In vitro synthesis of uniform poly(dG)-poly(dC) by Klenow exo- fragment of polymerase I.

In this paper, we describe a production procedure of the one-to-one double helical complex of poly(dG)-poly(dC), characterized by a well-defined length (up to 10 kb) and narrow size distribution of molecules. Direct evidence of strands slippage during poly(dG)-poly(dC) synthesis by Klenow exo(-) fragment of polymerase I is obtained by fluorescence resonance energy transfer (FRET). We show that the polymer extension results in an increase in the separation distance between fluorescent dyes attached to 5' ends of the strands in time and, as a result, losing communication between the dyes via FRET. Analysis of the products of the early steps of the synthesis by high-performance liquid chromatography and mass spectroscopy suggest that only one nucleotide is added to each of the strand composing poly(dG)-poly(dC) in the elementary step of the polymer extension. We show that proper pairing of a base at the 3' end of the primer strand with a base in sequence of the template strand is required for initiation of the synthesis. If the 3' end nucleotide in either poly(dG) or poly(dC) strand is substituted for A, the polymer does not grow. Introduction of the T-nucleotide into the complementary strand to permit pairing with A-nucleotide results in the restoration of the synthesis. The data reported here correspond with a slippage model of replication, which includes the formation of loops on the 3' ends of both strands composing poly(dG)-poly(dC) and their migration over long-molecular distances (microm) to 5' ends of the strands.

DNA polymerase from mesophilic and thermophilic bacteria. III. Lack of fidelity in the replication of synthetic polydeoxyribonucleotides by DNA polymerase from Bacillus licheniformis and Bacillus stearothermophilus.

1. DNA polymerase from the mesophile Bacillus licheniformis and the thermophile Bacillus stearothermophilus has been used to study the replication of poly(dA-dT)-poly(dA-dT) and poly(dC)-poly(dG) templates at 37, 45, and 55 degrees C. 2. Incorporation of non-complementary deoxyribonucleoside triphosphates (misincorporation) occurred with both enzymes and both templates. Non-specific incorporation (de novo polynucleotide synthesis, random attachment to existing strands, and tritium exchange of nucleotides) accounted for, at most, a small fraction of the total observed misincorporation. The error rates at 37 degrees C for the complete system were as follows:: B. licheniformis: dATP, 1/61; dCTP, 1/830; dGTP, 1/360; dTTP, 1/65; B. stearothermophilus: dATP, 1/68; dCTP, 1/1430; dGTP, 1/440; dTTP, 1/67. For both organisms, the error rate for dCTP and dGTP was independent of incubation temperature; the error rate for dATP and dTTP was 5-50-fold greater than that for dCTP or dGTP and increased significantly from 37 to 55 degrees C. 3. The ratio of dATP to dTTP incorporation with the poly(dA-dT)-poly-(dA-dT) template was independent of temperature and close to unity. The ratio of dCTP to dGTP incorporation with the poly(dC)-poly(dG) template decreased from approx. 0.2 to 0.05 for the mesophile and from approx. 0.06 to 0.03 for the thermophile as the temperature increased from 37 to 55 degrees C.

Template-directed synthesis on the oligonucleotide d(C7-G-C7).

When the deoxynucleotide template d(C7-G-C7) is incubated with the activated nucleotides 2-MeImpG and 2-MeImpC, a series of oligomers of G up to the sevenmer and a series of copolymers of composition GnC with n = 3 to 13 are formed. Oligomers GnC with n greater than 7 are completely degraded by pancreatic ribonuclease, establishing that they contain a 3' to 5' internucleotide bond between 5'-C and 3'-G within a sequence of the form (pG)ipC(pG)j. As expected, (pG)7-Cp and (pG)6-Cp are major hydrolysis products. Detailed analysis of the product distribution shows that a substantial fraction of the oligomeric products are of the type (pG)ipC(pG)j with i less than 7. This shows that product synthesis does not necessarily begin at the 3' terminus of the template. The significance of this finding in terms of the origin of molecular replication is discussed.

Difference in the mechanisms of poly(dT) synthesis by DNA polymerases beta and gamma.

Poly(dT) products which were synthesized depending on (rA)n . (dT)12-18 as a template . primer by mammalian DNA polymerases beta and gamma were analyzed by alkaline sucrose gradient centrifugation. The size of the population of poly(dT) chains synthesized by DNA polymerase beta increased slowly and consistently during incubation up to at least 30 min. On the other hand, the product size with DNA polymerase gamma reached the final size (7 s) within 5 min and the number of products increased during further incubation. Comparison of product number per enzyme molecule suggests that DNA polymerase beta acts on multiple primers in a distributive fashion while DNA polymerase gamma completes poly(dT) chains of large size in a one-by-one fashion.

[Unprimed synthesis of poly (d(A-T)), catalyzed by a preparation of Escherichia coli DNA polymerase I]. / Issledovanie prirody bezmatrichnogo sinteza d(A-T)-sopolimera, katalizirchemogo preparatami DNK-polimerazy I Escherichia coli.

The initial events of the de novo synthesis of poly[d(A-T)], catalyzed by preparations of E. coli DNA-polymerase I, were investigated. The data provide evidence that deoxynucleoside diphosphate: oligonucleotide deoxynucleotidyl transferase (dNDP-transferase), the enzyme which is able to catalyze unprimed polymerization of dNDP, participates in the process of initiation. This conclusion is based on the following data: 1) preincubation of E. coli DNA-polymerase I preparation with dADP and dTDT abolishes a lag-period in the poly[d(A-T)] synthesis; 2) dithiothreitol and N-ethylmaleinide, inhibitors of dNDP-transferase, inhibit de novo synthesis of [d(A-T)]-copolymer by preparations of E. coli DNA-polymerase I but do not effect primed synthesis ensured by this enzyme. High concentration of the substrate have similar effect. Using two-dimentional thin-layer chromatography and microcolumn chromatography on TEAE-cellulose we have shown that preliminary incubation of DNA-polymerase I preparations with dADP and dTDP results in the synthesis of short oligonucleotides (from di- to decanucleotides). Hydrolysis of these oligonucleotides with dilute sulfuric acid demonstrates that among the reaction products prevail oligoadenylates and oligothymidylates, but an appreciable amounts of heterooligomers including oligo[d(A-T)] were revealed as well. The model of so called de novo synthesis of regular polynucleotides is proposed, according to which dNDP-transferase, an accompanying enzyme in the preparations of DNA-polymerase I E. coli, is carrying out the synthesis of short oligonucleotides which form template-primer complexes repeatedly replicated by the DNA-polymerase I E. coli.

[Display of 8-hydroxy-GTP substrate properties of UTP in the reaction of polynucleotide synthesis catalyzed by RNA-polymerase from Escherichia coli in the presence of poly[d(AT).d(AT)] template]. / Proiavlenie 8-oksi-GTP substratnykh svoistv UTP v reaktsii polinukleotidnogo sinteza RNK-polimerazoi Escherichia coli na matritse poli[d(AT).d(AT)].

8-oxy-GTP was obtained via reaction of GTP with ascorbic acid and addition of hydrogen peroxide. 8-oxy-GTP is recognized and displays substrate properties of UTP on substitution of 8-oxy-GTP for UTP in polynucleotide synthesis catalyzed by E. coli RNA polymerase on a poly[d(A-T)].poly[d(A-T)] template. Such incorporation does not take place at equimolar quantities of GTP and 8-Br-GTP. The incorporation of 8-oxy-GTP instead of UTP, is 2.5-3 times higher upon replacement of Mg2+ by Mn2+ ions. The dinucleotide ApU serving as an initiator rises the incorporation level of 8-oxy-GTP both for Mg2+ and Mn2+ ions. 8-oxy-GTP slightly inhibits poly[r(A-U)] synthesis, but UTP strongly inhibits the incorporation of 8-oxy-GTP. [alpha-32P] 8-oxy-GTP is incorporated mainly instead of UTP, but it can be incorporated also during the substitution of 8-oxy-GTP for ATP.

[An improved chemico-enzymatic method of synthesizing long DNA segments]. / Usovershenstvovannyi khimiko-fermentativnyi metod sinteza protiazhennykh fragmentov DNK.

A simple and economy method of the biochemical assembling of long double-stranded DNA segments is described. A single-stranded polydeoxynucleotide 122 bases long representing a fragment of synthetic gene of human beta-interferon was assembled from three synthetic fragments 36 (two) and 50 bases long on four complementary 12-mers as templates. This single-stranded polynucleotide was converted, in the presence of DNA polymerase 1 and a 12-meric primer, in to the full-length double-stranded DNA (the beta-interferon gene segment). It was cloned into an E. coli plasmid vector pBR322 and its sequence confirmed.

Functionalization of DNA G-Wires for patterning and nanofabrication.

DNA structures made of guanine tetrads present remarkable properties and are thus first choice candidates for applications in nanofabrication. Starting from the work of Kotlyar et al., we report here that the klenow exo(-) fragment of DNA polymerase I can extend poly(dG)-poly(dC) from various 5'-modified (dG)(10(-))(dC)(10) templates. This allows the production of end-functionalized four-stranded wires (G-Wires) assembled from the folding of poly(dG) strands. G-Wires bearing thiol moieties can be easily combed on Au and Pt surfaces, whereas a 5' single-stranded overhang of a random sequence provides the unique possibility to assemble complex structures for nanoconstruction purposes.

De novo synthesis of a polymer of deoxyadenylate and deoxythymidylate by calf thymus DNA polymerase alpha.

In a reaction mixture containing calf thymus DNA polymerase alpha (DNA nucleotidyltransferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase; EC 2.7.7.7), calf thymus DNA unwinding protein, DNA, deoxyadenosine 5'-triphosphate and deoxythymidine 5'-triphosphate, a copolymer of deoxyadenylate and deoxythymidylate is synthesized after a lag period of 1-2 hr. In the presence of the four deoxyribonucleoside triphosphates only deoxyadenylate and deoxythymidylate are incorporated into the polymer and the rate of synthesis is decreased. The reaction variably occurs in the absence of DNA or DNA unwinding protein but with a greatly entended lag period. The optimal Mg2+ concentration for synthesis of the polymer of deoxyadenylate and deoxythymidylate is 1 mM, in contrast to an optimal Mg2+ concentration of 8 mM for DNA synthesis with activated DNA as template. Characterization of the product of de novo synthesis indicates that it is the alternating copolymer, poly(dA-dT).

Polynucleotides. L. Synthesis and properties of poly (2'-chloro-2'-deoxyadenylic acid) and poly (2'-bromo-2'-deoxyadenylic acid).

Poly (2'-chloro-2'-deoxyadenylic acid) and poly (2'-bromo-2'-deoxyadenylic acid) were synthesized from the corresponding diphosphates with the aid of polynucleotide phosphorylase from E. coli. UV, CD, acid titration and mixing with poly (U) were investigated. Comparing these properties with those of poly (A) and poly (2'-azido-2'-deoxyadenylic acid), it was found that 2''substituents exert significant effects on the thermal stability of these polynucleotides, though the overall conformational structure was not greatly changed.

Modified polynucleotides. I. Investigation of the enzymatic polymerization of 5-alkyl-dUTP-s.

The chemical synthesis of 5-alkyl-dUTP-s and their participation as substrates in poly[d(A-6)] primed polymerization reactions with dATP by E. coli DNA polymerase I enzyme has been described. In comparison with dTTP, at saturating substrate concentrations, the rate of hypochromic effect was found to be 17.3% higher for dUTP and was lower by 27.4% for 5-ethyl-dUTP, 29.5% for 5-n-propyl-dUTP, 31.4% for 5-n-butyl-dUTP and by 85.0% for 5-n-pentyl-dUTP. No hypochromic effect could be observed, however, with 5-iso-propyl-, 5-tert.butyl- and 5-n-hexyl-dUTP-s. Polydeoxynucleotides have also been isolated from the reaction mixture and some of their structural properties determined.

Polynucleotides. XLV Synthesis and properties of poly(2'-azido-2'-deoxyinosinic acid).

Poly (2'-azido-2'-deoxyinosinic acid), [poly (Iz)], was synthesized from 2'-azido-2'-deoxyinosine diphosphate by the action of polynucleotide phosphorylase. Poly (Iz) has UV absorption properties similar to poly (I) and hypochromicity of 11% at 0.15M Na+ and neutrality. In solutions of high Na+ ion concentration, poly (Iz) forms a multi-stranded complex and its Tm at 1.0M Na+ ion concentration was 43 degrees. Upon mixing with poly (C), poly (Iz) forms a 1:1 complex having a Tm lower than that of poly (I)-poly (C) complex in the same conditions. The effect of substitution at the 2'-position of the poly (I) strand was discussed in relation to the interferon-inducing activity.

O4-Methyl, -ethyl, or -isopropyl substituents on thymidine in poly(dA-dT) all lead to transitions upon replication.

In a previous paper, we reported that O4-methyl dTTP can be incorporated into poly(dA-dT) in place of thymidine without distortion of the helical structure, but on replication it could behave as deoxycytidine and misincorporate dGTP. Only weak interactions are possible for any O4-modified T X A pair. While O4-alkyl T X G pairing should be favored, experiments to detect the ability of Escherichia coli DNA polymerase I (pol I) to utilize the triphosphate as dCTP were ambiguous. dTTPs with larger alkyl groups (ethyl, isopropyl) have now been synthesized and tested for their recognition as dTTP by pol I. Enhanced steric hindrance could be expected, particularly for O4-isopropyl dTTP, which has a three-carbon branched chain. However, both compounds behaved qualitatively like O4-methyl dTTP, being incorporated into poly(dA-dT) and then directing deoxyguanosine misincorporation by pol I. Quantitative comparisons of mutagenicity were not possible because of the finding that, unlike polymers made with O4-methyl dTTP, those made with ethyl or isopropyl dTTP were resistant to hydrolysis by using a variety of nucleases. The frequent misincorporations of dGTP would be expected to produce transitions in vivo. O4-ethyldeoxythymidine is very poorly repaired in vivo, which would also be expected for repair of O4-isopropyldeoxythymidine. Therefore, under suitable conditions, these particular carcinogen products are likely to be initiators of carcinogenesis.

Base dynamics of nitroxide-labeled thymidine analogues incorporated into (dA-dT)n by DNA polymerase I from Escherichia coli.

Nitroxide-labeled thymidine substrates (dL) for Escherichia coli DNA polymerase I (pol I) were used to synthesize spin-labeled alternating double-stranded copolymers with (dA-dT)n as a template. All dL substrates use an alkane or alkene tether substituted into the 5-position of the pyrimidine ring to link a five- or six-membered ring nitroxide to the pyrimidine base. The kinetics of dL incorporation show some tether dependence with respect to tether length and tether geometry. The electron spin resonance (ESR) spectra of (dA-dT,dL)n duplexes directly formed by polymerization with pol I are compared with the ESR spectra of (dA)n(dT,dL)n duplexes, which are obtained after annealing of nitroxide-labeled single strands with complementary unlabeled single strands. The ESR spectra indicate that nitroxide-labeled analogues with tethers short enough to let the nitroxide ring reside in the major groove are excellent reporter groups for monitoring hybridization. A small difference between the ESR line shapes of the alternating duplexes (dA-dT,dL)n and the homopolymer duplexes (dA)n(dT,dL)n containing the same dL is detectable, suggesting the presence of subtle differences in the base dynamics between both systems. Computer simulation of the ESR spectra of the (dA-dT,dL)n duplexes was successful with the same motional model reported earlier [Kao, S.-C., & Bobst, A.M. (1985) Biochemistry 24, 5465-5469]. The thymidine motion arising from tilting and torsion of base pairs and base twisting in (dA-dT)n is similar to that in (dA)n(dT)n and is of the order of 4 ns.

Primed and unprimed synthesis of poly (dA-dT) by calf thymus DNA polymerase alpha.

The primed and unprimed synthesis of poly(dA-dA-dT) by calf thymus DNA polymerase alpha (DNA nucleotidyltransferase; deoxynucleoside triphosphate: DNA deoxynucleotidyltransferase EC 2.7.7.7) has been compared to replication of activated DNA. Synthesis of poly(dA-dT) by alpha-polymerase is both autocatalytic and exponential. The rate of synthesis of poly(dA-dT) is markedly affected by the Mg2+ concentration and has a higher temperature optimum than replication of activated DNA, implicating "slippage" as a necessary part of poly(dA-dT) replication. Calf thymus 24,000-dalton unwinding protein influences poly(dA-dT) synthesis by increasing both the exponential rate constant and the rate of linear synthesis. Single-stranded template poly(dA-dT) is provided alpha-polymerase by both "strand slippage" and melting by unwinding protein.

A study on the unprimed poly (dA-dT) synthesis catalyzed by preparations of E. coli DNA polymerase I.

Evidence was obtained indicating that the initiation of poly (dA-dT) de novo synthesis is provided by deoxynucleoside diphosphate: oligonucleotide deoxynucleotidyl transferase (dNDP-transferase present in preparations of E. coli DNA polymerase I and capable of catalyzing the unprimed polymerization of dNDP. dNDP-transferase synthesyzes short oligonucleotides which form template-primer complexes repeatedly replicated by DNA polymerase I. This conclusion was based on the following observations: the abolition of the lag period of poly (dA-dT) synthesis by preincubation of DNA-polymerase I preparations with dADP and dTDP; the presence of oligo (dA-dT) among the preincubation products; the suppressive effect of dithiothreitol and N-ethylmaleimide (inhibitors of dNDP-transferase) on the de novo, but not on the primed synthesis of poly (dA-dT), catalyzed by preparations of DNA-polymerase I.

Poly-2'-deoxy-2'-fluoro-cytidylic acid: enzymatic synthesis, spectroscopic characterization and interaction with poly-inosinic acid.

The polymerization of 2'deoxy-2'-fluoro-cytidine-diphosphate (dCflDP) by polynucleotide phosphorylase is barely detectable in the presence of Mg++ under usual experimental conditions for polymerization of nucleoside diphosphates. High concentrations of enzyme have to be used to accomplish the synthesis. Mn++ is a better activator than Mg++ for the reaction. cCflDP inhibits the polymerization of CDP and has a Km=8.8X10-3M, six times higher than CDP.- The polymer, poly (dCfl), ressembles in many respects poly(C), but not poly(dC): the acid selfstructure forms at similar pK's; interaction with poly(I) yields a 1:1 complex the CD spectrum of which is similar to that of poly(I).poly(C). Finally, the Tm's of poly(I).poly(dCfl) are comparable to those of poly(I).poly(C).

A method for the specific inhibition of poly[d(A-T)] synthesis using the A-T specific quinoxaline antibiotic TANDEM.

A serious problem in the replication of repeating-sequence DNa polymers using Escherichia coli DNA polymerase I arises from the fact that this polymerase has a very strong preference for the replication of poly[d(A-T)]. Thus reactions primed with DNA containing small amounts of contaminating poly[d(A-T)] will eventually result in complete domination of the synthesis by poly[d(A-T)]. This problem can be overcome by the addition to the reaction mixture of the synthetic quinoxaline antibiotic TANDEM which binds specifically to poly[d(A-T)] completely inhibiting its replication. Using thermal denaturation experiments it can be shown that TANDEM does not bind to most other synthetic DNA polymers (e.g., poly(dA) . poly(dT) and poly[d(A-T-C)] . poly[d(G-A-T)] and therefore their replication is not inhibited. The only exception we have encountered is poly[d(T-A-C)] . poly[d(G-T-A)] which does bind TANDEM and therefore the drug cannot be used during the synthesis of this polymer. The fact that poly[d(T-A-C)] . poly[d(G-T-A)] does bind TANDEM while poly[d(A-T-C)] . poly[d(G-A-T)] does not, suggests that the drug recognizes T-A rather than A-T sequences.

Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 12. Synthesis of a DNA duplex corresponding to a sequence of 23 nucleotide units adjoining the C-C-A end.

In continuing the work on the total synthesis of the gene for an Escherichia coli tyrosine suppressor tRNA (accompanying papers) and as a part of a study of the mechanism of transcription of this gene, a 23-nucleotide unit-long DNA corresponding to the previously determined (Loewen, P., Sekiya, T., and Khorana, H. G. (1974) J. Biol. Chem. 249, 217) sequence has been synthesized. The synthesis was carried out by dividing the total duplex into the following five deoxyribooligonucleotide segments, all of which were chemically synthesized: (a) the undecanucleotide, d(A-G-T-G-A-T-G-G-T-G-G); (b)the undecanucleotide, d(T-C-A-C-T-T-T-C-A-A-A); (c) the undecanucleotide, d(G-G-A-C-T-T-T-T-G-A-A); (d) the dodecanucleotide, d(A-G-T-C-C-C-T-G-A-A-C-T); and (e) the heptanucleotide, d(A-G-T-T-C-A-G). All the five synthetic oligonucleotides were characterized by chromatographic and radioactive fingerprinting methods after labeling the 5'-ends with a 32P-phosphate group. Synthesis of the double-stranded DNA duplex was completed by joining 5'-phosphorylated segments 1, 3, and 4 in the presence of segments 2 and 5 using T4-polynucleotide ligase. The DNA duplex was characterized.

Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 7. Enzymatic joining of the chemically synthesized segments to form a DNA duplex corresponding to the nucleotide sequence 1-26.

Duplex [I], which represents the nucleotide sequence 1-26 of the double-stranded DNA corresponding to the precursor for a tyrosine suppressor tRNA, has been synthesized by the enzymatic joining of five chemically synthesized deoxyribooligonucleotide segments. The synthesis was accomplished in two different ways. In a one-step synthesis, all of the five segments were used together: segments 2, 3, and 5 carried 5'-33P-labeled phosphate groups while segment 4 carried a 32P-phosphate group. An alternative, two-step method involved the joining of 5'-32P-phosphorylated segment 2 to segment 4 (carrying 5'-OH group or 5'-32P- or 33P-labeled phosphate group) in the presence of segment 3 followed by the joining of [5-32P]segment 5 in a second step. The duplex [I]' (segments 2 to 5) thus obtained was phosphorlated at the 5'-ends with polynucleotide kinase and then joined to segment 1 to give duplex [I] quantitatively. The preparative methods described have the desired flexibility for performing the subsequent operations necessary for the total synthesis of the structural gene for the tyrosine suppressor tRNA precursor.