Hairpin Duplex Research Articles

Affinity gel electrophoresis of nucleic acids: Nucleobase-selective separation of DNA and RNA on agarose-poly(9-vinyladenine) conjugated gel

Poly(9-vinyladenine) (PVAd) was immobilized within an agarose gel matrix to produce a novel affinity gel for the base-specific separation of nucleic acids by electrophoresis. The shape (single- or double-stranded) and base content of nucleic acids were specifically recognized by the affinity gel. Only single stranded DNA, of which the sequence is not regular enough to form a stable duplex hairpin structure, was selectively adsorbed over double-stranded DNA. Among five polynucleotides having different bases such as poly(A), poly(G), poly(C), poly(U) and poly(I), poly(U) and poly(I) were base-specifically adsorbed by PVAd, probably by hydrogen bond formation. The effect of the molecular mass and size of poly(9-vinyladenine) was also examined.

Homologous Recognition and Triplex Formation Promoted by RecA Protein between Duplex Oligonucleotides and Single-stranded DNA

RecA protein formed a stable triplex from a 33 bp duplex oligonucleotide and a circular plus strand of M13 DNA when a hairpin connection at the proximal end of the homologous duplex oligonucleotide blocked displacement of the 5′ end of its own plus strand. An oligonucleotide with a hairpin connection at the other end yielded five times fewer joints that survived deproteinization, and an ordinary duplex oligonucleotide yielded none. The stability of the three-stranded structure was not attributable to exonucleolytic nibbling of the 3′ end of the hairpin oligonucleotide, which could generate a region of stable duplex DNA. In the triplexes, the hairpin duplex became more accessible to copper phenanthroline, exhibited novel sites of cleavage by DNase I, and resisted digestion by Escherichia coli exonuclease I. The enzymatic methylation of only two residues at N-6 adenine and two at N-4 cytosine in the hairpin duplex prior to the pairing reaction lowered the tm of triplexes by 8 deg.C, whereas extensive methylation at N-7 guanine by dimethyl sulfate had no effect. These results are discussed in relation to possible models of triplex DNA.

DNA-induced dimerization of the Escherichia coli Rep helicase

The Escherichia coli Rep protein is a DNA helicase that is involved in DNA replication. We have examined the effects of DNA binding on the assembly state of the Rep protein using small-zone gel permeation chromatography and chemical crosslinking of the protein. Complexes of Rep protein were formed with short single-stranded and duplex hairpin oligodeoxynucleotides with lengths such that only a single Rep monomer could bind per oligodeoxynucleotide (i.e. 2 Rep monomers could not bind contiguously on the oligodeoxynucleotides). In the absence of DNA, Rep protein is monomeric (Mr 72,800) up to concentrations of at least 8 μm (monomer), even in the presence of its nucleotide cofactors (ATP, ADP, ATP-γ-S). However, the binding of Rep monomers to single-stranded (ss) oligodeoxynucleotides, d(pN)n (12 ≤ n ≤ 20), induces the Rep monomers to oligomerize. Upon treatment of the Rep-ss oligodeoxynucleotide complexes with the protein crosslinking reagent dimethyl-suberimidate (DMS) and subsequent removal of the DNA, crosslinked Rep dimers are observed, independent of oligodeoxynucleotide length (n ≤ 20). Furthermore, short duplex oligodeoxynucleotides also induce the Rep monomers to dimerize. Formation of the Rep dimers results from an actual DNA-induced dimerization, rather than the adventitious crosslinking of Rep monomers bound contiguously to a single oligodeoxynucleotide. The purified DMS-crosslinked Rep dimer shows increased affinity for DNA and retains DNA-dependent ATPase and DNA helicase activities, as shown by its ability to unwind M13 RF DNA in the presence of the bacteriophage f1 gene II protein. On the basis of these observations and since the dimer is the major species when Rep is bound to DNA, we suggest that a DNA-induced Rep dimer is the functionally active form of the Rep helicase.

DNA-induced dimerization of the Escherichia coli Rep helicase

The Escherichia coli Rep protein is a DNA helicase that is involved in DNA replication. We have examined the effects of DNA binding on the assembly state of the Rep protein using small-zone gel permeation chromatography and chemical crosslinking of the protein. Complexes of Rep protein were formed with short single-stranded and duplex hairpin oligodeoxynucleotides with lengths such that only a single Rep monomer could bind per oligodeoxynucleotide (i.e. 2 Rep monomers could not bind contiguously on the oligodeoxynucleotides). In the absence of DNA, Rep protein is monomeric ( M r 72,800) up to concentrations of at least 8 μ m (monomer), even in the presence of its nucleotide cofactors (ATP, ADP, ATP-γ-S). However, the binding of Rep monomers to single-stranded (ss) oligodeoxynucleotides, d(pN) n (12 ≤ n ≤ 20), induces the Rep monomers to oligomerize. Upon treatment of the Rep-ss oligodeoxynucleotide complexes with the protein crosslinking reagent dimethyl-suberimidate (DMS) and subsequent removal of the DNA, crosslinked Rep dimers are observed, independent of oligodeoxynucleotide length ( n ≤ 20). Furthermore, short duplex oligodeoxynucleotides also induce the Rep monomers to dimerize. Formation of the Rep dimers results from an actual DNA-induced dimerization, rather than the adventitious crosslinking of Rep monomers bound contiguously to a single oligodeoxynucleotide. The purified DMS-crosslinked Rep dimer shows increased affinity for DNA and retains DNA-dependent ATPase and DNA helicase activities, as shown by its ability to unwind M13 RF DNA in the presence of the bacteriophage f1 gene II protein. On the basis of these observations and since the dimer is the major species when Rep is bound to DNA, we suggest that a DNA-induced Rep dimer is the functionally active form of the Rep helicase.

Intramolecular triplex formation of the purine-purine-pyrimidine type

Six octadecamers with hairpin motifs have been synthesized and investigated for possible intramolecular triplex formation. Electrophoretic, hypochromic, and CD evidence suggest that d(CCCCTTTGGGGTTTGGGG) and d(GGGGTTTGGGGTTTCCCC) can form G.G.C intramolecular triplexes via double hairpin formation in neutral solutions, presumably with the terminal G tract folding back along the groove of the hairpin duplex. In contrast, d(GGGGTTTCCCCTTTGGGG) and the three corresponding 18-mers containing one G and two C tracts each forms a single hairpin duplex with a dangling single strand. The design of the sequences has led to the conclusion that the two G tracts are antiparallel to each other in such a triplex. Magnesium chloride titrations indicate that Mg2+ is not essential for such an intramolecular triplex formation. The main advantage of our constructs when compared to the intermolecular triplex formation is that the shorter triplex stem can be formed in a much lower DNA concentration. The merit of G.G.C triplex, in contrast to that of C+.G.C, lies in the fact that acidic condition is not required in its formation and will, thus, greatly expand our repertoire in the triplex strategy for the recognition and cleavage of duplex DNA. Spectral binding studies with actinomycin D (ACTD) and chromomycin A3 (CHR) as well as fluorescence lifetime measurements with ethidium bromide (EB) suggest that although hairpin duplexes bind these drugs quite well, the intramolecular triplexes bind poorly. Interestingly, the binding densities for the strong-binding hairpins obtained from Scatchard plots are about one ACTD molecule per oligomeric strand, whereas more than two drug molecules are found in the case of CHR, in agreement with the recent NMR studies indicating that CHR binds to DNA in the form of a dimer.

Helix-coil transition of parallel-stranded DNA. Thermodynamics of hairpin and linear duplex oligonucleotides.

The stabilities have been determined of different DNA double helices constructed with the two constituent strands in a parallel orientation. These molecules incorporate polarity-inverting loop structures (hairpins) or nucleotide sequences (duplexes) which impose the desired polarity on the two strands constituting the sugar-phosphate backbone. The hairpins consisted of d(A.T)n stems (n = 8 or 10) and either a 5'-p-5' linkage in a d(C)4 loop (ps-C8 and ps-C10) or a 3'-p-3' linkage in a d(G)4 loop (ps-G10). The linear duplexes had 21-nt (ps-C2.C3) and 25-nt (ps-D1.D2, ps-D3.D4) mixed A,T sequences and normal chemical linkages throughout. Reference molecules with normal antiparallel helical orientations (hairpins aps-C8, aps-C10, and aps-G10 and duplexes aps-C3.C7, aps-D1.D3, and aps-D2.D4) were also synthesized and studied. Hydrogen bonding in ps-DNA is via reverse Watson-Crick base pairs, and the various constructs display spectroscopic, chemical, biochemical, and electrophoretic properties distinct from those of their aps counterparts. For example, both the ps and aps molecules show a pronounced UV absorption hyperchromicity upon melting, but the spectral distribution is not the same. Thus, the difference spectra (ps-aps) in the native state are characterized by a positive peak at 252 nm, an isosbestic point at 267 nm, and a negative peak at 282 nm. Temperature-dependent absorbances were recorded at selected wavelengths and in the form of complete spectra to derive the thermodynamic parameters for the helix-coil transitions.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of nuclear proteins that specifically interact with adeno-associated virus type 2 inverted terminal repeat hairpin DNA

A palindromic hairpin duplex containing the inverted terminal repeat sequence of adeno-associated virus type 2 (AAV) DNA was used as a substrate in gel retardation assays to detect putative proteins that specifically interact with the AAV hairpin DNA structures. Nuclear proteins were detected in extracts prepared from human KB cells coinfected with AAV and adenovirus type 2 that interacted with the hairpin duplex but not in nuclear extracts prepared from uninfected, AAV-infected, or adenovirus type 2-infected KB cells. The binding was specific for the hairpin duplex, since no binding occurred with a double-stranded DNA duplex with the identical nucleotide sequence. Furthermore, in competition experiments, the binding could be reduced with increasing concentrations of the hairpin duplex but not with the double-stranded duplex DNA with the identical nucleotide sequence. S1 nuclease assays revealed that the binding was sensitive to digestion with the enzyme, whereas the protein-bound hairpin duplex was resistant to digestion with S1 nuclease. The nucleotide sequence involved in the protein binding was localized within the inverted terminal repeat of the AAV genome by methylation interference assays. These nuclear proteins may be likely candidates for the pivotal enzyme nickase required for replication or resolution (or both) of single-stranded palindromic hairpin termini of the AAV genome.

Telomeric DNA oligonucleotides form novel intramolecular structures containing guanine-guanine base pairs.

Structural properties of DNA oligonucleotides corresponding to the single-stranded molecular terminus of telomeres from several organisms were analyzed. Based on physical studies including nondenaturing polyacrylamide gel electrophoresis, absorbance thermal denaturation analysis, and 1H and 31P nuclear magnetic resonance spectroscopy, we conclude that these molecules can self-associate by forming non-Watson-Crick, guanine.guanine based-paired, intramolecular structures. These structures form below 40 degrees C at moderate ionic strength and neutral pH and behave like hairpin duplexes in nondenaturing polyacrylamide gels. Detailed analysis of the hairpin structure formed by the telomeric sequence from Tetrahymena, (T2G4)4, shows that it is a unique structure stabilized by hydrogen bonds and contains G residues in the syn conformation. We propose that this novel form of DNA is important for telomere function and sets a precedent for the biological relevance of non-Watson-Crick base-paired DNA structures.

The synthesis of DNA complementary to polyadenylate-containing RNA from Bacillus subtilis.

We had shown earlier (Gopalakrishna, Y., Langley, D., Sarkar, N. (1981) Nucleic Acid Res. 9, 3545-3554) that a substantial fraction of mRNA of various bacterial species carries 3'-terminal polyadenylate sequences. In this paper, we show that poly(A)-containing RNA from Bacillus subtilis can serve as template for the synthesis of complementary DNA by avian myeloblastosis virus reverse transcriptase, provided that oligodeoxythymidylate is added as primer. Poly(A)-RNA purified by affinity chromatography on oligo(dT)-cellulose was 20 times more effective as template for cDNA formation than total bacterial RNA, whereas rRNA was inactive. The average chain length of the cDNA was 400 nucleotides (range = 230-800 nucleotides), and 95% of the cDNA could be degraded by the single-strand specific S1 nuclease after denaturation. The small fraction (5%) that was resistant to S1 nuclease may represent duplex hairpin structures. Annealing with poly(A)-RNA protected cDNA from degradation by S1 nuclease, indicating that cDNA indeed contains nucleotide sequences complementary to poly(A)-RNA. These results constitute independent evidence that a large fraction (about 40%) of B. subtilis mRNA is polyadenylated. Moreover, the synthesis of cDNA to bacterial mRNA provides an important new tool for the study of bacterial mRNA structure.

About 30% of minute virus of mice RNA is spliced out following polyadenylation

MANY viral and eukaryotic messenger RNAs have been shown to contain blocks of sequence which, although adjacent to one another in the RNA, are copied from non-contiguous regions of the genome that are joined together post-transcriptionally. This phenomenon of RNA splicing has been shown to occur during the processing of several DNA and RNA tumour viruses1–7 and cellular8–10 mRNAs. The mechanism by which splicing is carried out has not been elucidated, and any direct approach is complicated by the elaborate splicing patterns found in the systems which have been examined so far. We have extended these studies of RNA splicing to a potentially simpler viral system, the minute virus of mice (MVM), a non-defective member of the parvovirus group. The members of this group are considered to be among the simplest of the animal viruses, and comprise an icosahedral virion made from three structural protein species, containing one linear single-stranded DNA chromosome of ∼1.5 × 106 molecular weight (for review see ref. 11). The MVM DNA molecule contains a stable hairpin duplex at its 5′-end and a 3′-terminal structure suitable for priming complementary strand synthesis in vitro12,13. Little is known about the transcription process in parvo viruses. The only cytoplasmic mRNA species found for adeno-associated virus (AAV), a member of the defective subgroup, is a transcript of 70% of the genome which haS been shown by in vitro translation to code for the three capsid proteins14. The one study of transcription carried out in the non-defective subgroup has identified an analogous RNA species in Kilham rat virus (KRV)-infected cell cytoplasm which is complementary to 70% of the viral strand and sediments as a single component at about 18S (ref. 15). We report here our attempts to determine the sequence arrangement of MVM RNA by examining, in the electron microscope, hybrids formed between the abundant species of nuclear and cytoplasmic poly(A)+ RNA and single-stranded genomic DNA. We have found that about 30% of MVM RNA is spliced out following polyadenylation.

The structure of Tetrahymena pyriformis mitochondrial DNA : II. The complex structure of strain GL mitochondrial DNA

1. 1. Isolated mtDNA from Tetrahymena pyriformis strain GL is a linear duplex molecule with an average molecular weight of 32.6 · 10 6 and without internal gaps or breaks. Denaturation of this DNA results in single strands with a duplex hairpin at one end. The length of this hairpin varies between 0 and 5 μm within one preparation. 2. 2. Under renaturation conditions the single strands with hairpins are able to circularize in two ways, depending on the length of the hairpin. Circularization is also observed after partial digestion with exonuclease III of native strain GL mtDNA. 3. 3. All these data fit a model (see Fig. 2) in which the DNA is heterogeneous in length at both ends. At the left end a 10-μm duplication-inversion is present; part of this duplication-inversion is complementary to a region at the right end of the molecule. 4. 4. The analogy between the structural peculiarities of strain GL mtDNA and of some linear viral DNAs is stressed.

DNA of minute virus of mice: self-priming, nonpermuted, single-stranded genome with a 5'-terminal hairpin duplex.

The genome of the nondefective parvovirus minute virus of mice (MVM) is a linear DNA molecular weight 1.48 x 10(6), which is single stranded for approximately 94% of its length. In contrast to the genomes from defective parvoviruses MVM DNA does not contain a detectable inverted terminal redundancy. A combination of enzymatic and physical techniques has shown that the molecule contains a stable hairpin duplex of approximately 130 base pairs located at the 5' terminus of the genome. MVM DNA is efficiently utilized as a template-primer by a number of DNA polymerases, including reverse transcriptases. Polymerases lacking 5' to 3' exonuclease activity yield a duplex DNA product with a molecular weight 1.96 times that of the viral genome, in which the newly synthesized complementary strand is covalently attached to the template. This duplex contains an internal "nick" that can be sealed by DNA ligase to produce a self-complementary single-strand circle. The MVM DNA duplex is cleaved twice by EcoR-RI restriction endonuclease to yield three distinct fragments in molar amounts. These results suggest that the initiation of DNA synthesis in vitro occurs at a point within 100 bases of the 3' end of the genome, using the 3' terminus of viral DNA as a primer, and that the sequence of nucleotides in the genome is not permuted.