory effects of the 5′‐triphosphates of (E)‐5‐(2‐bromovinyl)‐2′‐deoxyuridine and (E)‐5‐(2‐bromovinyl)‐1‐β‐D‐arabinofuranosyluracil on DNA polymerase γ

(E)-5-(2-Bromovinyl)-2'-deoxyuridine 5'-triphosphate (BrVdUTP) and (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil 5'-triphosphate (BrVarafUTP), which are known as specific inhibitors of herpes simplex viral (type 1 and 2) DNA polymerase, were found to be strong inhibitors of DNA polymerase gamma from human KB and murine myeloma cells. In fact BrVdUTP and BrVarafUTP were found to be stronger inhibitors of DNA polymerase gamma than of other DNA polymerases having viral (herpes simplex virus or retrovirus) origin or cellular (eukaryotic alpha and beta, or prokaryotic) origin. The mode of inhibition of DNA polymerase gamma by BrVdUTP and BrVarafUTP was competitive with respect to dTTP, the normal substrate. Whereas BrVdUTP was an efficient substrate for DNA polymerase gamma and other DNA polymerases that were examined, BrVarafUTP failed to serve as a substrate for DNA synthesis. Ki values for BrVdUTP (40 nM) and BrVarafUTP (7 nM) with DNA polymerase gamma, as determined with (rA)n.(dT) as the template.primer, were much smaller than the Km values for dTTP (0.16 microM and 0.71 microM for murine and human DNA polymerase gamma, respectively). Thus, the affinity of BrVdUTP or BrVarafUTP for DNA polymerase gamma was much stronger than that of dTTP.

DNA synthesis in vivo and in vitro in Escherichia coli irradiated with ultraviolet light.

DNA synthesis was followed in vivo and in permeable Escherichia coli after ultraviolet light irradiation, irradiation and incubation in a growth medium containing chloramphenicol and in unirradiated cells. In vitro, replicative type DNA synthesis was partially restored after incubation of cells in medium containing chloramphenicol, but not in vivo. The DNA was pulse-labeled in permeable cells in the presence of deoxyribonucleoside triphosphates and ribonucleoside triphosphates. dCTP was replaced by 5-Hg-dCTP as a substrate for DNA synthesis. Hg-DNA was separated from cellular nucleic acids on thiol-agarose affinity columns. The 5' termini of newly synthesized DNA were analyzed after treatment with alkaline phosphatase and rephosphorylation with polynucleotide kinase and [gamma-32P]ATP. DNA synthesis in unirradiated permeable E. coli represents a replicative process dependent on ATP and inhibited by novobiocin. About 70% of the nascent DNA carried terminally labeled RNA moiety at its 5' end. In vitro DNA synthesis in irradiated cells was suppressed and hardly influenced by the presence of ATP or novobiocin. The 5'-RNA content of this cell population was less than 5%.

Inhibition of replicative DNA synthesis in nuclei of Strongylocentrotus intermedium urchin embryo by 2′,3′-dideoxy-3′-aminonucleoside 5′-triphosphates

2′,3′-Dideoxy-3′-aminonucleoside 5′-triphosphates have been shown to be inhibitors of replicative DNA synthesis in isolated nuclei of sea urchin embryo. These compounds inhibit the Okazaki fragment synthesis. The effect of 2′,3′-dideoxy-3′-aminothymidine 5′-triphosphate and arabinothymidine 5′-triphosphate is reversible when adding the corresponding substrate for DNA synthesis, 2′-deoxythymidine 5′-triphosphate.

An antifolate-induced lesion in newly synthesized DNA

Antifolates cause cells to accumulate exogenous UdR as dUMP in drug-treated cells. This accumulation of intracellular dUMP has no effect on inhibition of UdR incorporation into DNA. On the other hand, dUMP accumulation does result in detectable dUTP being synthesized and utilized as substrate for DNA synthesis. Although in the presence of metoprine (DDMP), dUMP is incorporated into DNA instead of TMP, the natural product of UdR substrate in the absence of the drug, DNA synthesis is equally rate limited by exogenous UdR. However, when analyzed on alkaline sucrose gradients, DNA which has incorporated exogenous UdR in the presence of antifolate sediments at 4S or less. This newly synthesized DNA accumulates preferentially while either synthesis of larger DNA is blocked, or high molecular weight DNA is preferentially broken down as a result of DNA repair. These observations further increase the metabolic complexity of pathways that must be considered as underlying the differential toxicity of antifolates.