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At least 451 records · Page 25Linked to original sources
PubMed1986

A unique four-stranded model of a homologous recombination intermediate.

This paper proposes a model of four-stranded DNA synapsis during recombination between homologous segments of two DNA duplexes. The proposed intermediate is one of only two known models having relative chain orientations about the synaptic junction that are consistent with recent topological results on the integrative recombination of bacteriophage lambda. This model has the advantage of providing a mechanism for recognition of sequence homology between duplexes through specific hydrogen-bond formation; other models are discussed in comparison. The new model is based on an alternative family of DNA structures having chain directions opposite to those of the Watson-Crick family of structures. Idealized coordinates for generating both right- and left-handed forms of these alternative structures are presented for further study.

Bacteriophage lambda↗
PubMed1992

Purification and characterization of UL9, the herpes simplex virus type 1 origin-binding protein.

UL9, the origin-binding protein of herpes simplex virus type 1 (HSV-1), has been overexpressed in an insect cell overexpression system and purified to homogeneity. In this report, we confirm and extend recent findings on the physical properties, enzymatic activities, and binding properties of UL9. We demonstrate that UL9 exists primarily as a homodimer in solution and that these dimers associate to form a complex nucleoprotein structure when bound to the HSV origin of replication. We also show that UL9 is an ATP-dependent helicase, capable of unwinding partially duplex DNA in a sequence-independent manner. Although the helicase activity of UL9 is demonstrable on short duplex substrates in the absence of single-stranded DNA-binding proteins, the HSV single-stranded DNA-binding protein ICP8 (but not heterologous binding proteins) stimulates UL9 to unwind long DNA sequences of over 500 bases. We were not able to demonstrate unwinding of fully duplex DNA sequences containing the HSV origin of replication. However, in experiments designed to detect origin-dependent unwinding, we did find that UL9 wraps supercoiled DNA independent of sequence or ATP hydrolysis.

Animals↗
PubMed1978

Structure of recombinant plasmids containing synthetic human foetal globin gene sequences.

In vitro synthesized duplex DNA complementary to human foetal globin messenger RNA was integrated into bacterial plasmids and amplified by transformation of Escherichia coli. Recombinants carrying globin DNA were identified by hybridization of foetal globin messenger RNA to bacterial DNA in situ and by liquid hybridization of purified plasmids to specific globin complementary DNA probes. Heteroduplex mapping revealed either a simple insertion loop at the position of the EcoRI site of the parental plasmid DNA. We provide evidence to suggest that these deletions are the result of a site-specific nicking activity of the EcoRI preparations used in the formation of recombinant plasmids.

DNA Restriction Enzymes↗
PubMed1992

Comparative NMR study of A(n)-bulge loops in DNA duplexes: intrahelical stacking of A, A-A, and A-A-A bulge loops.

We have prepared a series of deoxyoligonucleotide duplexes of the sequence d(G-C-A-T-C-G-X-G-C-T-A-C-G).d(C-G-T-A-G-C-C-G-A-T-G-C), in which X represents either one (A), two (A-A), or three (A-A-A) unpaired adenine basis. Using two-dimensional proton and phosphorus NMR spectroscopy, we have characterized conformational features of these bulge-loop duplexes in solution. We find that Watson-Crick hydrogen bonding is intact for all 12 base pairs, including the GC bases that flank the bulge loop. Observation of NOE connectivities in both H2O and D2O allows us to unambiguously localize all of the bulged adenine residues to intrahelical positions within the duplex. This is in contrast to an earlier model for multiple-base bulge loops in DNA [Bhattacharyya, A., & Lilley, D. M. J. (1989) Nucleic Acids Res. 17, 6821-6840], in which all but the most 5' bulged base are looped out into solution. We find that insertion of two or three bases into the duplex results in the disruption of specific sequential NOEs for the base step across from the bulge loop site on the opposite strand. This disruption is characterized by a partial shearing apart of these bases, such that certain sequential NOEs for this base step are preserved. We observe a downfield-shifted phosphorus resonance, which we assign in the A-A-A bulge duplex to the 3' side of the last bulged adenine residue. Proton and phosphorus chemical shift trends within the An-bulge duplex series indicate that there is an additive effect on the structural perturbations caused by additional unpaired bases within the bulge loop. This finding parallels previous observations [Bhattacharyya, A., & Lilley, D. M. J. (1989) Nucleic Acids Res. 17, 6821-6840; Hsieh, C.-H., & Griffith, J. D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4833-4837] on the magnitude of the induced bending of DNA duplexes by multiple-base bulge loops.

Adenine↗
PubMed2003

Non-Watson-Crick basepairing and hydration in RNA motifs: molecular dynamics of 5S rRNA loop E.

Explicit solvent and counterion molecular dynamics simulations have been carried out for a total of >80 ns on the bacterial and spinach chloroplast 5S rRNA Loop E motifs. The Loop E sequences form unique duplex architectures composed of seven consecutive non-Watson-Crick basepairs. The starting structure of spinach chloroplast Loop E was modeled using isostericity principles, and the simulations refined the geometries of the three non-Watson-Crick basepairs that differ from the consensus bacterial sequence. The deep groove of Loop E motifs provides unique sites for cation binding. Binding of Mg(2+) rigidifies Loop E and stabilizes its major groove at an intermediate width. In the absence of Mg(2+), the Loop E motifs show an unprecedented degree of inner-shell binding of monovalent cations that, in contrast to Mg(2+), penetrate into the most negative regions inside the deep groove. The spinach chloroplast Loop E shows a marked tendency to compress its deep groove compared with the bacterial consensus. Structures with a narrow deep groove essentially collapse around a string of Na(+) cations with long coordination times. The Loop E non-Watson-Crick basepairing is complemented by highly specific hydration sites ranging from water bridges to hydration pockets hosting 2 to 3 long-residing waters. The ordered hydration is intimately connected with RNA local conformational variations.

Base Pair Mismatch↗
PubMed2005

Influence of initial charge state on fragmentation patterns for noncovalent drug/DNA duplex complexes.

The charge state-dependent dissociation of various DNA duplexes and drug/duplex complexes has been investigated using collisionally activated dissociation (CAD) in a quadrupole ion trap mass spectrometer (QIT-MS). Several non-self-complementary 14-residue oligonucleotides were employed, in addition to an array of known DNA-interactive ligands, including the intercalators daunomycin and nogalamycin, as well as the minor groove binding agents distamycin, netropsin, 4',6-diamidino-2-phenylindole, and Hoechst 33342. In general, the dissociation pathways exhibited by both the duplexes and the drug/duplex complexes were found to be markedly sensitive to initial charge state. Time- and activation voltage-independent duplex strand separation predominated for higher charge states, which was interpreted to be a result of internal Coulombic repulsion or partial unzipping in the interface, while time- and activation voltage-dependent covalent cleavage predominated for lower charge states. The identity of the drug and the sequence of the duplex were both found to affect the competition between different dissociation processes. The dissociation pathways for the lower charge state complexes are probably more reflective of specific drug-DNA interactions because Coulombic and/or conformational effects are less marked for these precursors.

Benzimidazoles↗
PubMed2005

Stability of 3' double nucleotide overhangs that model the 3' ends of siRNA.

Thermodynamic parameters are reported for duplex formation in 1 M NaCl for 16 RNA sequences, each containing a core tetramer duplex, GGCC, and a 3' overhang consisting of two bases. The results indicate additional double-helical stability is conferred by the double 3' terminal overhang relative to the single 3' terminal overhang. A nearest-neighbor analysis of the data indicates that the free energy contribution at 37 degrees C of the second base in the double 3' terminal overhang varies from 0 to 0.7 kcal/mol. The second base in the 3' double overhang can contribute nearly the same stability to a duplex as a base pair or a 3' dangling overhang. Stability contribution of a dangling base, two nucleotides removed from the 3' end of a duplex, is dependent upon both the identity of the base as well as that of the dangling base that it neighbors. A second dangling base only increases the stability of the duplex when it is neighboring a 3' purine dangling nucleotide. Furthermore, a second dangling pyrimidine provides a greater contribution to duplex stability than a purine. A nearest-neighbor model was developed to predict the influence of 3' double overhang on the stability of duplex formation. The model improves the prediction of free energy and melting temperature when tested against six sequences with different core duplexes.

Models, Molecular↗
PubMed2003

P-loop catalytically assisting the enzymatic cleavage of single-stranded DNA.

We demonstrated that a P-loop, a looped complex formed inside duplex DNA by adding peptide nucleic acids (PNA), acts catalytically as a template for enzymatic cleavage of single-stranded probe oligodeoxynucleotides (ODN). A PD-loop complex formed from P-loop and probe ODN was digested efficiently by a restriction enzyme, and the truncated probe ODN was released. The P-loop nicked by the enzyme can form PD-loop again with another probe ODN, and then assisted the enzymatic cleavage of an excess of probe ODN. In addition, by using dumbbell-formed ODN as a probe ODN, the efficiency of the P-loop-assisted ODN cleavage was enhanced considerably as compared with that of linear ODN. Thus, the method utilizing P-loop will make it possible to amplify the sequence information of duplex DNA via a catalytic cleavage of probe ODNs.

Base Sequence↗
PubMed1997

Sequence effects on the relative thermodynamic stabilities of B-Z junction-forming DNA oligomeric duplexes.

Circular dichroism (CD) and ultraviolet absorption techniques were employed in characterizing the sequence-dependent thermodynamic stabilities of B-Z junction-forming DNA duplexes. The Watson strand of the duplexes has the general sequence (5meC-G)4-NXYG-ACTG (where N = A or G and XY represents all permutations of pyrimidine bases). Duplexes were generated by mixing stoichiometric amounts of the complementary strands. Circular dichroism studies indicate that each duplex is fully right-handed at low salt (e.g., 115 mM Na+) but undergoes a salt-induced conformational transition to a structure that possesses both left- and right-handed conformations at high salt (4.5 M Na+), and hence a B-Z junction. Optical melting studies of the DNA duplexes at fixed DNA concentration with total Na+ concentration ranging from 15 mM to 5.0 M were determined. A nonlinear dependence of the melting temperature (Tm) on [Na+] was observed. Thermodynamic parameters at Na+ concentrations of 115 mM and 4.5 M with a wide range of DNA concentrations were determined from UV optical melting studies via construction of van't Hoff plots. A change of a single dinucleotide within these duplexes significantly affected the helix stabilities. The experimentally obtained free energies for the duplex to single-strand transitions were in close agreement with predicted values obtained from two different methods.

Base Sequence↗
PubMed1994

DNase I footprinting of triple helix formation at polypurine tracts by acridine-linked oligopyrimidines: stringency, structural changes and interaction with minor groove binding ligands.

We have investigated the binding of short (10 base) acridine-linked triplex-forming oligonucleotides to the target sequence A6G6.C6T6 by DNase I footprinting. Specific binding is detected at low pH (< 6.0) for 5'-Acr-T5C5 and 5'-Acr-5BrU5(5Me)C5. The sequence T5C5, lacking the acridine modification, binds less strongly, though specific binding is still evident. 5'-Acr-T5C5 produces footprints at slightly lower concentrations than 5'-Acr-5BrU5(5Me)C5. All three oligonucleotides produce enhanced DNase I digestion at the 3'-end of the target purine strand, suggesting that there is a DNA structural change at the triplex-duplex boundary. Target sequences AnG4A and TAC3Tn, containing one and two triplex mismatches, show no interaction with the acridine-free oligonucleotide, but bind the acridine-linked oligonucleotides. In these secondary binding modes the third strand is positioned so that the mismatches are located at the 3'-end of the oligonucleotide. Mithramycin and distamycin, binding in the minor groove to GC- and AT-rich sequences respectively, abolish triple helix formation.

Acridines↗
PubMed1991

Recognition and photo-induced cleavage and cross-linking of nucleic acids by oligonucleotides covalently linked to ellipticine.

Oligopyrimidines covalently linked to ellipticine derivatives form duplex and triplex structures with target single-stranded oligopurine sequences. They also bind to duplex DNA at homopurine-homopyrimidine sequences where they form local triple helices. Irradiation at wavelengths longer than 300 nm of the complex formed by an oligonucleotide-ellipticine conjugate with its target sequence induced (i) cleavage of the target at bases located in close proximity to the dye and (ii) cross-linking of the target sequence to the derivatized oligonucleotide. Both cross-linking and cleavage reactions decreased when temperature increased with a half-transition corresponding to the dissociation of the oligonucleotide-ellipticine conjugate from its target nucleic acid, demonstrating that the observed photochemical effects are dependent on hybrid formation. When the target was a double-stranded DNA, photochemical reactions were observed on both strands of the duplex. Photo-induced cross-linking was more efficient than cleavage when the target was single-stranded; the reverse was observed when the target was duplex DNA.

Base Sequence↗
PubMed1993

Sites of predicted stress-induced DNA duplex destabilization occur preferentially at regulatory loci.

This paper describes a computational method to predict the sites on a DNA molecule where imposed superhelical stresses destabilize the duplex. Several DNA sequences are analyzed in this way, including the pBR322 and ColE1 plasmids, bacteriophage f1, and the polyoma and bovine papilloma virus genomes. Superhelical destabilization in these molecules is predicted to occur at small numbers of discrete sites, most of which are within regulatory regions. The most destabilized sites include the terminator and promoter regions of specific plasmid operons, the LexA binding sites of genes under SOS control, the intergenic control region of bacteriophage f1, and the polyadenylylation sites in eukaryotic viruses. These results demonstrate the existence of close correspondences between sites of predicted superhelical duplex destabilization and specific types of regulatory regions. The use of these correspondences to supplement string-matching techniques in the search for regulatory loci is discussed.

Bacterial Proteins↗
PubMed1989

Improved chemistry for oligodeoxyribonucleotide synthesis substantially improves restriction enzyme cleavage of a synthetic 35mer.

Two DNA duplexes of identical sequence and 35 nt in length were synthesized by an original and a highly improved version of phosphoramidite chemistry. By base composition analysis, DNA synthesized by improved chemistry (termed DMTS-imp) contained no detectable modified bases while DNA synthesized by the original chemistry (termed DMTS-std) had a large number of modifications. Under optimal reaction conditions, HhaI and RsaI cleaved the DMTS-std duplex to 76-77% completion and the DMTS-imp duplex to 96-99% completion. Restriction analysis and piperidine treatment yielded estimates of approximately 3.0% modified nucleotides in DMTS-std and approximately 1.0% in DMTS-imp. Overall, the improvements in chemistry increased the restriction efficiency of synthetic DNA up to 10-fold.

Adenine↗
PubMed2003

Evaluation of the RNA determinants for bacterial and yeast RNase III binding and cleavage.

Bacterial double-stranded RNA-specific RNase III recognizes the A-form of an RNA helix with little sequence specificity. In contrast, baker yeast RNase III (Rnt1p) selectively recognizes NGNN tetraloops even when they are attached to a B-form DNA helix. To comprehend the general mechanism of RNase III substrate recognition, we mapped the Rnt1p binding signal and directly compared its substrate specificity to that of both Escherichia coli RNase III and fission yeast RNase III (PacI). Rnt1p bound but did not cleave long RNA duplexes without NGNN tetraloops, whereas RNase III indiscriminately cleaved all RNA duplexes. PacI cleaved RNA duplexes with some preferences for NGNN-capped RNA stems under physiological conditions. Hydroxyl radical footprints indicate that Rnt1p specifically interacts with the NGNN tetraloop and its surrounding nucleotides. In contrast, Rnt1p interaction with GAAA-capped hairpins was weak and largely unspecific. Certain duality of substrate recognition was exhibited by PacI but not by bacterial RNase III. E. coli RNase III recognized RNA duplexes longer than 11 bp with little specificity, and no specific features were required for cleavage. On the other hand, PacI cleaved long, but not short, RNA duplexes with little sequence specificity. PacI cleavage of RNA stems shorter than 27 bp was dependent on the presence of an UU-UC internal loop two nucleotides upstream of the cleavage site. These observations suggest that yeast RNase IIIs have two recognition mechanisms, one that uses specific structural features and another that recognizes general features of the A-form RNA helix.

Base Sequence↗
PubMed1998

What affects the effect of 2'-alkoxy modifications? 1. Stabilization effect of 2'-methoxy substitutions in uniformly modified DNA oligonucleotides.

The thermostability of hybrid duplexes with uniformly 2'-methoxy modified DNA strands (D'R and RD'), their unmodified DNA:RNA counterparts (DR and RD), and corresponded RNA:RNA (RR) duplexes for six sequences with different GC and deoxypyrimidine (dPy) content was measured. The linear correlation between the total stabilization effect of 2'-methoxy modifications (Delta DeltaG(o)37(D'R-DR)) and the relative stability of corresponding unmodified hybrids compared to the RR counterparts (Delta DeltaG(o)37(RR-DR)) suggests that the initial conformational and the thermodynamic state of the "parent" unmodified hybrid governs the effect of 2'-methoxy (and may be other 2'-alkoxy) modifications whose mechanism of action includes an S --> N conformational shift resulting in an RNA-like A-form duplex. We also found a correlation between the "hydrophobic" part of the total effect (Delta DeltaG(o)37(D'R-RR)) and the dA fraction in the modified DNA strand, suggesting that the "hydrophobic" effect of the 2'-methoxy groups results mainly from intraresidue steric effects increasing rigidity of the modified sugar rings. The correlations observed enabled us to predict the stability of hybrids with 2'-methoxy modified DNA strands for any sequence except for sequences with (dU)10 and (dA)10 strings.

Circular Dichroism↗
PubMed1980

Replication of the plasmid pBR322 under the control of a cloned replication origin from the single-stranded DNA phage M13.

The replication origins of viral and complementary strands of bacteriophage M13 DNA are contained within a 507-nucleotide intergenic region of the viral genome. Chimeric plasmids have been constructed by inserting restriction endonuclease fragments of the M13 intergenic region into the plasmid pBR322. Replication of these hybrid plasmids, under conditions not permissive for the plasmid replicon, depends on specific segments of the M13 origin region and on the presence of M13 helper virus. Thus M13-infected polA- Escherichia coli can be transformed to ampicillin resistance by hybrid plasmids that have a functional M13 origin. Cells transformed to drug resistance by plasmids bearing M13 origin sequences contain the duplex chimeric DNA at high copy number but do not accumulate significant amounts of single-stranded plasmid DNA. Rare transducing phages carrying single-stranded chimeric DNA are produced and can be detected by their ability to transduce cells to ampicillin resistance. Plasmids containing a 270-nucleotide fragment from the gene II-proximal half of the intergenic region produce transformants at high frequency under nonpermissive conditions. A central Hae III fragment, Hae III-G, containing the nucleotide sequence coding for the RNA primer for the complementary strand and the nicking site for gene II protein, is sufficient for plasmid replication in M13-infected polA- cells but not for high frequency transformation. Additional sequence information on the gene II side of the Hae III-G fragment is necessary for efficient transformation by the plasmid DNA.

Coliphages↗