A new approach to DNA synthesis from synthetic oligonucleotides. Synthesis of DNA coding for the repeated antigenic determinant of foot and mouth disease virus

A rapid method for assembly of DNA from synthetic oligodeoxynucleotides has been developed which involves separate ligation of top- and bottom-strand oligonucleotides followed by filling in 3'-ends of the duplex formed, blunt end cloning into a specialized vector pBBV, and recovery of the synthetic DNA from the recombinant plasmid by means of restriction nuclease BbvII. The method allows for many oligonucleotides to be ligated at once, with no intermediates being isolated, and any DNA to be recovered on cloning, no matter what the sequences of its termini are. Ten oligodeoxynucleotides (I)-(X) have been chemically synthesised and used to prepare, by this method, a 60-membered duplex with complementary tetranucleotide 5'-protrusions (DNA I) which comprises the cDNA sequence 3397-3456 of foot and mouth disease virus (FMDV) strain O1K. Self-ligation of the duplex in the head-to-tail manner yielded 120 to 900 bp long synthetic DNAs (DNA II-DNA XV) coding for oligomers of the major antigenic determinant (the amino acid sequence 141-160 of protein VP1) of FMDV. The synthetic hexamer (DNA VI) was fused to gene lacZ' on plasmid pBBV21 and expressed in E. coli. The fusion was found to complement the lacZ deletion M15, from which it follows that the fused protein associated with the alpha-deficient beta-galactosidase to yield a tetramer carrying, on its N-termini, 24 antigenic determinants of FMDV.     

Synthesis of a 33-member polynucleotide containing the "core" att site of phage lambda DNA and its cloning

Two polynucleotides containing 33 monomeric units were synthesized by a solid-phase phosphotriester method. These polynucleotides form a duplex with protruding 5'-ends, which allows to clone the duplex in EcoRI site of a cloning vehicle. Each polynucleotide was purified by electrophoresis in polyacrylamide gel, and the duplex obtained was cloned in EcoRI site of pUR 222 plasmid DNA. The structure of the cloned duplex containing the "core" att site of phage lambda was confirmed by sequencing.     

Hybrid gene synthesis: its application to the assembly of DNA sequences encoding the human parathyroid hormones and analogues

Bypassing any intermediate steps of purification and gene assembly, several synthetic oligonucleotides constituting a DNA duplex with a small base-mismatching region were phosphorylated, annealed, and ligated directly into a linearized plasmid vector. After transformation in bacteria, the two plasmid strands individually yielded two different plasmids bearing altered versions of the same gene. Via this approach, DNA coding sequences of the human parathyroid hormone and analogues were synthesized and cloned in Escherichia coli.     

Interaction of the antitumour alkaloid coralyne with duplex deoxyribonucleic acid structures: spectroscopic and viscometric studies.

The interaction of coralyne, an antitumour alkaloid with natural and synthetic duplex DNAs was investigated under conditions where the drug existed fully as a true monomer for the first time using spectrophotometric, spectrofluorimetric, circular dichroic and viscometric techniques. The absorption spectrum of coralyne monomer showed hypochromic and bathochromic effects on binding to duplex DNAs. This effect was used to determine the binding parameters of coralyne. The binding constants for four natural DNAs and four synthetic polynucleotides obtained from spectrophotometric titration, according to an excluded site model, using McGhee-von Hippel analysis, were all in the range of (0.38-9.8) x 10(5) M-1, and showed a relatively high specificity for the GC rich ML DNA and the alternating GC polynucleotide. The binding of coralyne decreased with increasing ionic strength, indicating that the binding affinity has a strong electrostatic component. Coralyne stabilized all the DNAs against thermal strand separation. The intense steady state fluorescence of coralyne was effectively quenched on binding to DNAs and the quantitative data on the Stern-Volmer quenching constant obtained was sequence dependent, being maximum with the GC rich DNA and alternating GC polymer. Circular dichriosm studies further evidenced for a strong perturbation of the B-conformation of DNAs consequent to coralyne binding with the concomitant development of extrinsic circular dichroic bands for the bound drug molecules suggesting their strong intercalated geometry in duplex DNAs. Further tests of intercalation using viscosity measurements on linear and covalently closed plasmid DNA conclusively proved the strong intercalation of coralyne in duplex DNA. Binding of the closely related natural alkaloid, berberine under these conditions showed considerably lower affinity to duplex DNAs in all experiments. Taken together, these results suggest that coralyne binds strongly to duplex DNAs by a mechanism of intercalation with specificity towards alternating GC duplex structure.     

Extent of single-stranded DNA required for efficient TraI helicase activity in vitro

The IncF plasmid protein TraI functions during bacterial conjugation as a site- and strand-specific DNA transesterase and a highly processive 5' to 3' DNA helicase. The N-terminal DNA transesterase domain of TraI localizes the protein to nic and cleaves this site within the plasmid transfer origin. In the cell the C-terminal DNA helicase domain of TraI is essential for driving the 5' to 3' unwinding of plasmid DNA from nic to provide the strand destined for transfer. In vitro, however, purified TraI protein cannot enter and unwind nicked plasmid DNA and instead requires a 5' tail of single-stranded DNA at the duplex junction. In this study we evaluate the extent of single-stranded DNA adjacent to the duplex that is required for efficient TraI-catalyzed DNA unwinding in vitro. A series of linear partial duplex DNA substrates containing a central stretch of single-stranded DNA of defined length was created and its structure verified. We found that substrates containing >or=27 nucleotides of single-stranded DNA 5' to the duplex were unwound efficiently by TraI, whereas substrates containing 20 or fewer nucleotides were not. These results imply that during conjugation localized unwinding of >20 nucleotides at nic is necessary to initiate unwinding of plasmid DNA strands.     

A general method for inserting specific DNA sequences into cloning vehicles

A general method has been developed to introduce any double-stranded DNA molecule into cloning vehicles at different restriction endonuclease sites. In this method a chemically synthesized decadeoxyribonucleotide duplex, containing a specific restriction endonuclease sequence, is joinlex DNA is cut by the same restriction endonuclease to generate the cohesive ends. It is then inserted into the restriction endonuclease cleavage site of the cloning vehicle. To demonstrate the feasibility of this new method, we have inserted separately the synthetic lac operator DNA at the Bam I and HindIII cleavage sites of the plasmid pMB9 DNA.     

Hemicatenanes form upon inhibition of DNA replication

Plasmid DNA incubated in interphase Xenopus egg extracts is normally assembled into chromatin and then into synthetic nuclei which undergo one round of regulated replication. During a study of restriction endonuclease cut plasmid replication intermediates (RIs) by the Brewer–Fangman 2D gel electrophoresis technique, we have observed the formation of a strong spike of X-shaped DNA molecules in extracts that otherwise yield very little or no RIs. Formation of these joint molecules is also efficiently induced in replication-competent extracts upon inhibition of replication fork progression by aphidicolin. Although their electrophoretic properties are quite similar to those of Holliday junctions, 2D gels of doubly cut plasmids show that these junctions can link two plasmid molecules at any pair of DNA sequences, with no regard for sequence homology at the branch points. Neutral–neutral–alkaline 3D gels show that the junctions only contain single strands of parental size and no recombinant strands. A hemicatenane, in which one strand of a duplex is wound around one strand of another duplex, is the most likely structure to account for these observations. The mechanism of formation of these novel joint DNA molecules and their biological implications are discussed.     

The DinG protein from Escherichia coli is a structure-specific helicase

The Escherichia coli DinG protein is a DNA damage-inducible member of the helicase superfamily 2. Using a panel of synthetic substrates, we have systematically investigated structural requirements for DNA unwinding by DinG. We have found that the helicase does not unwind blunt-ended DNAs or substrates with 3'-ss tails. On the other hand, the 5'-ss tails of 11-15 nucleotides are sufficient to initiate DNA duplex unwinding; bifurcated substrates further facilitate helicase activity. DinG is active on 5'-flap structures; however, it is unable to unwind 3'-flaps. Similarly to the homologous Saccharomyces cerevisiae Rad3 helicase, DinG unwinds DNA.RNA duplexes. DinG is active on synthetic D-loops and R-loops. The ability of the enzyme to unwind D-loops formed on superhelical plasmid DNA by the E. coli recombinase RecA suggests that D-loops may be natural substrates for DinG. Although the availability of 5'-ssDNA tails is a strict requirement for duplex unwinding by DinG, the unwinding of D-loops can be initiated on substrates without any ss tails. Since DinG is DNA damage-inducible and is active on D-loops and forked structures, which mimic intermediates of homologous recombination and replication, we conclude that this helicase may be involved in recombinational DNA repair and the resumption of replication after DNA damage.     

Application of synthetic oligonucleotides to the diagnosis of human genetic diseases

Under appropriate conditions synthetic oligonucleotide hybridization probes display essentially absolute hybridization specificity. That is, every nucleotide must form a Watson-Crick base pair in order that the probe forms a stable duplex. All of the non-Watson-Crick base pairs, including G-T, have a destabilizing effect. Thus, it is possible to choose stringent conditions of hybridization such that, while a perfectly matched duplex between an oligonucleotide and complementary DNA will form, duplexes mismatched at one or more position will not. Mutations in a single base in the DNA sequence of a gene can and do result in genetic diseases. The hybridization of oligonucleotides to the region of DNA containing these base changes would be affected by the mutations and thus, oligonucleotide hybridization provides a means of detecting single base changes. In an attempt to develop a non-radioactive method for the detection of human genetic diseases, we have prepared biotinylated-oligonucleotides by an enzymatic method. An oligonucleotide probe (23-mer) containing a single biotinylated deoxyuridine residue at the 3'-terminus was prepared by a primer extention reaction using E. coli DNA polymerase I (Klenow fragment). The probe could be specifically and tightly bound with Avidin D in 1 M NaCl. It could be hybridized to a plasmid DNA containing a perfectly matched complementary sequence, but not to a DNA containing 5 non-consecutive non-complementary bases. The hybridized biotinylated probe could be visualized by Avidin D and biotinylated alkaline phosphatase, even when 1.8 ng of the plasmid DNA (0.5 fmol) was used. A general approach to the enzymatic synthesis of oligonucleotides containing a single biotinylated deoxyuridine at the 3' end is described.     

Characterization of an endonuclease associated with the drug resistance plasmid pKM101.

An endonuclease was detected in strains of Salmonella typhimurium containing the drug resistance plasmid pKM101. The enzyme was not detectable in strains lacking this plasmid, but it was present in strains containing mutants of pKM101 that were no longer able to enhance host cell mutagenesis. The endonuclease had a molecular weight of roughly 75,000 and, at pH 7.0, was equally active on single-stranded and duplex deoxyribonucleic acid (DNA). The reaction with single-stranded DNA was optimal at pH 5.5, whereas with duplex DNA the optimum was pH 6.8. The enzyme required a divalent cation for activity, and it had no detectable exonuclease activity with single-stranded or duplex DNA. The endonuclease extensively degraded DNA with no apparent base specificity, forming 5'-phosphomonoester termini. Although characterization of the endonuclease has not revealed its function, the enzyme does not appear to be a restriction endonuclease.     

DNA binding and alkylation by the "left half" of azinomycin B

Azinomycin B (also known as carzinophilin A) contains two electrophilic functional groups-an epoxide and an aziridine residue-that react with nucleophilic sites in duplex DNA to form cross-links at 5'-dGNT and 5'-dGNC sequences. Although the aziridine residue of azinomycin is undoubtedly required for cross-link formation, analogues containing an intact epoxide group but no aziridine residue retain significant biological activity. Azinomycin epoxide analogues (e.g., 5 and 6) are of interest due to their potent biological activity and because there is evidence that azinomycin may decompose in vivo to yield such compounds. To investigate the chemical events underlying the toxicity of azinomycin epoxides, DNA binding and alkylation by synthetic analogues of azinomycin B (6, 8, and 9) that comprise the naphthalene-containing "left half" of the antibiotic have been investigated. The epoxide-containing analogue of azinomycin (6) efficiently alkylates guanosine residues in duplex DNA. DNA alkylation by 6 is facilitated by noncovalent binding of the compound to the double helix. The results of UV-vis absorbance, fluorescence spectroscopy, DNA winding, viscometry, and equilibrium dialysis experiments indicate that the naphthalene group of azinomycin binds to DNA via intercalation. Equilibrium dialysis experiments provide an estimated binding constant of (1.3 +/- 0.3) x 10(3) M(-)(1) for the association of a nonalkylating azinomycin analogue (9) with duplex DNA. The DNA-binding and alkylating properties of the azinomycin epoxide 6 provide a basis for understanding the cytotoxicity of azinomycin analogues which contain an epoxide residue but no aziridine group and may provide insight into the mechanisms by which azinomycin forms interstrand DNA cross-links.     

Evidence for Conservative (Two-Progeny) DNA Double-Strand Break Repair

The double-strand break repair models for homologous recombination propose that a double-strand break in a duplex DNA segment is repaired by gene conversion copying a homologous DNA segment. This is a type of conservative recombination, or two-progeny recombination, which generates two duplex DNA segments from two duplex DNA segments. Transformation with a plasmid carrying a double-strand gap and an intact homologous DNA segment resulted in products expected from such conservative (two-progeny) repair in Escherichia coli cells with active E. coli RecE pathway (recBC sbcA) or with active bacteriophage λ Red pathway. Apparently conservative double-strand break repair, however, might result from successive events of nonconservative recombination, or one-progeny recombination, which generates only one recombinant duplex DNA segment from two segments, involving multiple plasmid molecules. Contribution of such intermolecular recombination was evaluated by transformation with a mixture of two isogenic parental plasmids marked with a restriction site polymorphism. Most of the gap repair products were from intramolecular and, therefore, conservative (two-progeny) reaction under the conditions chosen. Most were conservative even in the absence of RecA protein. The double-strand gap repair reaction was not affected by inversion of the unidirectional replication origin on the plasmid. These results demonstrate the presence of the conservative (two-progeny) double-strand break repair mechanism. These experiments do not rule out the occurrence of nonconservative (one-progeny) recombination since we set up experimental conditions that should favor detection of conservative (two-progeny) recombination.     

Intramolecular compactization of circular DNA in interaction with dansyl hydrazide trivaline leading to a formation of a new type of structure--triple rings

Compactization of supercoiled circular plasmid pBR322 caused by interaction with synthetic oligopeptide dansyl hydrazide trivaline capable of beta-structure formation was studied by electron microscopy. The results show that at rising input peptide concentration circular DNA molecules undergo intramolecular structural transition with the formation of compact ring structures. The compact ring structures are formed by the fiber having the thickness of 60 A. The analysis of morphology of intermediate structures and the contour length measurements enable us to conclude that 60 A-fiber contains three lying side-by-side and interwound double-stranded DNA segments. Thus, the compact ring structures are addressed to as triple rings. The triple ring have one special point, where the triple region ends are locked by a duplex DNA segment. The mechanisms responsible for the triple ring formation may be of importance for DNA and chromatin compactization processes in vivo.     

Open strands adjacent to iterons promote the binding of the replication initiator protein (Rep) of pSC101 to the unit sequence of the iterons in vitro

The purified dimeric form of the Rep protein, a replication initiator protein of the plasmid pSC101, has a low affinity for repeated sequences, iterons, in the replication origin of the plasmid, and higher affinities for two inverted repeats in the operator region of the rep gene resulting in its functioning as an autorepressor. Studies of binding to various synthetic DNA have established that Rep can bind to duplex iteron-sequence carrying open (non-complementary) strands at one end proximal to the rep gene. Open strands at the opposite end of the iteron have no effect on Rep-binding. One open strand seems to be required in a sequence-specific fashion. A randomly sequenced duplex DNA with the open strands cannot bind to Rep but can function as a significant competitor. This suggests that Rep has some affinity for the open strands and forms a stable complex with the adjacent iteron. The mutated Rep protein, Rep1, which causes an increase in the plasmid copy number in vivo, has equally high affinity for the iteron with the open strands as wild type Rep, though it has a lower affinity for the inverted repeats than the wild type. The Rep dimer might bind to these DNA sequences with different modes.     

Inhibition of poly(ADP-ribose)polymerase binding to DNA by thymidine dimer

The ability of poly(ADP-ribose)polymerase to bind damaged DNA was assessed by electrophoretic mobility shift assay. DNA binding domain of poly(ADP-ribose)polymerase (PARPDBD) binds to synthetic deoxyribonucleotide duplex 10-mer. However, the synthetic deoxyribonucleotide duplex containing cys-syn thymidine dimer which produces the unwinding of DNA helix structure lost its affinity to PARPDBD. It was shown that the binding of PARPDBD to the synthetic deoxyribonucleotide duplex was not affected by O6-Me-dG which causes only minor distortion of DNA helix structure. This study suggests that the stabilized DNA helix structure is important for poly(ADP-ribose)polymerase binding to DNA breaks, which are known to stimulate catalytic activity of poly(ADP-ribose)polymerase.     

Development of bis-locked nucleic acid (bisLNA) oligonucleotides for efficient invasion of supercoiled duplex DNA

In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasion into duplex DNA (DSI). We thus report on the development of a clamp type of LNA ON—bisLNA—with capacity to bind and invade into supercoiled double-stranded DNA. The bisLNA links a triplex-forming, Hoogsteen-binding, targeting arm with a strand-invading Watson–Crick binding arm. Optimization was carried out by varying the number and location of LNA nucleotides and the length of the triplex-forming versus strand-invading arms. Single-strand regions in target duplex DNA were mapped using chemical probing. By combining design and increase in LNA content, it was possible to achieve a 100-fold increase in potency with 30% DSI at 450 nM using a bisLNA to plasmid ratio of only 21:1. Although this first conceptual report does not address the utility of bisLNA for the targeting of DNA in a chromosomal context, it shows bisLNA as a promising candidate for interfering also with cellular genes.     

DNA base excision repair in human malaria parasites is predominantly by a long-patch pathway

Mammalian cells repair apurinic/apyrimidinic (AP) sites in DNA by two distinct pathways: a polymerase beta (pol beta)-dependent, short- (one nucleotide) patch base excision repair (BER) pathway, which is the major route, and a PCNA-dependent, long- (several nucleotide) patch BER pathway. The ability of a cell-free lysate prepared from asexual Plasmodium falciparum malaria parasites to remove uracil and repair AP sites in a variety of DNA substrates was investigated. We found that the lysate contained uracil DNA glycosylase, AP endonuclease, DNA polymerase, flap endonuclease, and DNA ligase activities. This cell-free lysate effectively repaired a regular or synthetic AP site on a covalently closed circular (ccc) duplex plasmid molecule or a long (382 bp), linear duplex DNA fragment, or a regular or reduced AP site in short (28 bp), duplex oligonucleotides. Repair of the AP sites in the various DNA substrates involved a long-patch BER pathway. This biology is different from mammalian cells, yeast, Xenopus, and Escherichia coli, which predominantly repair AP sites by a one-nucleotide patch BER pathway. The apparent absence of a short-patch BER pathway in P. falciparum may provide opportunities to develop antimalarial chemotherapeutic strategies for selectively damaging the parasites in vivo and will allow the characterization of the long-patch BER pathway without having to knock-out or inactivate a short-patch BER pathway, which is necessary in mammalian cells.     

Nonhomologous DNA end joining of synthetic hairpin substrates in Xenopus laevis egg extracts.

Processes of DNA end joining are assumed to play a major role in the elimination of DNA double-strand breaks (DSB) in higher eucaryotic cells. Linear plasmid molecules terminated by nonhomologous restriction ends are the typical substrates used in the analysis of joining mechanisms. However, due to their limited structural variability, DSB ends generated by restriction cleavage cover probably only part of the total spectrum of naturally occurring DSB termini. We therefore devised novel DNA substrates consisting of synthetic hairpin-shaped oligonucleotides which permit the construction of blunt ends and 5'- or 3'-protruding single-strands (PSS) of arbitrary sequence and length. These substrates were tested in extracts of Xenopus laevis eggs known to efficiently join linear plasmids bearing nonhomologous restriction termini (Pfeiffer and Vielmetter, 1988). Sequences of hairpin junctions indicate that the short hairpins are joined by the same mechanisms as the plasmid substrates. However, the bimolecular DNA end joining reaction was only detectable when both hairpin partners had a minimal duplex stem length of 27bp and their PSS-tails did not exceed 10nt.     

Simple and rapid preparation of gapped plasmid DNA for incorporation of oligomers containing specific DNA lesions

The cytotoxicity, mutagenicity, and carcinogenicity of DNA base lesions are largely determined by the responses of cellular DNA repair proteins, DNA polymerases, and signaling pathways. Elucidation of these processes is thus of high biochemical interest. Such studies increasingly rely on DNA substrates containing specific lesions at defined locations. Although short synthetic DNA oligomers have frequently proved useful, circular plasmid substrates are preferable for much biochemical work, and essential for in vivo studies. However, the complexity of current approaches for preparing such substrates and limitations inherent in the procedures have posed problems. We present here a simple, highly versatile procedure for preparing gapped duplex plasmids, into which oligomers incorporating specific lesions can easily be inserted. Endonuclease N.BstNBI was used to nick twice the same strand of a pUC19-derived plasmid (pUC19HBDa), at two GAGTCNNNN sequences separated by 22 bases. Removal of the 22-nt oligomer and further purification produced a highly pure gapped plasmid. To illustrate application of this procedure, 22-nt oligonucleotides containing a single uracil residue were ligated into the gapped molecules. The pUC19HB(Da) plasmid can be modified to accept almost any DNA-lesion-containing oligomer. Using this new approach to incorporate specific DNA lesions into popular reporter genes will facilitate in vivo study of cellular responses to DNA damage.