Cytosolic localization and in vitro assembly of human de novo thymidylate synthesis complex.

De novo thymidylate synthesis is a crucial pathway for normal and cancer cells. Deoxythymidine monophosphate (dTMP) is synthesized by the combined action of three enzymes: serine hydroxymethyltransferase (SHMT1), dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS), with the latter two being targets of widely used chemotherapeutics such as antifolates and 5-fluorouracil. These proteins translocate to the nucleus after SUMOylation and are suggested to assemble in this compartment into the thymidylate synthesis complex. We report the intracellular dynamics of the complex in cancer cells by an in situ proximity ligation assay, showing that it is also detected in the cytoplasm. This result indicates that the role of the thymidylate synthesis complex assembly may go beyond dTMP synthesis. We have successfully assembled the dTMP synthesis complex in vitro, employing tetrameric SHMT1 and a bifunctional chimeric enzyme comprising human thymidylate synthase and dihydrofolate reductase. We show that the SHMT1 tetrameric state is required for efficient complex assembly, indicating that this aggregation state is evolutionarily selected in eukaryotes to optimize protein-protein interactions. Lastly, our results regarding the activity of the complete thymidylate cycle in vitro may provide a useful tool with respect to developing drugs targeting the entire complex instead of the individual components.

The catalytic activity of serine hydroxymethyltransferase is essential for de novo nuclear dTMP synthesis in lung cancer cells.

Cancer cells reprogramme one-carbon metabolism (OCM) to sustain growth and proliferation. Depending on cell demands, serine hydroxymethyltransferase (SHMT) dynamically changes the fluxes of OCM by reversibly converting serine and tetrahydrofolate (THF) into 5,10-methylene-THF and glycine. SHMT is a tetrameric enzyme that mainly exists in three isoforms; two localize in the cytosol (SHMT1/SHMT2α) and one (SHMT2) in the mitochondria. Both the cytosolic isoforms can also translocate to the nucleus to sustain de novo thymidylate synthesis and support cell proliferation. Finally, the expression levels of the different isoforms are regulated to a certain extent by a yet unknown crosstalk mechanism. We have designed and fully characterized a set of three SHMT1 mutants, which uncouple the oligomeric state of the enzyme from its catalytic activity. We have then investigated the effects of the mutations on SHMT1 nuclear localization, cell viability and crosstalk in lung cancer cells (A549; H1299). Our data reveal that in these cell lines de novo thymidylate synthesis requires SHMT1 to be active, regardless of its oligomeric state. We have also confirmed that the crosstalk between the cytosolic and mitochondrial SHMT actually takes place and regulates the expression of the two isoforms. Apparently, the crosstalk mechanism is independent from the oligomeric state and the catalytic activity of SHMT1. DATABASE: Structural data are available in the PDB under the accession number 6FL5.

Structural and functional roles of dynamically correlated residues in thymidylate kinase.

Thymidylate kinase is an important enzyme in DNA synthesis. It catalyzes the conversion of thymidine monophosphate to thymidine diphosphate, with ATP as the preferred phosphoryl donor, in the presence of Mg2+. In this study, the dynamics of the active site and the communication paths between the substrates, ATP and TMP, are reported for thymidylate kinase from Thermus thermophilus. Conformational changes upon ligand binding and the path for communication between the substrates and the protein are important in understanding the catalytic mechanism of the enzyme. High-resolution X-ray crystal structures of thymidylate kinase in apo and ligand-bound states were solved. This is the first report of structures of binary and ternary complexes of thymidylate kinase with its natural substrates ATP and ATP-TMP, respectively. Distinct conformations of the active-site residues, the P-loop and the LID region observed in the apo and ligand-bound structures revealed that their concerted motion is required for the binding and proper positioning of the substrate TMP. Structural analyses provide an insight into the mode of substrate binding at the active site. The residues involved in communication between the substrates were identified through network analysis using molecular-dynamics simulations. The residues identified showed high sequence conservation across species. Biochemical analyses show that mutations of these residues either resulted in a loss of activity or affected the thermal stability of the protein. Further, molecular-dynamics analyses of mutants suggest that the proper positioning of TMP is important for catalysis. These data also provide an insight into the phosphoryl-transfer mechanism.

Thymidylate synthase prompts metastatic progression through the dTMP associated EMT process in pancreatic ductal adenocarcinoma.

As a fundamental metabolic enzyme, anti-Thymidylate synthase (TS) strategy has been shown to be an effective therapy for human cancers. However, the genuine effects of TS in pancreatic ductal adenocarcinoma (PDA) are still conflicting. We systemically assessed the prognostic value and whether TS associated with malignant progression in PDA. Protein and mRNA expression level of TS were evaluated in en bloc PDA samples, the prognostic effect of TS expressed in cytoplasm or cytonuclear was determined separately in the first time. The impact of TS on tumor cell behaviors was assessed in in vitro assays, and the TS associated metastatic potential was further determined in two different PDA metastatic models. The retrospective clinical analysis firstly demonstrated that tumor cytonuclear TS expression was positively correlated with lymphatic metastasis and negatively correlated with the overall survival (OS) in PDA patients. The subsequent experiments further confirmed that TS depletion can effectively abate EMT (epithelial to mesenchymal) process in in vitro and decline most of the metastatic lesions in two different PDA mice models, and the deoxythymidine monophosphate (dTMP) biosynthesis malfunction resulted imbalanced dNTP pools may be the fundamental causation. Collectively, the present study suggested the prospective strategy of combined anti-TS scheme for metastatic PDA, and we strongly suggest further clinical standardization research with a large cohort to verify the prognostic value and the therapeutic potential of TS in PDA.

High-level QM/MM calculations support the concerted mechanism for Michael addition and covalent complex formation in thymidylate synthase.

Thymidylate synthase (TS) is a promising cancer target, due to its crucial function in thymine synthesis. It performs the reductive methylation of 2'-deoxyuridine-5'-phosphate (dUMP) to thymidine-5'-phosphate (dTMP), using N-5,10-methylene-5,6,7,8-tetrahydrofolate (mTHF) as a cofactor. After the formation of the dUMP/mTHF/TS noncovalent complex, and subsequent conformational activation, this complex has been proposed to react via nucleophilic attack (Michael addition) by Cys146, followed by methylene-bridge formation to generate the ternary covalent intermediate. Herein, QM/MM (B3LYP-D/6-31+G(d)-CHARMM27) methods are used to model the formation of the ternary covalent intermediate. A two-dimensional potential energy surface reveals that the methylene-bridged intermediate is formed via a concerted mechanism, as indicated by a single transition state on the minimum energy pathway and the absence of a stable enolate intermediate. A range of different QM methods (B3LYP, MP2 and SCS-MP2, and different basis sets) are tested for the calculation of the activation energy barrier for the formation of the methylene-bridged intermediate. We test convergence of the QM/MM results with respect to size of the QM region. Inclusion of Arg166, which interacts with the nucleophilic thiolate, in the QM region is important for reliable results; the MM model apparently does not reproduce energies for distortion of the guanidinium side chain correctly. The spin component scaled-Møller-Plessett perturbation theory (SCS-MP2) approach was shown to be in best agreement (within 1.1 kcal/mol) while the results obtained with MP2 and B3LYP also yielded acceptable values (deviating by less than 3 kcal/mol) compared with the barrier derived from experiment. Our results indicate that using a dispersion-corrected DFT method, or a QM method with an accurate treatment of electron correlation, increases the agreement between the calculated and experimental activation energy barriers, compared with the semiempirical AM1 method. These calculations provide important insight into the reaction mechanism of TS and may be useful in the design of new TS inhibitors.

Enzymatic Manufacture of Deoxythymidine-5'-Triphosphate with Permeable Intact Cells of E. coli Coexpressing Thymidylate Kinase and Acetate Kinase.

A one-pot process of enzymatic synthesis of deoxythymidine-5'-triphosphate (5'-dTTP) employing whole cells of recombinant Escherichia coli coexpressing thymidylate kinase (TMKase) and acetate kinase (ACKase) was developed. Genes tmk and ack from E. coli were cloned and inserted into pET28a(+), and then transduced into E. coli BL21 (DE3) to form recombinant strain pTA in which TMKase and ACKase were simultaneously overexpressed. It was found that the relative residual specific activities of TMKase and ACKase, in pTA pretreated with 20 mM ethylene diamine tetraacetic acid (EDTA) at 25°C for 30 min, were 94% and 96%, respectively. The yield of 5'-dTTP reached above 94% from 5 mM deoxythymidine 5'-monophosphate (5'-dTMP) and 15 mM acetyl phosphate catalyzed with intact cells of pTA pretreated with EDTA. The process was so effective that only 0.125 mM adenosine-5'- triphosphate was sufficient to deliver the phosphate group from acetyl phosphate to dTMP and dTDP.

Targeting the de novo biosynthesis of thymidylate for the development of a PET probe for pancreatic cancer imaging.

The development of cancer-specific probes for imaging by positron emission tomography (PET) is gaining impetus in cancer research and clinical oncology. One of the hallmarks of most cancer cells is incessant DNA replication, which requires the continuous synthesis of nucleotides. Thymidylate synthase (TSase) is unique in this context because it is the only enzyme in humans that is responsible for the de novo biosynthesis of the DNA building block 2'-deoxy-thymidylate (dTMP). TSase catalyzes the reductive methylation of 2'-deoxy-uridylate (dUMP) to dTMP using (R)-N(5),N(10)-methylene-5,6,7,8-tetrahydrofolate (MTHF) as a cofactor. Not surprisingly, several human cancers overexpress TSase, which makes it a common target for chemotherapy (e.g., 5-fluorouracil). We envisioned that [(11)C]-MTHF might be a PET probe that could specifically label cancerous cells. Using stable radiotracer [(14)C]-MTHF, we had initially found increased uptake by breast and colon cancer cell lines. In the current study, we examined the uptake of this radiotracer in human pancreatic cancer cell lines MIAPaCa-2 and PANC-1 and found predominant radiolabeling of cancerous versus normal pancreatic cells. Furthermore, uptake of the radiotracer is dependent on the intracellular level of the folate pool, cell cycle phase, expression of folate receptors on the cell membrane, and cotreatment with the common chemotherapeutic drug methotrexate (MTX, which blocks the biosynthesis of endogenous MTHF). These results point toward [(11)C]-MTHF being used as PET probe with broad specificity and being able to control its signal through MTX co-administration.

Raltitrexed's effect on the development of neural tube defects in mice is associated with DNA damage, apoptosis, and proliferation.

The causal metabolic pathway and the underlying mechanism between folate deficiency and neural tube defects (NTDs) remain obscure. Thymidylate (dTMP) is catalyzed by thymidylate synthase (TS) using the folate-derived one-carbon unit as the sole methyl donor. This study aims to examine the role of dTMP biosynthesis in the development of neural tube in mice by inhibition of TS via a specific inhibitor, raltitrexed (RTX). Pregnant mice were intraperitoneally injected with various doses of RTX on gestational day 7.5, and embryos were examined for the presence of NTDs on gestational day 11.5. TS activity and changes of dUMP and dTMP levels were measured following RTX treatment at the optimal dose. DNA damage was determined by detection of phosphorylated replication protein A2 (RPA2) and γ-H2AX in embryos with NTDs induced by RTX. Besides, apoptosis and proliferation were also analyzed in RTX-treated embryos with NTDs. We found that NTDs were highly occurred by the treatment of RTX at the optimal dose of 11.5 mg/kg b/w. RTX treatment significantly inhibited TS activity. Meanwhile, dTMP was decreased associated with the accumulation of dUMP in RTX-treated embryos. Phosphorylated RPA2 and γ-H2AX were significantly increased in RTX-treated embryos with NTDs compared to control. More apoptosis and decreased proliferation were also found in embryos with NTDs induced by RTX. These results indicate that impairment of dTMP biosynthesis caused by RTX led to the development of NTDs in mice. DNA damage and imbalance between apoptosis and proliferation may be potential mechanisms.

Azoxymethane-induced colon carcinogenesis in mice occurs independently of de novo thymidylate synthesis capacity.

Folate metabolism affects DNA synthesis, methylation, mutation rates, genomic stability, and gene expression, which are altered in colon cancer. Serine hydroxymethyltransferase 1 (SHMT1) regulates thymidylate (dTMP) biosynthesis and uracil accumulation in DNA, and as such affects genome stability. Previously, we showed that decreased SHMT1 expression in Shmt1 knockout mice (Shmt1(-/+)) or its impaired nuclear localization, as occurs in mice over-expressing an Shmt1 transgene (Shmt1(tg+)), results in elevated uracil incorporation into DNA, which could affect colon cancer risk. We used these 2 models to determine the effect of altered SHMT1 expression and localization, and its interaction with folate insufficiency, on azoxymethane (AOM)-induced colon cancer in mice. Shmt1(-/+) and Shmt1(tg+) mice were weaned to a control or folate-and-choline-deficient (FCD) diet and fed the diet for 28 or 32 wk, respectively. At 6 wk of age, mice were injected weekly for 6 wk with 10 mg/kg AOM (w/v in saline). Colon uracil concentrations in nuclear DNA were elevated 2-7 fold in Shmt1(-/+) and Shmt1(tg+) mice. However, colon tumor incidence and numbers were not dependent on SHMT1 expression in Shmt1(-/+) or Shmt1(-/-) mice. The FCD diet reduced tumor load independent of Shmt1 genotype. In contrast, Shmt1(tg+) mice exhibited a 30% reduction in tumor incidence, a 50% reduction in tumor number, and a 60% reduction in tumor load compared with wild-type mice independent of dietary folate intake. Our data indicate that uracil accumulation in DNA does not predict tumor number in AOM-mediated carcinogenesis. Furthermore, enrichment of SHMT1 in the cytoplasm, as observed in Shmt1(tg+) mice, protects against AOM-mediated carcinogenesis independent of its role in nuclear de novo dTMP biosynthesis.

Site-directed mutagenesis maps interactions that enhance cognate and limit promiscuous catalysis by an alkaline phosphatase superfamily phosphodiesterase.

Catalytic promiscuity, an evolutionary concept, also provides a powerful tool for gaining mechanistic insights into enzymatic reactions. Members of the alkaline phosphatase (AP) superfamily are highly amenable to such investigation, with several members having been shown to exhibit promiscuous activity for the cognate reactions of other superfamily members. Previous work has shown that nucleotide pyrophosphatase/phosphodiesterase (NPP) exhibits a >106-fold preference for the hydrolysis of phosphate diesters over phosphate monoesters, and that the reaction specificity is reduced 10³-fold when the size of the substituent on the transferred phosphoryl group of phosphate diester substrates is reduced to a methyl group. Here we show additional specificity contributions from the binding pocket for this substituent (herein termed the R' substituent) that account for an additional ~250-fold differential specificity with the minimal methyl substituent. Removal of four hydrophobic side chains suggested on the basis of structural inspection to interact favorably with R' substituents decreases phosphate diester reactivity 104-fold with an optimal diester substrate (R' = 5'-deoxythymidine) and 50-fold with a minimal diester substrate (R' = CH3). These mutations also enhance the enzyme's promiscuous phosphate monoesterase activity by nearly an order of magnitude, an effect that is traced by mutation to the reduction of unfavorable interactions with the two residues closest to the nonbridging phosphoryl oxygen atoms. The quadruple R' pocket mutant exhibits the same activity toward phosphate diester and phosphate monoester substrates that have identical leaving groups, with substantial rate enhancements of ~10¹¹-fold. This observation suggests that the Zn²âº bimetallo core of AP superfamily enzymes, which is equipotent in phosphate monoester and diester catalysis, has the potential to become specialized for the hydrolysis of each class of phosphate esters via addition of side chains that interact with the substrate atoms and substituents that project away from the Zn²âº bimetallo core.

cCMP, cUMP, cTMP, cIMP and cXMP as possible second messengers: development of a hypothesis based on studies with soluble guanylyl cyclase α(1)ß(1).

Adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate are second messengers that regulate multiple physiological functions. The existence of additional cyclic nucleotides in mammalian cells was postulated many years ago, but technical problems hampered development of the field. Using highly specific and sensitive mass spectrometry methods, soluble guanylyl cyclase has recently been shown to catalyze the formation of several cyclic nucleotides in vitro. This minireview discusses the broad substrate-specificity of soluble guanylyl cyclase and the possible second messenger roles of cyclic nucleotides other than adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate. We hope that this article stimulates productive and critical research in an area that has been neglected for many years.

A highly sensitive capillary electrophoresis method using p-nitrophenyl 5'-thymidine monophosphate as a substrate for the monitoring of nucleotide pyrophosphatase/phosphodiesterase activities.

A highly sensitive capillary electrophoresis method has been developed to monitor the activity of nucleotide pyrophosphatases/phosphodiesterases (NPPs) and screen for NPP inhibitors. In this method, p-nitrophenyl 5'-thymidine monophosphate (p-Nph-5'-TMP) was used as an artificial substrate, and separation of reaction products was performed on a dynamically coated capillary. We found that the optimal capillary electrophoresis (CE) conditions were as follows: fused-silica capillary (20cm effective length×75.5µm (id)), electrokinetic injection for 60s, 70mM phosphate buffer containing polybrene 0.002%, pH 9.2, constant current of -80µA, constant capillary temperature of 15°C and detection at 400nm. To allow precise quantification, 2-methyl-4,6-dinitrophenol (dinitrocresol) was applied as an internal standard. The limit of detection (LOD) and the limit of quantification (LOQ) were 137 and 415nM, respectively. This new method was shown to be over 8-fold more sensitive than the conventional spectrophotometric assays and 16-fold more than the previously reported CE procedure, and the results (K(m) values for NPP1 and NPP3, K(i) values for standard inhibitors) obtained were in accordance with previous literature data. Therefore, this new method is an improvement of actual techniques and could be used as a quick and standard analytical technique for the identification and characterization of NPP inhibitors.

[Stoichiometric model of folate-dependent metabolism of one-carbon units in human placenta].

The work is dedicated to creation of the mathematical model of folate-dependent one-carbon unit metabolism (FOCM) and study of its function in human placenta under homocysteine load and the most common mutations in the genes of methylenetetrahydrofolate reductase (MTHFR) and cystathionine beta-synthase (CBS). In the model we have taken into account specific features of placental expression of genes that encode enzymes of FOCM. Using software tools Metatool and COBRAToolbox we have identified key metabolites, elementary modes and metabolic fluxes through different reactions of the system. It is shown that the most vulnerable links in the system are the folate cycle and synthesis of precursors of nucleic acids, inosine monophosphate and thymidyne monophosphates, which are changing in the broad range from significant inhibition to activation depending on the imposed conditions. The most stable links in the system are the reactions of glutathione and taurine synthesis. Simulation results coincide with the results obtained in similar experimental conditions. Under certain imposed conditions non-obvious relationships between the system links are revealed, and this becomes the basis for a purposeful test of predictions generated by the model.

Enantiomeric specificity of biologically significant Cu(II) and Zn(II) chromone complexes towards DNA.

Novel chiral Schiff base ligands (R)/(S)-2-amino-3-(((1-hydroxypropan-2-yl)imino)methyl)-4H-chromen-4-one (L(1) and L(2)) derived from 2-amino-3-formylchromone and (R/S)-2-amino-1-propanol and their Cu(II)/Zn(II) complexes (R1, S1, R2, and S2) were synthesized. The complexes were characterized by elemental analysis, infrared (IR), hydrogen ((1) H) and carbon ((13)C) nuclear magnetic resonance (NMR), electrospray ionization-mass spectra (ESI-MS), and molar conductance measurements. The DNA binding studies of the complexes with calf thymus were carried out by employing different biophysical methods and molecular docking studies that revealed that complexes R1 and S1 prefers the guanine-cytosine-rich region, whereas R2 and prefers the adenine-thymine residues in the major groove of DNA. The relative trend in K(b) values followed the order R1>S1>R2>S2. This observation together with the findings of circular dichroic and fluorescence studies revealed maximal potential of (R)-enantiomeric form of complexes to bind DNA. Furthermore, the absorption studies with mononucleotides were also monitored to examine the base-specific interactions of the complexes that revealed a higher propensity of Cu(II) complexes for guanosine-5'-monophosphate disodium salt, whereas Zn(II) complexes preferentially bind to thymidine-5'-monophosphate disodium salt. The cleavage activity of R1 and R2 with pBR322 plasmid DNA was examined by gel electrophoresis that revealed that they are good DNA cleavage agents; nevertheless, R1 proved to show better DNA cleavage ability. Topoisomerase II inhibitory activity of complex R1 revealed that the complex inhibits topoisomerase II catalytic activity at a very low concentration (25 µM). Furthermore, in vitro antitumor activity of complexes R1 and S1 were screened against human carcinoma cell lines of different histological origin.

Liquid chromatography-tandem mass spectrometry method for quantification of thymidine kinase activity in human serum by monitoring the conversion of 3'-deoxy-3'-fluorothymidine to 3'-deoxy-3'-fluorothymidine monophosphate.

Thymidine kinase 1 (TK1) is an enzyme involved in DNA synthesis whose activity in serum is indicative of tumor proliferation and the severity of blood malignancies. 3'-deoxy-3'-fluorothymidine (FLT), a specific exogenous substrate for TK1, is phosphorylated by TK1 in the presence of a phosphorylating buffer, therefore the conversion of FLT to 3'-deoxy-3'-fluorothymidine monophosphate (FLT-MP) can be measured to assess serum TK1 activity. Here we describe a liquid chromatography-MS/MS (LC-MS/MS) method for quantification of FLT and FLT-MP from serum using protein precipitation and column switching followed by detection on an Applied Biosystems SCIEX API 4000 QTrap mass spectrometer. The method was linear over the range of 0.5-500 ng/mL for FLT and 2.5-2000 ng/mL for FLT-MP with a mean correlation coefficient of 0.9964 and 0.9935 for FLT and FLT-MP, respectively. The lower limit of quantification was 0.5 ng/mL for FLT and 2.5 ng/mL for FLT-MP. Intra-assay accuracy and inter-assay accuracy was within ±12% for both FLT and FLT-MP. Intra-assay precision was 2.8% to 7.7% for FLT and 3.3% to 5.8% for FLT-MP. Inter-assay precision was 4.6% to 14.9% for FLT and 4.9% to 14.6% for FLT-MP. Serum TK1 activity was measured in serum from hepatocellular carcinoma patients and age-matched controls under standardized conditions. Elevated TK1 activity was detected in 26.3% of hepatocellular carcinoma samples compared to controls. This method provides a robust alternative to radiometric and immunochemical assays of serum TK1 activity.

Novel positron emission tomography tracer distinguishes normal from cancerous cells.

Development of tumor-specific probes for imaging by positron emission tomography has broad implications in clinical oncology, such as diagnosis, staging, and monitoring therapeutic responses in patients, as well as in biomedical research. Thymidylate synthase (TSase)-based de novo biosynthesis of DNA is an important target for drug development. Increased DNA replication in proliferating cancerous cells requires TSase activity, which catalyzes the reductive methylation of dUMP to dTMP using (R)-N(5),N(10)-methylene-5,6,7,8-tetrahydrofolate (MTHF) as a cofactor. In principle, radiolabeled MTHF can be used as a substrate for this reaction to identify rapidly dividing cells. In this proof-of-principle study, actively growing (log phase) breast cancer (MCF7, MDA-MB-231, and hTERT-HME1), normal breast (human mammary epithelial and MCF10A), colon cancer (HT-29), and normal colon (FHC) cells were incubated with [(14)C]MTHF in culture medium from 30 min to 2 h, and uptake of radiotracer was measured. Cancerous cell lines incorporated significantly more radioactivity than their normal counterparts. The uptake of radioactively labeled MTHF depended upon a combination of cell doubling time, folate receptor status, S phase percentage, and TSase expression in the cells. These findings suggest that the recently synthesized [(11)C]MTHF may serve as a new positron emission tomography tracer for cancer imaging.

Synthesis of new chiral heterocyclic Schiff base modulated Cu(II)/Zn(II) complexes: their comparative binding studies with CT-DNA, mononucleotides and cleavage activity.

New Schiff base ligand L derived from the condensation reaction of 2-amino-3-formylchromone with (R)-2-amino-2-phenylethanol was synthesized and characterized which involves combination element of ammine functionality and naturally occurring heterocyclic chromone, 4H-benzopyran-4-one. Subsequently, their complexes 1 and 2 with Cu(NO3)2 and Zn(NO3)2, respectively were prepared. The DNA binding studies of the ligand L and complexes 1 and 2 with CT-DNA as compared to classical anticancer drug cisplatin were carried out by employing different optical methods viz, UV-vis, fluorescence, circular dichroism and viscosity measurements. Furthermore, the absorption studies, ¹H and ³¹P with mononucleotides were also monitored to examine the base specific interactions of the transition metal complexes which revealed a higher propensity of copper(II) complex 1 for 5'-GMP while for zinc(II) complex 2 towards 5'-TMP involving groove binding mechanism of the complexes towards DNA. The complex 1 exhibits a remarkable DNA cleavage activity with pBR322 DNA in presence of different activators and cleavage reaction involves various oxygen species suggesting the involvement of active oxygen species for the DNA scission.

Characterization of the TDP-D-ravidosamine biosynthetic pathway: one-pot enzymatic synthesis of TDP-D-ravidosamine from thymidine-5-phosphate and glucose-1-phosphate.

Ravidomycin V and related compounds, e.g., FE35A-B, exhibit potent anticancer activities against various cancer cell lines in the presence of visible light. The amino sugar moieties (D-ravidosamine and its analogues, respectively) in these molecules contribute to the higher potencies of ravidomycin and analogues when compared to closely related compounds with neutral or branched sugars. Within the ravidomycin V biosynthetic gene cluster, five putative genes encoding NDP-D-ravidosamine biosynthetic enzymes were identified. Through the activities of the isolated enzymes in vitro, it is demonstrated that ravD, ravE, ravIM, ravAMT and ravNMT encode TDP-D-glucose synthase, TDP-4-keto-6-deoxy-D-glucose-4,6-dehydratase, TDP-4-keto-6-deoxy-D-glucose-3,4-ketoisomerase, TDP-3-keto-6-deoxy-D-galactose-3-aminotransferase, and TDP-3-amino-3,6-dideoxy-D-galactose-N,N-dimethyl-transferase, respectively. A protocol for a one-pot enzymatic synthesis of TDP-D-ravidosamine has been developed. The results presented here now set the stage to produce TDP-D-ravidosamine routinely for glycosylation studies.

Coordinated leading and lagging strand DNA synthesis by using the herpes simplex virus 1 replication complex and minicircle DNA templates.

The origin-specific replication of the herpes simplex virus 1 genome requires seven proteins: the helicase-primase (UL5-UL8-UL52), the DNA polymerase (UL30-UL42), the single-strand DNA binding protein (ICP8), and the origin-binding protein (UL9). We reconstituted these proteins, excluding UL9, on synthetic minicircular DNA templates and monitored leading and lagging strand DNA synthesis using the strand-specific incorporation of dTMP and dAMP. Critical features of the assays that led to efficient leading and lagging stand synthesis included high helicase-primase concentrations and a lagging strand template whose sequence resembled that of the viral DNA. Depending on the nature of the minicircle template, the replication complex synthesized leading and lagging strand products at molar ratios varying between 1:1 and 3:1. Lagging strand products (∼0.2 to 0.6 kb) were significantly shorter than leading strand products (∼2 to 10 kb), and conditions that stimulated primer synthesis led to shorter lagging strand products. ICP8 was not essential; however, its presence stimulated DNA synthesis and increased the length of both leading and lagging strand products. Curiously, human DNA polymerase α (p70-p180 or p49-p58-p70-p180), which improves the utilization of RNA primers synthesized by herpesvirus primase on linear DNA templates, had no effect on the replication of the minicircles. The lack of stimulation by polymerase α suggests the existence of a macromolecular assembly that enhances the utilization of RNA primers and may functionally couple leading and lagging strand synthesis. Evidence for functional coupling is further provided by our observations that (i) leading and lagging strand synthesis produce equal amounts of DNA, (ii) leading strand synthesis proceeds faster under conditions that disable primer synthesis on the lagging strand, and (iii) conditions that accelerate helicase-catalyzed DNA unwinding stimulate decoupled leading strand synthesis but not coordinated leading and lagging strand synthesis.