The use of terminal blocking groups for the specific joining of oligonucleotides in RNA ligase reactions containing equimolar concentrations of acceptor and donor molecules.
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Under the conditions that RNA ligase converts the tetranucleotide, pA-A2-A, to larger polynucleotides, no such polymerization can be detected with the derivative, pA-A2-A(MeOEt), that possesses a terminal 2'-0-(alpha-methoxyethyl) group. The protection against self condensation offered by the methoxyethyl group in this system allows the specific joining of donor and acceptor oligonucleotides in reaction mixtures containing equimolar concentrations of the two species. Thus, the enzyme, together with ATP, converts equimolar quantities of A-A2-A and pA-A2-A(MeOEt) to A-A6-A(MeOEt) in 55% yield, while a similar reaction with A-A2-A and pU-U2-U(MeOEt) results in a 40% yield of A-A3-U3-U(MeOEt). The intermediate in these ligations is a disubstituted pyrophosphate composed of the donor molecule and the adenylate moiety deriving from ATP. In the case of the intermediate arising from the blocked adenosine tetranucleotide, the assigned structure, A5'pp5'A-A2-A(MeOEt), has been confirmed by chemical synthesis. The pyrophosphate derivative is able to participate in joining reactions in the absence of ATP. These observations constitute an efficient approach to the synthesis of larger polynucleotides from a specific series of oligonucleotide blocks since (i), the methoxyethyl group can be easily introduced into each oligonucleotide using the single addition reaction catalyzed by polynucleotide phosphorylase in the presence of a 2'-0-(alpha-methoxyethyl)nucleoside 5'-diphosphate, and (ii), the blocking group may be readily removed under mild conditions after each successive ligation reaction. Two other octanucleotides, I-I2-A-U3-U and U-U2-C-I3-A, have also been synthesized by this method, and these molecules correspond (with I substituting for G) to sequences appearing near the 3' terminus of the 6S RNA transcribed from phage lambda DNA. The terminal 3'-phosphate group serves equally well as a blocking group for specific ligation reactions in that the ligase converts equimolar amounts of A-A2-A and pA-A2-Ap to A-A6-Ap in 50% yield. (+info)
Construction of a double-stranded deoxyribonucleotide sequence of 45 base pairs designed to code for S-peptide 2-14 of bovine ribonuclease A.
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An artificial DNA duplex, each strand consisting of 45 monomers, is constructed from chemically synthesized deoxyribooligonucleotides. The resulting bihelical polymer may code for a modified S-peptide of Ribonuclease A. This is the first synthetic duplex designed to code for a eukaryotic message. (+info)
Genetic evidence for an additional function of phage T4 gene 32 protein: interaction with ligase.
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Gene 32 of bacteriophage T4 is essential for DNA replication, recombination, and repair. In an attempt to clarify the role of the corresponding gene product, we have looked for mutations that specifically inactivate one but not all of its functions and for compensating suppressor mutations in other genes. Here we describe a gene 32 ts mutant that does not produce progeny, but in contrast to an am mutant investigated by others, is capable of some primary and secondary DNA replication and of forming "joint" recombinational intermediates after infection of Escherichia coli B at the restrictive temperature. However, parental and progeny DNA strands are not ligated to covalently linked "recombinant" molecules, and single strands of vegetative DNA do not exceed unit length. Progeny production as well as capacity for covalent linkage in this gene 32 ts mutant are partially restored by additional rII mutations. Suppression by rII depends on functioning host ligase [EC 6.5.1.2; poly(deoxyribonucleotide):poly(deoxyribonucleotide) ligase (AMP-forming, NMN-forming)]. This gene 32 ts mutation (unlike some others) in turn suppresses the characteristic plaque morphology of rII mutants. We conclude that gene 32 protein, in addition to its role in DNA replication and in the formation of "joint" recombinational intermediates, interacts with T4 ligase [EC 6.5.1.1; poly(deoxyribonucleotide):poly(deoxyribonucleotide) ligase (AMP-forming)] when recombining DNA strands are covalently linked. The protein of the mutant that we describe here is mainly defective in this interaction, thus inactivating T4 ligase in recombination. Suppressing rII mutations facilitate substitution of host ligase. There is suggestive evidence that these interactions occur at the membrane. (+info)
Action of nicking-closing enzyme on supercoiled and nonsupercoiled closed circular DNA: formation of a Boltzmann distribution of topological isomers.
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Highly purified nicking-closing enzyme from mouse cells in 20-fold enzyme/substrate excess converts closed circular native PM2, ColE1, and Minicol DNA into limit product sets of DNAs. Each set has a mean degree of supercoiling of approximately zero. The individual species in the sets differ by deltatau = +/-1, +/-2, etc., and the relative masses fit a Boltzmann distribution. It was also demonstrated that "nonsupercoiled" closed circular duplex molecules serve as substrates for the nicking-closing enzyme, and that a distribution of topological isomers is generated. Polynucleotide ligase, acting on nicked circular DNA, forms under the same conditions, the same set of closed DNAs. The latter enzyme freezes the population into sets of molecules otherwise in configurational equilibrium in solution. (+info)
A complex ligase ribozyme evolved in vitro from a group I ribozyme domain.
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Like most proteins, complex RNA molecules often are modular objects made up of distinct structural and functional domains. The component domains of a protein can associate in alternative combinations to form molecules with different functions. These observations raise the possibility that complex RNAs also can be assembled from preexisting structural and functional domains. To test this hypothesis, an in vitro evolution procedure was used to isolate a previously undescribed class of complex ligase ribozymes, starting from a pool of 10(16) different RNA molecules that contained a constant region derived from a large structural domain that occurs within self-splicing group I ribozymes. Attached to this constant region were three hypervariable regions, totaling 85 nucleotides, that gave rise to the catalytic motif within the evolved catalysts. The ligase ribozymes catalyze formation of a 3',5'-phosphodiester linkage between adjacent template-bound oligonucleotides, one bearing a 3' hydroxyl and the other a 5' triphosphate. Ligation occurs in the context of a Watson-Crick duplex, with a catalytic rate of 0.26 min(-1) under optimal conditions. The constant region is essential for catalytic activity and appears to retain the tertiary structure of the group I ribozyme. This work demonstrates that complex RNA molecules, like their protein counterparts, can share common structural domains while exhibiting distinct catalytic functions. (+info)
A host-specific function is required for ligation of a wide variety of ribozyme-processed RNAs.
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Hepatitis delta virus (HDV) replicates its circular RNA genome via a rolling circle mechanism. During this process, cis-acting ribozymes cleave adjacent upstream sequences and thereby resolve replication intermediates to unit-length RNA. The subsequent ligation of these 5'OH and 2',3'-cyclic phosphate termini to form circular RNA is an essential step in the life cycle of the virus. Here we present evidence for the involvement of a host activity in the ligation of HDV RNA. We used both HDV and hammerhead ribozymes to generate a panel of HDV and non-HDV RNA substrates that bear 5' hydroxyl and 2', 3'- cyclic phosphate termini. We found that ligation of these substrates occurred in host cells, but not in vitro or in Escherichia coli. The host-specific ligation activity was capable of joining RNA in both bimolecular and intramolecular reactions and functioned in a sequence-independent manner. We conclude that mammalian cells contain a default pathway that efficiently circularizes ribozyme processed RNAs. This pathway could be exploited in the delivery of stable antisense and decoy RNA to the nucleus. (+info)
Evidence for an intermediate with a single-strand break in the reaction catalyzed by the DNA untwisting enzyme.
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The DNA untwisting enzyme relaxes covalently closed circylar DNAs by the sequential breaking (nicking) and closure of one strand of the duplex. The use of highly purified enzyme from rat liver nuclei at very high protein concentrations has permitted the detection of the nicked intermediate in the reaction. The nicking of closed circular simian virus 40 DNA was measured by alkaline sucrose gradient sedimentation or by equilibrium centrifugation in CsCl gradients containing propidium diiodide. The following observations support the hypothesis that the nicked DNA represents an intermediate in the untwisting reaction. The extent of nicking does not increase with time. Nicking is observed in the range of salt concentrations where the enzyme is active (0.01-0.25 M KCl), but is not observed at 0.50 Mkdl, where enzyme activity is undetectable. The nicked DNA that is generated during the reaction carried out in low salt rapidly disappears if the KCl concentration is raised to 0.50 M. At constant enzyme concentration, the number of nicks in the reaction mixture is independent of DNA concentration in the range from 3 to 14 mug/ml. The addition of an excess of unlabeled DNA to a reaction initially containing labeled nicked DNA partially chases the label from the nicked intermediate into covalently closed circular DNA. (+info)
Restriction assay for integrative recombination of bacteriophage lambda DNA in vitro: requirement for closed circular DNA substrate.
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A novel assay has been developed for in vitro genetic recombination of DNA. Substrate and product DNAs are cleaved with a restriction endonuclease and the resulting fragments are separated by electrophoresis in agarose gels. The substrate DNA has been chosen so that the recombination to be studied deletes a segment of DNA. The remaining DNA gives rise to a unique restriciton fragment, as does the DNA segment that has been removed. The method provides a convenient and physical, rather than genetic, assessment of the conversion of parental to recombinant DNA. This method has been applied to an in vitro system that carries out integrative recombination of bacteriophage lambda. We find that, different molecular forms of DNA tested, closed circular DNA is the only efficient substrate. Linear DNA and three kinds of circular DNA containing interruptions are at best very poor substrates. The implications of this surprising result are discussed. In addition, we show that the in vitro recombination system completes the breaking and rejoining steps of recombination. No stable DNA intermediates involving chiasmata or broken end structures are found. (+info)