[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.

Surface-initiated growth of poly d(A-T) by Taq DNA polymerase.

In this paper, we report surface-initiated d(A-T) polymerization by Taq DNA polymerase as a method for constructing DNA-tethered surfaces using an enzyme. The enzymatic polymerization was conducted successfully via two steps: tethering of oligo d(A-T)s onto the surface presenting carboxylic acids by amide coupling and surface-initiated polymerization using Taq DNA polymerase. In this enzymatic polymerization process, the design and construction of carboxylic acid-presenting surfaces were found to be an important factor: DNA growth did not occur on the gold surface coated only with the self-assembled monolayer (SAM) of 16-mercaptohexadecanoic acid (MHDA), but effectively proceeded on the surfaces presenting mixed SAMs of MHDA and 1-pentadecanethiol. The coupling of oligo d(A-T)s and the subsequent DNA polymerization reaction were characterized by polarized infrared external reflectance spectroscopy, ellipsometry, X-ray photoelectron spectroscopy, and atomic force microscopy.

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.

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.

Pure yeast RNA polymerase B (II) initiates transcription at specific points on supercoiled yeast DNA.

Pure yeast RNA polmymerase B (II) can selectively initiate abortive transcription on a supercoiled recombinant plasmid DNA carrying yeast DNA in the presence of low concentrations of ribonucleoside triphosphates and Mn2+. Five major products ranging between 60 and 150 nucleotides were characterized by hybridization. Three of them originate from the vector pBR322 and two from the yeast DNA insert. Based on a RNA primer extension reaction with recombinant M13 DNAs as template, a method allowing the mapping of the short abortive RNA products has been developed. An initiation site within the yeast DNA insert has thus precisely been mapped. The DNA sequence in this region was determined and showed several relevant features. The in vitro initiation site is preceded by a potential TATATATA box at -40 base pairs and at -105 by the sequence GTTAATCT similar to the consensus sequence GCTCAATCT usually found around 80 base pairs upstream from the cap site. Large blocks of alternated purine pyrimidine residues are found in this region as for several known yeast promotors. The 5' end of the RNA initiated from this site contains several potential signals for the initiation of translation. The possibility that a B to Z transition of DNA could be important for the interaction of the RNA polymerase with its template is discussed.

Deoxyuridine triphosphate pools after polyoma virus infection.

The synthesis of polyoma DNA in virus-infected 3T6 mouse fibroblasts is discontinuous with the intermediate formation of short Okazaki fragments. Hydroxyurea, an inhibitor of the enzyme ribonucleotide reductase, inhibits polyoma DNA synthesis, as measured by incorporation of radioactive thymidine. In the inhibited state, almost all incorporation occurs into short fragments. We investigated to what extent formation of short DNA fragments might be the result of incorporation of deoxyuridine triphosphate (dUTP) into DNA, followed by excision and repair reactions. We devised a sensitive enzymatic method for measuring dUTP in cell extracts which allows the determination of the dUTP pool when this pool amounts to between 0.1 and 2% of the dTTP pool. No dUTP was detected in growing mouse fibroblasts. After infection with polyoma virus cell extracts contained 0.4% dUTP (of dTTP) at the peak of DNA synthesis. Addition of hydroxyurea at this point led to a disappearance of dUTP. We conclude that dUTP incorporation can contribute only minimally to the generation of short fragments during polyoma DNA synthesis.

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.

N4-Methoxydeoxycytidine triphosphate is in the imino tautomeric form and substitutes for deoxythymidine triphosphate in primed poly d[A-T] synthesis with E. coli DNA polymerase I.

N4- Methoxydeoxycytidine triphosphate ( mo4dCTP ) substitutes for dTTP in poly d[A-T] synthesis with E. coli DNA polymerase I (Pol I). In parallel experiments using as template-primer, poly d[G-C], no incorporation of [14C] mo4dC was detected. This indicates that this deoxy derivative acts as the imino tautomer, as previously found for the riboderivative . Nearest neighbor analysis of transcripts of poly d[A-T] containing mo4dC shows that the derivative substitutes for only one base. In replication, singlestranded mo4dC -containing polymers gave little misincorporation, including that of dATP which can hydrogen-bond to mo4dC in the imino form, if the methoxy group is anti to the N-3. It is therefore assumed that the methoxy group is constrained anti in a polymer such as d[A-T], but can be in the syn form in singlestranded polymers and not recognized by DNA polymerase. mo4dC destabilizes the poly d[A-T] helix, as indicated by a lowered and less cooperative melting. Steric factors such as adjacent base displacement were invoked for similar findings with the doublestranded r( U61 , mo4C39 ) X r(A).

Primer/template-independent synthesis of poly d(A-T) by Taq polymerase.

Taq DNA polymerase polymerized dATP and dTTP to poly d(A-T) without requiring added primer/template in the temperature range of 60-70 degrees C. Tth DNA polymerase also catalyzed the reaction, while delta Tth, Vent, Vent(exo-), Pfu, Ultma, BcaBEST, and KOD DNA polymerases did not. The reaction was distinct from the template-nonrequiring terminal deoxynucleotidyl transferase reaction which absolutely required primers.

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.

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.

A DNA glycosylase from Escherichia coli that releases free urea from a polydeoxyribonucleotide containing fragments of base residues.

A poly (dA, [2-14C]dT) copolymer has been synthesized using terminal deoxynucleotidyltransferase. Treatment of the polydeoxyribonucleotide with potassium permanganate converts the thymine residues to urea and N-substituted urea derivatives, while the adenine residues are resistant to oxidation. This damaged polymer has been annealed with an equimolar amount of poly (dT) to generate a double-stranded polydeoxyribonucleotide containing scattered fragmented base residues, which are radioactively labeled selectively. On incubation of the latter with crude cell extracts from E. coli, free urea is released by a DNA glycosylase activity. The enzyme has been partly purified, and appears to be different from previously studied DNA glycosylase. It shows a strong preference for a double-stranded substrate, exhibits no cofactor requirement, and has a molecular weight of 20000 - 25000. Since fragmentation of pyrimidine residues is a major type of base lesion introduced in DNA by exposure to ionizing radiation, it seems likely this DNA glycosylase is active in repair of X-ray-induced lesions.

Distamycin paradoxically stimulates the copying of oligo(dA).poly(dT) by DNA polymerases.

Distamycin A, a polypeptide antibiotic, binds to dA.dT-rich regions in the minor groove of B-DNA. By virtue of its nonintercalating binding, distamycin acts as a potent inhibitor of the synthesis of DNA both in vivo and in vitro. Here we report that distamycin paradoxically stimulates Escherichia coli DNA polymerase I (pol I), its large (Klenow) fragment, and bacteriophage T4 DNA polymerase to copy oligo(dA).poly(dT) in vitro. It is found that distamycin increases the maximum velocity (Vmax) of the extension of the oligo(dA) primer by pol I without affecting the Michaelis constant (Km) of the primer. Gel electrophoresis of the extended primer indicates that the antibiotic specifically increases the rate of addition of the first three dAMP residues. Lastly, in the presence of both distamycin and the oligo(dT)-binding protein factor D, which increases the processivity of pol I, a synergistic stimulation of polymerization is attained. Taken together, these results suggest that distamycin stimulates synthesis by increasing the rate of initiation of oligo(dA) extension. The stimulatory effect of distamycin is inversely related to the stability of the primer-template complex. Thus, maximum stimulation is exerted at elevated temperatures and with shorter oligo(dA) primers. That distamycin increases the thermal stability of [32P](dA)9.poly(dT) is directly demonstrated by electrophoretic separation of the hybrid from dissociated [32P](dA)9 primer. It is proposed that by binding to the short primer-template duplex, distamycin stabilizes the oligo(dA).poly(dT) complex and, therefore, increases the rate of productive initiations of synthesis at the primer terminus.