Inhibition of klenow fragment (exo-) catalyzed DNA polymerization by (5R)-5,6-dihydro-5-hydroxythymidine and structural analogue 5,6-dihydro-5-methylthymidine

Oligonucleotides containing 5R-5,6-dihydro-5-hydroxythymidine (5R-3) and structural analogue 5,6-dihydro-5-methylthymidine (9) at defined sites were chemically synthesized via a method that obviates the use of NH4OH. Oligonucleotides prepared by this method were used to examine the effects of 5R-3 and 9 on the fidelity of Klenow (exo-) in vitro. The presence of lesions 5R-3 and 9 in DNA templates was shown to inhibit polymerization of primers hybridized to these templates. Inhibition was observed for both translesional synthesis and extension one nucleotide past the lesion, with the latter being more pronounced. The fidelity of Klenow (exo-) was reduced only slightly when utilizing substrates containing either dihyropyrimidine nucleotide. These results provide the first experimental verification of computational studies carried out on the effects of 3 on DNA templates, and are consistent with a structural model in which the C5-methyl group of 5R-3 adopts a pseudoaxial orientation resulting in a disruption in base stacking.     

Self-complementary oligodeoxyribonucleotides containing 2'-O-(anthraquinone-2-methyl)adenosine

Incorporation of an intercalating agent into an oligodeoxynucleotide (ODN) has the potential to enhance binding affinity upon duplex formation, to increase ODN hydrophobicity, and to enhance resistance to nuclease hydrolysis. Site-specific intercalation has been achieved through the synthesis of 2'-O-(anthraquinone-2-methyl)adenosine(rA*) and its incorporation into the palindromic dodecanucleotide d(CGCrA*CATGTGCG). Melting temperature, CD spectra, 1D and 2D (DQF-COSY and NOESY) NMR spectra, and molecular models were obtained and compared with the unmodified dodecamer. The data clearly establish that intercalation of the anthraquinone ring into a predominantly B-type helix occurs between the A4-T9 and C5-G8 base pairs, significantly stabilizing the duplex and enhancing the hydrophobicity of the ODN.     

Mechanism-based inhibition of C5-cytosine DNA methyltransferases by 2-H pyrimidinone

DNA duplexes in which the target cytosine base is replaced by 2-H pyrimidinone have previously been shown to bind with a significantly greater affinity to C5-cytosine DNA methyltransferases than unmodified DNA. Here, it is shown that 2-H pyrimidinone, when incorporated into DNA duplexes containing the recognition sites for M.HgaI-2 and M.MspI, elicits the formation of inhibitory covalent nucleoprotein complexes. We have found that although covalent complexes are formed between 2-H pyrimidinone-modified DNA and both M.HgaI-2 and M.MspI, the kinetics of complex formation are quite distinct in each case. Moreover, the formation of a covalent complex is still observed between 2-H pyrimidinone DNA and M.MspI in which the active-site cysteine residue is replaced by serine or threonine. Covalent complex formation between M.MspI and 2-H pyrimidinone occurs as a direct result of nucleophilic attack by the residue at the catalytic position, which is enhanced by the absence of the 4-amino function in the base. The substitution of the catalytic cysteine residue by tyrosine or chemical modification of the wild-type enzyme with N-ethylmaleimide, abolishes covalent interaction. Nevertheless the 2-H pyrimidinone-substituted duplex still binds to M.MspI with a greater affinity than a standard cognate duplex, since the 2-H pyrimidinone base is mis-paired with guanine.     

Identification of a subdomain within DNA-(cytosine-C5)-methyltransferases responsible for the recognition of the 5' part of their DNA target

In previous work on DNA-(cytosine-C5)-methyltransferases (C5-MTases), domains had been identified which are responsible for the sequence specificity of the different enzymes (target-recognizing domains, TRDs). Here we have analyzed the DNA methylation patterns of two C5-MTases containing reciprocal chimeric TRDs, consisting of the N- and C-terminal parts derived from two different parental TRDs specifying the recognition of 5'-CC(A/T)GG-3' and 5'-GCNGC-3'. Sequences recognized by these engineered MTases were non-symmetrical and degenerate, but contained at their 5' part a consensus sequence which was very similar to the 5' part of the target recognized by the parental TRD which contributed the N-terminal moiety of the chimeric TRD. The results are discussed in connection with the present understanding of the mechanism of DNA target recognition by C5-MTases. They demonstrate the possibility of designing C5-MTases with novel DNA methylation specificities.     

Guanine tetraplex formation by short DNA fragments containing runs of guanine and cytosine

Using CD spectroscopy, guanine tetraplex formation was studied with short DNA fragments in which cytosine residues were systematically added to runs of guanine either at the 5' or 3' ends. Potassium cations induced the G-tetraplex more easily with fragments having the guanine run at the 5' end, which is just an opposite tendency to what was reported for (G+T) oligonucleotides. However, the present (G+C) fragments simultaneously adopted other conformers that complicated the analysis. We demonstrate that repeated freezing/thawing, performed at low ionic strength, is a suitable method to exclusively stabilize the tetraplex in the (G+C) DNA fragments. In contrast to KCl, the repeated freeze/thaw cycles better stabilized the tetraplex with fragments having the guanine run on the 3' end. The tendency of guanine blocks to generate the tetraplex destabilized the d(G5).d(C5) duplex whose strands dissociated, giving rise to a stable tetraplex of (dG5) and single-stranded (dC5). In contrast to d(G3C3) and d(G5C5), repeated freezing/thawing induced the tetraplex even with the self-complementary d(C3G3) or d(C5G5); hence the latter oligonucleotides preferred the tetraplex to the apparently very stable duplex. The tetraplexes only included guanine blocks while the 5' end cytosines interfered neither with the tetraplex formation nor the tetraplex structure.     

Inhibition of DNA cytosine methyltransferase by chemopreventive selenium compounds, determined by an improved assay for DNA cytosine methyltransferase and DNA cytosine methylation

The organoselenium compounds benzyl selenocyanate (BSC) and 1,4-phenylenebis(methylene)selenocyanate (p-XSC), as well as sodium selenite, are effective chemopreventive agents for various chemically induced tumors in animal models at both the initiation and postinitiation stages. The mechanisms involved at the postinitiation stage are not clear. Because several lines of evidence indicate that inhibition of excess DNA (cytosine-5)-methyltransferase (Mtase) may be a sufficient factor for the suppression or reversion of carcinogenesis, we examined the effects of sodium selenite, BSC, p-XSC and benzyl thiocyanate (BTC), the sulfur analog of BSC, on Mtase activity in nuclear extracts of human colon carcinomas, and of p-XSC on the Mtase activity of HCT116 human colon carcinoma cells in culture. For this purpose, we developed an improved Mtase assay, in which the incorporation of the methyl-[3H] group from S-adenosyl[methyl-3H]methionine into deoxycytidine of poly(dI-dC)-poly(dI-dC), is specifically determined by HPLC with radioflow detection after enzymatic hydrolysis, enhancing specificity and reliability. In a variation, using SssI methyltransferase and labeled S-adenosylmethionine, the overall methylation status of DNA in various tissues can also be compared. Selenite, BSC and p-XSC inhibited Mtase extracted from a human colon carcinoma with IC50s of 3.8, 8.1 and 5.2 microM, respectively; BTC had no effect. p-XSC also inhibited the Mtase activity and growth of human colon carcinoma HCT116 cells, with an IC50 of approximately 20 microM. The improved Mtase assay should prove to be a reliable method for screening potential Mtase inhibitors, especially using cells in culture. We suggest that inhibition of Mtase may be a major mechanism of chemoprevention by selenium compounds at the postinitiation stage of carcinogenesis.     

Synthesis of modified oligodeoxyribonucleotides containing 9-(2'-amino-2'-deoxy-beta-D-arabinofuranosyl)adenine

A method for preparing a modified derivative of 2'-amino-2'-deoxy-arabino-adenosine ready for directed insertion into an oligonucleotide chain during solid-phase synthesis was elaborated. A series of the title oligonucleotides (6-25-mers) containing a 2'-amino-2'-deoxy-arabino-adenosine fragment were prepared. A high reactivity of the 2'-amino group during the acylation with carboxylic acid anhydrides was demonstrated. It was shown that the insertion of the modified fragments into oligonucleotides did not inhibit the formation of the DNA duplex.     

Efficacy of 5,6-dihydro-5-azathymidine against cutaneous herpes simplex virus in hairless mice

5,6-Dihydro-5-azathymidine, administered subcutaneously, was active both prophylactically and therapeutically against cutaneous herpesvirus infection of hairless mice. Activity was comparable to that obtained with adenine arabinoside.     

A comparative study of the thermal stability of oligodeoxyribonucleotides containing 5-substituted 2'-deoxyuridines

Two series of modified oligonucleotides based on the self-complementary dodecamer d(CGCTAATTAGCG) were synthesized. The first contained the -C identical withCCH2R linker at C5 of deoxyuridine at position 4 (T*) of d(CGCT*AATTAGCG) and the second contained the -SR linker. The goal of the study was to evaluate and compare these two types of side chains for suitability as tethers for linking reporter groups to oligonucleotides. Our primary concern was how these tethers would effect duplex stability. The modified nucleosides were synthesized by palladium-mediated coupling reactions between the substituted alkyne and 5'-(4, 4'-dimethoxytrityl)-5-iodo-2'-deoxyuridine and between a disulfide and 5-chloromercurio-2'-deoxyuridine. The C5 deoxyuridine side chains evaluated included C identical with CCH3, C identical with CCH2NHC(O)CH3, C identical with CCH2N(CH3)2, C identical with CCH2N-HC(O)C5H4N, C identical with CCH2NHC(O)C10H15, SCH3, SC6H5 and SCH2CH2NHC(O)CH3. The nucleosides containing these substituents were incorporated into oligo-deoxyribonucleotides by standard phosphoramidite methodology. Melting studies demonstrated that the sequence containing the C identical with CCH3side chain had the highest T m value (59.1 degrees C) in comparison with the control sequence (T m = 55.2 degrees C) and that any additional substituent on C3 of the propynyl group lowered the T m value relative to propynyl. Nevertheless, even the most destabilizing substituent, adamantylcarbamoyl, yielded an oligodeoxyribonucleotide that dissociated with a T m of 54 degrees C, which is only 1.2 degrees C less than the control sequence. In contrast, the thioether substituents led to lower T m values, ranging from as low as 45.1 degrees C for SPh up to 52.2 degrees C for SMe. Replacing the methyl of the SMe substituent with a CH2CH2NHC(O)CH3 tether led to no further reduction in melting temperature. The T m value of the CH2CH2NHC(O)CH3-containing oligonucleotide was less than the natural sequence by 1.6 degrees C/substituent. This is sufficiently small that it is anticipated that the C5 thioether linkage may be as useful as the acetylenic linkage for tethering reporter groups to oligonucleotides. More importantly, the thioether linkage provides a means to position functional groups to interact specifically with opposing complementary (target) sequences.     

Studies on the mechanism of action of 1-beta-D-arabinofuranosyl-5-azacytosine (fazarabine) in mammalian lymphoblasts

Fazarabine has shown activity in the panel of 60 cultured human tumor lines of the National Cancer Institute. COMPARE analyses relating correlation coefficients of other anticancer drugs with those of fazarabine suggest that this agent operates through a similar mode of action to that of cytarabine. Studies have been carried out both in culture and in vivo to examine the mechanism of action of fazarabine in P388 murine and Molt-4 human lymphoblasts. Authentic fazarabine nucleotide standards were prepared by chemical and enzymatic methods and characterized on HPLC by comparison to related pyrimidine nucleoside-5'-phosphates as well as by enzymatic digestion. Fazarabine inhibited the incorporation of labeled thymidine into DNA without influencing the synthesis of RNA or protein. Deoxycytidine overcomes this inhibition of DNA synthesis and also prevents the cytotoxicity of the drug to lymphoblasts, probably by competing for fazarabine uptake and metabolism. Fazarabine was rapidly phosphorylated in both cell lines; in P388 cells it was incorporated into DNA, where it continued to undergo the same type of ring opening and degradation as the free nucleoside. Alkaline elution studies demonstrated that exposure to the agent resulted in the formation of alkaline labile sites. Fazarabine also inhibited the methylation of deoxycytidine residues in DNA, but this effect was less pronounced than that produced by 5-azacytidine. Taken together, these studies suggest that fazarabine probably acts by arresting the synthesis and/or altering the structural integrity or functional competence of DNA.     

Mutagenicity of nitric oxide is not caused by deamination of cytosine or 5-methylcytosine in double-stranded DNA

Several human tumors of diverse histological origin have a high incidence of C:G to T:A transition mutations at methylated CpG sites in tumor suppressor genes. We used a sensitive genetic assay to examine the ability of nitric oxide (NO), a physiological intra- and intercellular messenger molecule, to promote these transitions by deaminating cytosine (C) or methylcytosine (5mC) in double-stranded DNA. Exposure of a test double-stranded plasmid containing C or 5mC at the target site to NO in phosphate-buffered solution at pH 7.4 followed by transformation into Escherichia coli ung- strain to avoid repair of U did not result in a significant increase in reversion frequency. In addition, exposure of E. coli transformed with the target plasmid to an NO-releasing spermine-NO complex during log-phase growth did not result in larger numbers of revertants, whereas Salmonella typhimurium strain TA1535 showed a dose-responsive increase in reversion frequency when treated in the same way. We conclude that genotoxicity of NO is not caused by deamination of C or 5mC to U or T, respectively, in double-stranded DNA. This is supported by the finding that extracts of TA1535 contained high uracil-DNA glycosylase activity, suggesting that the difference in mutagenesis between the strains is not due to a lack of uracil repair. Therefore, mutational hot-spots seen in human tumor tissues at CpG sites are probably not due to the action of NO at 5mC.     

Interaction of the recombinant human methylpurine-DNA glycosylase (MPG protein) with oligodeoxyribonucleotides containing either hypoxanthine or abasic sites

Methylpurine-DNA glycosylases (MPG proteins, 3-methyladenine-DNA glycosylases) excise numerous damaged bases from DNA during the first step of base excision repair. The damaged bases removed by these proteins include those induced by both alkylating agents and/or oxidizing agents. The intrinsic kinetic parameters (k(cat) and K(m)) for the excision of hypoxanthine by the recombinant human MPG protein from a 39 bp oligodeoxyribonucleotide harboring a unique hypoxanthine were determined. Comparison with other reactions catalyzed by the human MPG protein suggests that the differences in specificity are primarily in product release and not binding. Analysis of MPG protein binding to the 39 bp oligodeoxyribonucleotide revealed that the apparent dissociation constant is of the same order of magnitude as the K(m) and that a 1:1 complex is formed. The MPG protein also forms a strong complex with the product of excision, an abasic site, as well as with a reduced abasic site. DNase I footprinting experiments with the MPG protein on an oligodeoxyribonucleotide with a unique hypoxanthine at a defined position indicate that the protein protects 11 bases on the strand with the hypoxanthine and 12 bases on the complementary strand. Competition experiments with different length, double-stranded, hypoxanthine-containing oligodeoxyribonucleotides show that the footprinted region is relatively small. Despite the small footprint, however, oligodeoxyribonucleotides comprising <15 bp with a hypoxanthine have a 10-fold reduced binding capacity compared with hypoxanthine-containing oligodeoxyribonucleotides >20 bp in length. These results provide a basis for other structural studies of the MPG protein with its targets.     

(R)-5-fluoro-5,6-dihydrouracil: kinetics of oxidation by dihydropyrimidine dehydrogenase and hydrolysis by dihydropyrimidine aminohydrolase

The biologically active isomer of 5-fluoro-5,6-dihydrouracil [(R)-5-fluoro-5,6-dihydrouracil, R-FUH2] was synthesized to study the kinetics of its enzymatic oxidation and hydrolysis by homogeneous dihydropyrimidine dehydrogenase (DPDase) and dihydropyrimidine aminohydrolase (DPHase), respectively. DPDase catalyzed the slow oxidation of R-FUH2 at pH 8 and 37 degrees with a Km of 210 microM and a kcat of 0.026 sec-1 at a saturating concentration of NADP+. The catalytic efficiency (kcat/Km) of DPDase for R-FUH2 was 1/14th of that for 5,6-dihydrouracil (UH2). In the opposite direction, DPDase catalyzed the reduction of 5-fluorouracil (FU) with a Km of 0.70 microM and a kcat of 3 sec-1 at a saturating concentration of NADPH. Thus, DPDase catalyzed the reduction of FU 30,000-fold more efficiently than the oxidation of R-FUH2. In contrast to the slow oxidation of R-FUH2 by DPDase, R-FUH2 was hydrolyzed very efficiently by DPHase with a Km of 130 microM and a kcat of 126 sec-1. The catalytic efficiency of DPHase for the hydrolysis of R-FUH2 was approximately twice that for the hydrolysis of UH2. Because R-FUH2 is hydrolysis of R-FUH2 was approximately twice that for the hydrolysis of UH2. Because R-FUH2 is hydrolyzed considerably more efficiently than it is oxidized and because the activity of DPHase was 250- to 500-fold greater than that of DPDase in bovine and rat liver, the hydrolytic pathway should predominate in vivo.     

Conformational and electronic properties of the two cis (5S,6R) and (5R,6S) diastereoisomers of 5,6-dihydroxy-5,6-dihydrothymidine: X-ray and theoretical studies

For the first time, raloxifene or alendronate was administered to rats immediately after ovariectomy for 10 months and compared with estrogen to elucidate mechanisms behind the raloxifene effects observed in nonreproductive and reproductive tissues. Specifically, 75-day-old rats were randomly selected as sham controls (Sham), ovariectomized controls (Ovx) or ovariectomized rats treated with fully efficacious doses of raloxifene (RA), 17 alpha-ethynyl estradiol (EE2) or alendronate (ABP). Lumbar vertebrae and proximal tibiae were examined by computed tomography (QCT) and by histomorphometry. Histomorphometry showed differences in bone architecture between groups when QCT densities were similar, but tibial trabecular bone analysis by QCT correlated with histomorphometry with r = .86 to .93, depending on the parameter. Both techniques confirmed that Ovx had substantially less bone than Sham, with greater loss of trabecular bone in the proximal tibia than vertebrae. Both techniques showed that RA had effects similar to but not identical with EE2 in preventing bone loss in vertebrae and tibiae. ABP partially prevented loss of bone in L-5, but was not significantly different from Ovx in the proximal tibia. This may be caused by ABP suppression of bone apposition, beyond effects observed for EE2 or RA. RA appeared to be more similar to EE2 because ABP significantly depressed bone formation (bone formation rate, mineral apposition rate) to below RA or EE2 levels, especially in L-5. Mechanical loading to failure of L-6 vertebrae showed a rank order of vertebral strength of Sham > RA > EE2 > Ovx > ABP, although significant differences were not observed between treatment groups. These data show that ABP suppression of bone formation can affect bone quality with long-term treatment. In other tissues, RA had minimal uterine effects, while significantly lowering serum cholesterol to below EE2-treated levels. Both EE2 and RA rats had significantly lower body weights than the other groups. ABP had no effect on serum lipids, uterine weight or body weight. Therefore, RA appears to have a broader range of desirable effects on bone, body weight, uteri and cholesterol than ABP or EE2 in ovariectomized rats.     

Molecular dynamics studies of axis bending in d(G5-(GA4T4C)2-C5) and d(G5-(GT4A4C)2-C5): effects of sequence polarity on DNA curvature

Gel retardation studies and other experiments indicate that DNA sequences containing the d(GA4T4C)n motif are curved, whereas those of identical composition but with a reverse sequence polarity, the d(GT4A4C)n motif, are straight. Hydroxyl radical cleavage experiments show that d(GA4T4C)n shows a unique signature, whereas d(GT4A4C)n behaves normally. To explain these results at a molecular level, molecular dynamics (MD) simulations were performed on the DNA duplexes d(G5-(GA4T4C)2-C5) and d(G5-(GT4A4C)2-C5) to 3.0 and 2.5 ns, respectively. The MD simulations are based on the Cornell force field implemented in the AMBER 4.1 modeling package and performed in a neutral solution of anionic DNA with K+, Cl- and Mg2+ at concentrations roughly comparable to a ligase buffer. Long range interactions were treated by the particle mesh Ewald method. Analysis of the results shows that the calculated dynamical structure of d(G5-(GA4T4C)2-C5) exhibits strong gross curvature, consistent with the observed behavior. The most significant locus of curvature in the MD structure is found at the central C15-G16 step, with an average roll angle of 12.8(+/-6.40)deg. The d(G5-(GT4A4C)2-C5) MD structure exhibited significantly less gross curvature. Analysis of results indicates that the reduction in gross curvature in the d(G5-(GT4A4C)2-C5) trajectory originates from the effect of the T10-A11 and T20-A21 steps, which showed average roll angles of 12.5(+/-5)deg. These three steps, T10-A11, C15-G16 and T20-A21, are half-helix turns away from one another, and their contributions to concerted bending cancel out. The A-tracts in the MD structure are essentially straight. The dynamical structure of d(G5-(GA4T4C)2-C5) exhibited minor groove deformation comprised of expansion at the 5' end of A-tracts and progressive narrowing towards the 3' end, consistent with and elaborating the interpretation of hydroxyl radical chemical probing results.     

Effect of selective cytosine methylation and hydration on the conformations of DNA triple helices containing a TTTT loop structure by FT-IR spectroscopy

5-Methylcytosines have been introduced into triplex-forming-oligonucleotides and shown to extend the pH range over which a triplex forms with a homopurine-homopyrimidine tract of duplex DNA. As a host strand, an oligodeoxypyrimidine with a base sequence of 5'-d(TC)3T4(CT)3 ([CC]) was designed to form a hairpin triplex with a 5'-d-A(GA)2G ([AG6]) purine strand at acidic pH (Tsay, et al., (1995) J. Biomol. Str. Dyn., 13, 1235-1245). We here present results obtained by FT-IR spectroscopy concerning the conformation of the hairpin triplex as a function of the selective substitution of cytosines by 5-methylcytosines in the host strand. Namely, cytosines are substituted by 5-methylcytosines in either the 3'-pyrimidine portion ([CM]) or the 5'-pyrimidine portion ([MC]) or in both ([MM]) of the host strand. The acidic-induced transitions of the equimolar mixtures of the purine target with either of the four pyrimidine oligomers gives rise to different apparent pK values, i.e., [MM].[AG6] (6.2) > [MC].[AG6] (6.0) > [CM].[AG6] (5.7) > [CC].[AG6] (5.2) > single-stranded oligopyrimidines (4.6 +/- 0.2), indicating that cytosine methylation expands the pH range compatible with the hairpin triplex formation regardless of whether the substitution is in the 5'-pyrimidine (Hoogsteen) portion or in the 3'-pyrimidine (Watson-Crick) portion. Thermal denaturation profiles indicated that all the triplexes denatured in a monophasic manner in the pH range of 4.0 to 7.0, and that cytosine methylations in any position of the 16-base pyrimidine oligomer increase the stability of the hairpin triplex DNA. IR spectra recorded in D2O and H2O solutions revealed that cytosine methylation does not significantly influence the conformation of triplex DNA in solution, i.e., all the four triplexes accept a similar sugar conformation, and predominately take on a S-type sugar pucker with a relative proportion of two S-type sugars for one N-type. Furthermore, we also investigated the effect of relative humidity (RH) on the conformation of triplex MC.AG6 in hydrated films, and found that the conformational change induced by the decrease of RH, from predominant S-type to primary N-type sugar pucker, might first occur in the purine strand at 86% RH.     

Structures of oligonucleotides containing 5-(methoxymethyl)-2'-deoxyuridine determined by NMR spectroscopy

Base pairing of 5-(methoxymethyl)-2'-deoxyuridine (MMdU) opposite either adenine or guanine in a seven base pair oligonucleotide duplex has been studied by NMR spectroscopy. When paired with A, we observe that the MMdU.A base pair adopts Watson-Crick geometry. The methoxymethyl substituent is not held in a fixed conformation and may rotate around the C5-CH2 and CH2-O bonds. Examination of the potential energy as a function of rotation around these bonds indicates the presence of four low energy conformations. No hydrogen bonding is indicated for the methoxymethyl substituent, and the four potential minima result from reduced steric clash. For the MMdU.G base pair, the two bases adopt a wobble geometry which does not change with increasing solvent pH. Similarly, we find four low energy conformations for the methoxymethyl substituent in the major groove of the DNA helix.