Extension of the range of DNA sequences available for triple helix formation: stabilization of mismatched triplexes by acridine-containing oligonucleotides

Triple helix formation usually requires an oligopyrimidine*oligopurine sequence in the target DNA. A triple helix is destabilized when the oligopyrimidine*oligopurine target contains one (or two) purine*pyrimidine base pair inversion(s). Such an imperfect target sequence can be recognized by a third strand oligonucleotide containing an internally incorporated acridine intercalator facing the inverted purine*pyrimidine base pair(s). The loss of triplex stability due to the mismatch is partially overcome. The stability of triplexes formed at perfect and imperfect target sequences was investigated by UV thermal denaturation experiments. The stabilization provided by an internally incorporated acridine third strand oligonucleotide depends on the sequences flanking the inverted base pair. For triplexes containing a single mismatch the highest stabilization is observed for an acridine or a propanediol tethered to an acridine on its 3'-side facing an inverted A*T base pair and for a cytosine with an acridine incorporated to its 3'-side or a guanine with an acridine at its 5'-side facing an inverted G*C base pair. Fluorescence studies provided evidence that the acridine was intercalated into the triplex. The target sequences containing a double base pair inversion which form very unstable triplexes can still be recognized by oligonucleotides provided they contain an appropriately incorporated acridine facing the double mismatch sites. Selectivity for an A*T base pair inversion was observed with an oligonucleotide containing an acridine incorporated at the mismatched site when this site is flanked by two T*A*T base triplets. These results show that the range of DNA base sequences available for triplex formation can be extended by using oligonucleotide intercalator conjugates.     

Triplex formation at physiological pH by oligonucleotides incorporating 5-Me-dC-(N4-spermine)

Oligonucleotide (ODN) directed triplex formation has therapeutic importance and depends on Hoogsteen hydrogen bonds between a duplex DNA and a third strand. While T*A:T triplets are formed at neutral pH, C+*G:C are favoured at acidic pH. Herein it is shown that 18-mer ODN containing spermine conjugated to 5-Me-dC at N4 (1-5), form triplexes with complementary 24-mer duplex 8:9 at neutral pH (7.3, 100 mM NaCl). Under such conditions, control ODN's carrying dC (6) or 5-Me-dC (7) did not show any triple helix formation. Remarkably, the triplexes from spermine-conjugates (1-5) have foremost stability at neutral pH (7.1), unlike the behavior of normal ODN's where optimal stability is at acidic pH (5.5). These results have importance in designing oligonucleotides for antigene applications.     

Chemical and enzymatic properties of bridging 5'-S-phosphorothioester linkages in DNA

We describe physicochemical and enzymatic properties of 5' bridging phosphorothioester linkages at specific sites in DNA oligonucleotides. The susceptibility to hydrolysis at various pH values is examined and no measurable hydrolysis is observed at pH 5-9 after 4 days at 25 degrees C. The abilities of three 3'- and 5'-exonuclease enzymes to hydrolyze the DNA past this linkage are examined and it is found that the linkage causes significant pauses at the sulfur linkage for T4 DNA polymerase and calf spleen phosphodiesterase, but not for snake venom phosphodiesterase. Restriction endonuclease (Nsi I) cleavage is also attempted at a 5'-thioester junction and strong resistance to cleavage is observed. Also tested is the ability of polymerase enzymes to utilize templates containing single 5'-S-thioester linkages; both Klenow DNA polymerase and T7 RNA polymerase are found to synthesize complementary strands successfully without any apparent pause at the sulfur linkage. Finally, the thermal stabilities of duplexes containing such linkages are measured; results show that T m values are lowered by a small amount (2 degrees C) when one or two thioester linkages are present in an otherwise unmodified duplex. The chemical stability and surprisingly small perturbation by the 5' bridging sulfur make it a good candidate as a physical and mechanistic probe for specific protein or metal interactions involving this position in DNA.     

The chi psi subunits of DNA polymerase III holoenzyme bind to single-stranded DNA-binding protein (SSB) and facilitate replication of an SSB-coated template

A complex of the chi and psi proteins is required to confer resistance to high levels of glutamate on the DNA polymerase III holoenzyme-catalyzed reaction (Olson, M., Dallmann, H. G., and McHenry, C. (1995) J. Biol. Chem. 270, 29570-29577). We demonstrate that this salt resistance also requires templates to be coated with the Escherichia coli single-stranded DNA-binding protein (SSB). We show that this is the result of a direct chipsi-SSB interaction that is strengthened approximately 1000-fold when SSB is bound to DNA. On model oligonucleotide templates, DNA polymerase III core is inhibited by SSB. We show that the minimal polymerase assembly that will synthesize DNA on SSB-coated templates is polymerase III-tau-psi chi. gamma, the alternative product of the dnaX gene, will not replace tau in this reaction, indicating that tau's unique ability to bind to DNA polymerase III holding chipsi in the same complex is essential. All of our findings are consistent with chipsi strengthening DNA polymerase III holoenzyme interactions with the SSB-coated lagging strand at the replication fork, facilitating complex assembly and elongation.     

Recognition of sequence-directed DNA structure by the Klenow fragment of DNA polymerase I

Time-resolved fluorescence spectroscopy was used to investigate the influence of sequence-directed DNA structure upon the interaction between the Klenow fragment of DNA polymerase I and a series of defined oligonucleotide primer/templates. 17/27-mer (primer/template) oligonucleotides containing a dansyl fluorophore conjugated to a modified deoxyuridine residue within the primer strand were used as substrates for binding to Klenow fragment. The time-resolved fluorescence anisotropy decay of the dansyl probe was analyzed in terms of two local environments, either solvent-exposed or buried, corresponding to primer/templates positioned with the primer 3' terminus in the polymerase site or the 3'-5' exonuclease site of the enzyme, respectively. Equilibrium constants for partitioning of DNA between the two sites were evaluated from the anisotropy decay data for primer/templates having different (A + T)-rich sequences flanking the primer 3' terminus. Primer/templates with AAAATG/TTTTAC and CGATAT/GCTATA terminal sequences (the nucleotides on the left refer to the last six bases at the 3' end of the primer, and the nucleotides on the right are the corresponding bases in the template) were bound mostly at the polymerase site. The introduction of single mismatches opposite the primer 3' terminus of these DNA substrates increased their partitioning into the 3'-5' exonuclease site, in accord with the results of an earlier study [Carver, T.E., Hochstrasser, R.A., and Millar, D.P. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10670-10674]. In contrast, a primer/template with the terminal sequence CAATTT/GTTAAA, containing an A-tract element AATTT, exhibited a surprising preference for binding at the 3'-5' exonuclease site, despite the absence of mismatched bases in the DNA substrate. Interruption of the A-tract with a single AG step, to give the terminal sequence CAGTTT/GTCAAA, reversed the effect of the A-tract, causing the DNA to partition in favor of the polymerase site. Moreover, the presence of a single mismatch opposite the primer 3' terminus was also sufficient to reverse the effect of the A-tract, resulting in a distribution of DNA between polymerase and 3'-5' exonuclease sites that was similar to that observed for the other mismatched DNA substrates. Taken together, these results suggest that the A-tract adopts an unusual conformation that is disruptive to binding at the polymerase site. The effect of the A-tract on binding of DNA to the polymerase site is discussed in terms of the unusual helix structural parameters associated with these sequence elements and the difference between the local geometry of the A-tract and the conformation adopted by duplex DNA within the polymerase cleft. The results of this study show that in addition to base mismatches, Klenow fragment can also recognize irregularities in the helix geometry of perfectly base-paired DNA.     

Dual recognition of double-stranded DNA by 2'-aminoethoxy-modified oligonucleotides: the solution structure of an intramolecular triplex obtained by NMR spectroscopy

The solution structure of an intramolecular triple helical oligonucleotide has been solved by NMR. The third strand of the pyrimidine x purine x pyrimidine triplex is composed of 2'-aminoethoxy-modified riboses, whereas the remaining part of the nucleic acid is DNA. The structure around the aminoethoxy modification was obtained with the help of selective isotope labeling in conjunction with isotope-editing experiments. Dinucleotide steps and interstrand connectivities, as well as the complete backbone conformation of the triplex, were derived from J-couplings, NOEs, and 31P chemical shifts. The structure of this triplex, solved by distance geometry, explains the extraordinary stability and increase in rate of triplex formation induced by 2'-aminoethoxy-modified oligonucleotides: apart from the formation of seven base triples, a well-defined hydrogen-bonding network is formed across the Crick-Hoogsteen groove involving the amino protons of the aminoethoxy moieties and the phosphates of the purine strand of the DNA. The modified strand adopts a conformation which is close to an A-type helix, whereas the DNA duplex conformation is best described as an unwound B-type helix. The groove dimensions and helical parameters of the 2'-aminoethoxy-modified rY x dRdY triplex are surprisingly well conserved in comparison with DNA triplexes.     

Neural activity related to reaching and grasping in rostral and caudal regions of rat motor cortex

We have analyzed the mutational spectra produced during in vitro DNA synthesis by DNA polymerase alpha-primase and DNA polymerase beta. The polymerase mutation frequency as measured in the in vitro herpes simplex virus thymidine kinase (HSV-tk) forward assay was increased when reactions utilized single-stranded DNA templates randomly modified by 20 mM N-ethyl-N-nitrosourea (ENU), relative to solvent-treated templates. A 20- to 50-fold increase in the frequency of G-->A transition mutations was observed for both polymerases, as expected due to mispairing by O6-ethylguanine lesions. Strikingly, ENU treatment of the template also resulted in a five- to 12-fold increased frequency of frameshift errors at heteropolymeric (non-repetitive) template sequences produced by polymerase beta and polymerase alpha-primase, respectively. The increased proportion of frameshift mutations at heteropolymeric sequences relative to homopolymeric (repetitive) sequences produced by each polymerase in response to ENU damage was statistically significant. For polymerase alpha-primase, one-base deletion errors at template guanine residues was the second most frequent mutational event, observed at a frequency only four-fold lower than the G-->A transition frequency. In the polymerase beta reactions, the frequency of insertion errors at homopolymeric (repetitive) sequences was increased six-fold using alkylated templates, relative to solvent controls. The frequency of such insertion errors was only three-fold lower than the frequency of G-->A transition errors by polymerase beta. Although ENU is generally regarded as a potent base substitution mutagen, these data show that monofunctional alkylating agents are capable of inducing frameshift mutations in vitro. Alkylation-induced frameshift mutations occur in both repetitive and non-repetitive DNA sequences; however, the mutational specificity is dependent upon the DNA polymerase.     

Coralyne has a preference for intercalation between TA.T triples in intramolecular DNA triple helices

Intercalating ligands may improve both the stability and sequence specificity of triple helices. Numerous intercalating drugs have been described, including coralyne, which preferentially binds triple helices, though its sequence specificity has been reported to be low [Lee,J.S., Latimer,L.J.P. and Hampel,K.J. (1993) Biochemistry , 32, 5591-5597]. In order to analyse the sequence preferences of coralyne we have used a combination of DNase I footprinting, UV melting, UV-visible spectrophotometry, circular dichroism and NMR spectroscopy to examine defined intermolecular triplexes and intramolecular triplexes linked either by hexaethylene glycol chains or by octandiol chains. DNase I footprinting demonstrated that coralyne has a moderate preference for triplexes over duplexes, but a substantial preference for TA.T triplets compared with CG. C+triplets. The drug was found to have essentially no effect on the melting temperatures of duplexes of the kind d(A)n.d(T)n or d(GA)n.d(TC)n. In contrast, it increased the T m for triplexes of the kind d(T)nd(A)n.dTn, but had little effect on the stability of d(TC)nd(GA).d(CT)n at either low or high pH. On binding to DNA triplexes, there is a large change in the absorption spectrum of coralyne and also a substantial fluorescence quenching that can be attributed to intercalation. The changes in the optical spectra have been used for direct titration with DNA. For triplexes d(T)6d(A)6.d(T)6, the Kd at 298 K was 0.5-0.8 microM. In contrast, the affinity for d(TC) nd(GA)n.d(CT)n triplexes was 6- to 10-fold lower and was characterized by smaller changes in the absorption and CD spectra. This indicates a preference for intercalation between TAT triples over CG.C+/TA.T triples. NMR studies confirmed interaction by intercalation. However, a single, secondary binding was observed at high concentrations of ligand to the triplex d(AGAAGA-L-TCTTCT-L-TCTTCT), presumably owing to the relatively low difference in affinity between the TA.T site and the competing, neighbouring sites.     

Residues at the carboxy terminus of T4 DNA polymerase are important determinants for interaction with the polymerase accessory proteins

Three T4 DNA polymerase accessory proteins (44P/62P and 45P) stimulate the polymerase (pol) activity and the 3'-5' exonuclease (exo) activity of T4 DNA polymerase (43P) on long, double-stranded DNA substrates. The 44P/62P "clamp loader" facilitates the binding of 45P, the "sliding clamp", to DNA that is primed for replication. Using a series of truncated 43P mutants, we identified a region at the extreme carboxy terminus of the DNA polymerase that is required for its interaction with accessory proteins. Truncation mutants of 43P lacking the carboxy-terminal 3, 6, or 11 residues retained full pol and exo activity on short synthetic primer-templates. However, the ability of the accessory proteins to enhance these activities on long double-stranded DNA templates was drastically reduced, and the extent of the reduction in activity was greater as more residues were deleted. One of the truncation mutants (N881), which had 17 residues removed from the carboxy terminus, showed reduced binding affinity and diminished pol activity but enhanced exo activity upon incubation with a small primer-template. The exo activity of the N881 mutant, on short, single-stranded DNA was unchanged, however, compared to the wild-type enzyme. These results are consistent with inferences drawn from the crystal structure of a DNA polymerase from a related T-even phage, RB69, where the carboxy-terminal 12 residues (equivalent to the 11 residues of 43P from phage T4) protrude from the thumb domain and are free to interact with complementary surfaces of the accessory proteins. The structural integrity of the thumb region in the N881 mutant is probably perturbed and could account for its reduced binding affinity and pol activity when incubated with short, double-stranded DNA substrates.     

悬浮聚合酚醛树脂基制动材料的摩擦学性能

采用悬浮聚合法制备热固性酚醛树脂,通过红外光谱分析和热重分析对该悬浮聚合酚醛树脂(suspension-polymerization phenolic resin,缩写为SPPR)的结构和热学性能进行分析。用模压成形工艺制备树脂基制动复合材料,利用JF150D-II型摩擦磨损试验机测试该材料与HT250灰铸铁在100～350℃盘面温度范围内的滑动摩擦性能。结果表明,SPPR的热分解温度为396℃,在400℃时质量损失率为12.7%,具有较好的热稳定性。经过正交试验得到树脂基制动复合材料的优化配方,制备的复合材料和HT250灰铸铁对摩,在100～350℃升温过程中,摩擦因数平均值为0.41,波动值为0.07,350℃的摩擦因数和最大摩擦因数的比值(μ350℃/μmax)为1。和国内外优质产品相比,该复合材料的摩擦因数适中,磨损率较低,摩擦稳定性和抗热衰退性能优异。     

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.     

Template specific inhibitor of mammalian DNA polymerases

A study of the inhibition of mouse cellular DNA polymerases by poly-nucleotides and their vinyl analogs is presented. Poly(dT)-directed poly(dA) synthesis by representatives of all three classes of cellular DNA polymerase could be completely inhibited by poly(9-vinyladenine), although higher concentrations were required in the case of the gamma class enzyme. Studies on the mechanism of the inhibition using the alpha class DNA polymerase and different templates showed that the enzyme activity was inhibited in all cases where base-pairing between the vinyl polymer and the template occurred; poly(9-vinyladenine) did not interfere with the replication of templates to which it does not bind. The inhibition occurred shortly after addition of poly(9-vinyladenine) to ongoing reactions, yet the enzyme was not displaced from the template - primer complex.     

Information transfer from peptide nucleic acids to RNA by template-directed syntheses

Peptide nucleic acids (PNAs) are uncharged analogs of DNA and RNA in which the ribose-phosphate backbone is substituted by a backbone held together by amide bonds. PNAs are interesting as models of alternative genetic systems because they form potentially informational base paired helical structures. A PNA C10 oligomer has been shown to act as template for efficient formation of oligoguanylates from activated guanosine ribonucleotides. In a previous paper we used heterosequences of DNA as templates in sequence-dependent polymerization of PNA dimers. In this paper we show that information can be transferred from PNA to RNA. We describe the reactions of activated mononucleotides on heterosequences of PNA. Adenylic, cytidylic and guanylic acids were incorporated into the products opposite their complement on PNA, although less efficiently than on DNA templates.     

Dimerization of 8-OH-DPAT increases activity at serotonin 5-HT1A receptors

We have identified and partially purified two DNA polymerase activities from purified Trypanosoma brucei mitochondrial extracts. The DNA polymerase activity eluted from the single-stranded DNA agarose column at 0.15 M KCl (polymerase M1) was significantly inhibited by salt concentrations greater than 100 mM, utilized Mg2+ in preference to Mn2+ as a cofactor on deoxyribonucleotide templates with deoxyribose primers, and in the presence of Mn2+ favored a ribonucleotide template with a deoxyribose primer. A 44 kDa peptide in this fraction crossreacted with antisera against the Crithidia fasciculata beta-like mitochondrial polymerase. In activity gels the catalytic peptide migrated at an apparent molecular weight of 35 kDa. The DNA polymerase activity present in the 0.3 M KCl DNA agarose fraction (polymerase M2) exhibited optimum activity at 120-180 mM KCl, used both Mg2+ and Mn2+ as cofactors, and used deoxyribonucleotide templates primed with either deoxyribose or ribose oligomers. Activity gel assays indicate that the native catalytic peptide(s) is approximately 80 kDa in size. The two polymerases showed different sensitivities to several inhibitors: polymerase M1 shows similarities to the Crithidia fasciculata beta-like mitochondrial polymerase while polymerase M2 is a novel, salt-activated enzyme of higher molecular weight.     

Control of mutation frequency by bacteriophage T4 DNA polymerase. II. Accuracy of nucleotide selection by the L88 mutator, CB120 antimutator, and wild type phage T4 DNA polymerases

The ts CB1200 (antimutator) mutation in bacteriophage T4 DNA polymerase increases the accuracy of DNA replication since it results in a decrease in the frequency of mutations in other phage genes. The CB120 polymerases differs from the wild type enzyme in the slow rate at which it copies templates where primer extension requries displacement of polynucleotides base-paired to the template strand, even in the presence of the T4 DNA unwinding protein (gene 32-protein). The ratio of nucleotides turned over (DNA-dependent conversion of deoxynucleoside triphosphate to deoxynucleoside monophosphate) to nucleotides stably incorporated into product is 10 to 100 times higher with the mutant than wild type enzyme, depending on the DNA used as the template. This high turnover rate may increase the efficiency of removal of noncomplementary nucleotides by the antimutator enzyme and is in agreement with the findings of Muzyczka et al, (Muzyczka, N., Poland, R. L., and Bessman, M. J. (1972) J. Biol, Cehm. 247, 7116-7122) with the L141 and L42 antimutator T4 DNA polymerases. Since the 3'- to 5'-exonuclease activity of the CB120 mutant polymerase is not higher than that of the wild type enzyme, it is suggested that the high turnover rate may result from increased opportunity to remove newly incorporated nucleotides due to the slow rate at which the mutant enzyme moves to the next template nucleotide. In the accompanying paper we show that the CB120 antimutator polymerase also initially selects incorrect nucleotides for incorporation less frequently than the wild type enzyme. Thus this antimutator polymerase appears to have both greater accuracy in nucleotide selection and an enhanced ability to remove incorrect nucleotides.     

Polymerase recognition of synthetic oligodeoxyribonucleotides incorporating degenerate pyrimidine and purine bases

A universal base that is capable of substituting for any of the four natural bases in DNA would be of great utility in both mutagenesis and recombinant DNA experiments. This paper describes the properties of oligonucleotides incorporating two degenerate bases, the pyrimidine base 6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one and the purine base N6-methoxy-2,6-diaminopurine, designated P and K, respectively. An equimolar mixture of the analogues P and K (called M) acts, in primers, as a universal base. The thermal stability of oligonucleotide duplexes were only slightly reduced when natural bases were replaced by P or K. Templates containing the modified bases were copied by Taq polymerase; P behaved as thymine in 60% of copying events and as cytosine in 40%, whereas K behaved as if it were guanine (13%) or adenine (87%). The dUTPase gene of Caenorhabditis elegans, which we have found to contain three nonidentical homologous repeats, was used as a model system to test the use of these bases in primers for DNA synthesis. A pair of oligodeoxyribonucleotides, each 20 residues long and containing an equimolar mixture of P and K at six positions, primed with high specificity both T7 DNA polymerase in sequencing reactions and Taq polymerase in PCRs; no nonspecific amplification was obtained on genomic DNA of C. elegans. Use of P and K can significantly reduce the complexity of degenerate oligonucleotide mixtures, and when used together, P and K can act as a universal base.