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PubMed1990

Drug binding by branched DNA molecules: analysis by chemical footprinting of intercalation into an immobile junction.

Branched DNA structures interact with drugs differently from unbranched control duplexes of similar sequence. A specific interaction between the reagent (methidiumpropyl-EDTA).Fe(II) [MPE.Fe(II)] and a branched DNA molecule formed from 16-mer oligonucleotide strands has been reported [Guo, Q., Seeman, N. C., & Kallenbach, N. R. (1989) Biochemistry 28, 2355-2359]. The structure of the branched molecule is thought to be made up of two double-helical stacking domains with an overall twofold symmetry across the branch site. The MPE-Fe(II) interaction occurs predominantly at or adjacent to the branch site and is eliminated by a second intercalator, propidium iodide. Further studies on the nature and properties of this site are presented here. Comparison of the patterns of scission of linear duplex and branched tetramer by EDTA.Fe(II), MPE.Fe(II), and Cu(I)-(o-phenanthroline)2 [(OP)2Cu(I)] provides a higher resolution picture of the site of enhanced binding. In particular, the sensitive footprinting afforded by (OP)2Cu(I) allows us to localize the major site of preferential interaction with propidium precisely to the branch point itself, with a roughly twofold symmetric pattern of cuts resulting. In detail, the differential pattern with respect to each duplex control is distinct for each arm of the junction. Excess propidium results in apparent reversal of the crossover isomer of the junction, indicating a possible additional avenue for the action of drugs in biological systems--effects on the products of recombination.

Base Sequence↗

PubMed2005

pentafluorophenyl-phenyl interactions in biphenyl-DNA.

We prepared and investigated oligonucleotide duplexes of the sequence d(GATGAC(X)nGCTAG).d(CTAGC(Y)nGTCATC), in which X and Y designate biphenyl- (bph) and pentafluorobiphenyl- ((5F)bph) C-nucleotides, respectively, and n varies from 0-4. These hydrophobic base substitutes are expected to adopt a zipperlike, interstrand stacking motif, in which not only bph/bph or (5F)bph/(5F)bph homo pairs, but also (5F)bph/bph mixed pairs can be formed. By performing UV-melting curve analysis we found that incorporation of a single (5F)bph/(5F)bph pair leads to a duplex that is essentially as stable as the unmodified duplex (n=0), and 2.4 K more stable than the duplex with the nonfluorinated bph/bph pair. The T(m) of the mixed bph/(5F)bph pair was in between the T(m) values of the respective homo pairs. Additional, unnatural aromatic pairs increased the T(m) by +3.0-4.4 K/couple, irrespective of the nature of the aromatic residue. A thermodynamic analysis using isothermal titration calorimetry (ITC) of a series of duplexes with n=3 revealed lower (less negative) duplex formation enthalpies (DeltaH) in the (5F)bph/(5F)bph case than in the bph/bph case, and confirmed the higher thermodynamic stability (DeltaG) of the fluorinated duplex, suggesting it to be of entropic origin. Our data are compatible with a model in which the stacking of (5F)bph versus bph is dominated by dehydration of the aromatic units upon duplex formation. They do not support a model in which van der Waals dispersive forces (induced dipoles) or electrostatic (quadrupole) interactions play a dominant role.

Biphenyl Compounds↗

PubMed1992

Cleavage of oligoribonucleotides by a ribozyme derived from the hepatitis delta virus RNA sequence.

A self-cleaving RNA sequence from hepatitis delta virus was modified to produce a ribozyme capable of catalyzing the cleavage of RNA in an intermolecular (trans) reaction. The delta-derived ribozyme cleaved substrate RNA at a specific site, and the sequence specificity could be altered with mutations in the region of the ribozyme proposed to base pair with the substrate. A substrate target size of approximately 8 nucleotides in length was identified. Octanucleotides containing a single ribonucleotide immediately 5' to the cleavage site were substrates for cleavage, and cleavage activity was significantly reduced only with a guanine base at that position. A deoxyribose 5' to the cleavage site blocked the reaction. These data are consistent with a proposed secondary structure for the self-cleaving form of the hepatitis delta virus ribozyme in which a duplex forms with sequences 3' to the cleavage site, and they support a proposed mechanism in which cleavage involves attack on the phosphorus at the cleavage site by the adjacent 2'-hydroxyl group.

Base Sequence↗

PubMed1992

Thermodynamics of DNA branching.

Branched DNA molecules arise transiently as intermediates in genetic recombination or on extrusion of cruciforms from covalent circular DNA duplexes that contain palindromic sequences. The free energy of these structures relative to normal DNA duplexes is of interest both physically and biologically. Oligonucleotide complexes that can form stable branched structures, DNA junctions, have made it possible to model normally unstable branched states of DNA such as Holliday recombinational intermediates. We present here an evaluation of the free energy of creating four-arm branch points in duplex DNA, using a system of two complementary junctions and four DNA duplexes formed from different combinations of the same set of eight 16-mer strands. The thermodynamics of formation of each branched structure from the matching pair of intact duplexes have been estimated in two experiments. In the first, labeled strands are allowed to partition between duplexes and junctions in a competition assay on polyacrylamide gels. In the second, the heats of forming branched or linear molecules from the component strands have been determined by titration microcalorimetry at several temperatures. Taken together these measurements allow us to determine the standard thermodynamic parameters for the process of creating a branch in an otherwise normal DNA duplex. The free energy for reacting two 16-mer duplexes to yield a four-arm junction in which the branch site is incapable of migrating is + 1.1 (+/- 0.4) kcal mol-1 (at 18 degrees C, 10 mM-Mg2+). Analysis of the distribution of duplex and tetramer products by electrophoresis confirms that the free energy difference between the four duplexes and two junctions is small at this temperature. The associated enthalpy change at 18 degrees C is +27.1 (+/- 1.3) kcal mol-1, while the entropy is +89 (+/- 30) cal K-1 mol-1. The free energy for branching is temperature dependent, with a large unfavorable enthalpy change compensated by a favorable entropy term. Since forming one four-stranded complex from two duplexes should be an entropically unfavorable process, branch formation is likely to be accompanied by significant changes in hydration and ion binding. A significant apparent delta Cp is also observed for the formation of one mole of junction, +0.97 (+/-0.05) kcal deg-1 mol-1.

Base Composition↗

PubMed1998

Scission of DNA at a preselected sequence using a single-strand-specific chemical nuclease.

BACKGROUND: We were interested in developing a protocol for cleaving large DNAs specifically. Previous attempts to develop such methods have failed to work because of high levels of nonspecific background scission. RESULTS: R-loop formation was chosen for sequence-specific targeting, a method of hybridization whereby an RNA displaces a DNA strand of identical sequence in 70% formamide using Watson-Crick base-pairing, leading to a three-stranded structure. R-loops are stabilized in aqueous solution by modifying the bases with chemical reagents. The R-loop was cleaved using a novel nuclease prepared from the Thr48-->Cys mutant of the single-strand-specific M-13 gene V protein (GVP), which was alkylated with 5-(iodoacetamido-beta-alanyl)1,10-phenanthroline. The cleavage products of the pGEM plasmid were cloned in to the pCR 2.1-TOPO vector. Adenovirus 2 DNA (35.8 kb; tenfold larger than the pGEM plasmid) was also cleaved quantitatively at a preselected sequence. CONCLUSIONS: A new method for cleaving duplex DNA at any preselected sequence was developed. The cleavage method relies on the chemical conversion of M-13 GVP into a nuclease, reflecting GVP's specificity for single-stranded DNA. The GVP chimera is the first example of a semisynthetic secondary structure specific nuclease. The chemical nuclease activity of 1,10-phenanthroline-copper is uniquely suited to this technique because it oxidizes the deoxyribose moiety without generating diffusible intermediates, providing clonable DNA fragments. The protocol could be useful in generating large DNA fragments for mapping the contiguity of probes or defining the exon-intron structure of transcription units.

Adenoviridae↗

PubMed1998

Insights into the dynamic nature of DNA duplex structure via analysis of nuclear Overhauser effect intensities.

Sequence-dependent structures of DNA duplexes in solution can be reliably determined using NMR data if care is taken to determine restraint bounds accurately. This entails use of complete relaxation matrix methods to analyze nuclear Overhauser effect (NOE) spectroscopic cross-peak intensities, yielding accurate distance restraints. NMR studies of various DNA duplexes have suggested that there may be some limited internal motions. First, it is typically not possible to reconcile all vicinal proton coupling constants in deoxyribose rings with a single conformer. In addition, with the increased accuracy of interproton distance measurements afforded by the complete relaxation matrix algorithm MARDIGRAS, we find inconsistencies in certain distances which can most readily be ascribed to limited conformational flexibility, since conformational averaging is nonlinear. As base-sugar interproton distances depend on both sugar pucker and glycosidic torsion angle chi, motion involving these structural variables should be reflected by experimental data. Possible motional models have been considered to account for all of the data for three DNA duplexes. Analysis of intraresidue base-sugar interproton NOE bounds patterns suggests a motional model with individual sugars in equilibrium between S (2'-endo) and N (3'-endo) conformations, with S being preferred. As sugar repuckering is correlated with changes in glycosidic torsion angle chi, different sugar conformers imply different values for chi, but this is insufficient to account for all data. A two-state jump between anti and syn glycosidic conformers was considered, but it was incapable of accounting for all data. However, a model with restricted diffusion (rocking) about the glycosidic bond in addition to sugar repuckering was capable of accommodating all experimental data. This motional model is in qualitative agreement with experimental 13C relaxation-derived order parameter values in a DNA duplex.

Base Composition↗

PubMed2002

Conformational changes of a benzo[a]pyrene diol epoxide-N(2)-dG adduct induced by a 5'-flanking 5-methyl-substituted cytosine in a (Me)CG double-stranded oligonucleotide sequence context.

Mutations in p53 genes are one of the most common genetic alterations in human cancers. A disproportionate number of mutations are found in certain codons of the p53 gene, mostly at CpG dinucleotide sequences, which are highly methylated in human tissues. The reactivities of the mutagenic metabolite of benzo[a]pyrene, the bay region diol epoxide r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), to yield adducts with guanine at the exocyclic amino group (e.g., trans-anti-BPDE-N(2)-dG, or G*), are enhanced when the cytosine in CpG sequences in DNA is methylated at its 5-position ((Me)CpG). However, methylation may also affect the characteristics of these adducts, and we have therefore investigated whether adduct conformations are different in double-stranded DNA in methylated (Me)CpG* and in unmethylated CpG* sequence contexts in the oligonucleotide model system duplex 5'-d(CCAT[(5X)C]GCTACC).d(GGTAGCGATGG) with X = H or -CH(3). The (-)-trans-adduct exhibits a striking conformational change from a minor groove structure external to the DNA duplex in the unmethylated CpG* sequence, to an intercalative conformation in the (Me)CG* sequence context. In contrast, the conformation of the stereoisomeric (+)-trans-adduct is predominantly of the minor groove type in both the methylated and unmethylated sequences. These results indicate that methylation of CpG sequences may affect not only chemical reactivities of chemically reactive intermediates with DNA, but also the conformational properties of the DNA adducts formed. Thus, both factors must be considered in evaluating the effects of cytosine methylation in CpG sequences on the biological consequences of the DNA adducts formed.

5-Methylcytosine↗

PubMed1991

Real time automated simultaneous double-stranded DNA sequencing using two-color fluorophore labeling.

We determined the base sequences of both strands of duplex DNA simultaneously with an automated DNA sequencer. Primers for each strand were labeled with two different dyes and chain extension reactions for both strands produced four DNA families: "A", "C", "G", and "T". An image splitting-prism and band-pass filters were mounted on a real time DNA sequencer to detect fluorescences from the two different dyes separately. As a result, one thousand bases of a clone could be determined at the same time.

Automation↗

PubMed1997

The mouse Clc1/myotonia gene: ETn insertion, a variable AATC repeat, and PCR diagnosis of alleles.

Myotonias are muscle diseases in which the function of the muscular chloride channel ClC-1 is impaired. Null alleles of the corresponding Clc1 gene on mouse chromosome (Chr) 6 provide animal models for human myotonias. It was shown that the allele adr (Clc1adr) is due to an insertion of an ETn type transposon that is transcribed and leads to multiple splicing events; the allele mto (Clc1adr-mto) involves a stop codon near the N-terminus. We have determined the genomic organization of the mouse Clc1 gene and the sequence requirements for the transposon insertion in the Clc1adr allele. The mouse Clc1 gene is composed of 23 exons, ranging from 39 to 372 bp, and spans approximately 23 kb of genomic DNA. The exon/intron organization is highly homologous to that of the human CLCN1 gene; the homology of the coding sequence is 97% to rat and 89% to human. In the adr allele the ETn transposon is inserted into intron 12, the largest intron. Whereas the 5' and 3' LTR sequences of the ETn transposon are homologous to those reported for other insertional mutations of the mouse, no consensus motif for an insertion target site could be defined. On the basis of flanking sequences, we provide duplex PCR diagnoses for the adr, adr-mto, and wild-type alleles of Clc1. Close to the 3' end of intron 12, a tetranucleotide repeat (AATC)n was found that is polymorphic between mouse species Mus musculus, M. molossinus, M. castaneus, and M. spretus, and can thus be used for chromosomal mapping studies.

Alleles↗

PubMedDate not supplied

Characterization of a 12-mer duplex d(GGCGGAGTTAGG).d(CCTAACTCCGCC) containing a highly reactive (+)-CC-1065 sequence by 1H and 31P NMR, hydroxyl-radical footprinting, and NOESY restrained molecular dynamics calculations.

The solution structure of the GC-rich non-self-complementary DNA 12-mer duplex (I), which contains a (+)-CC-1065 highly reactive bonding sequence 5'AGTTA* (where * denotes the [formula: see text] covalent modification site), has been examined thoroughly by one- and two-dimensional proton and phosphorus NMR spectroscopy, hydroxyl-radical footprinting, and NOESY restrained molecular mechanics and dynamics calculations. The assignments of the nonexchangeable proton resonances (except some of the H5' and H5" protons due to severe resonance overlap), phosphorus resonances, and the exchangeable resonances (except amino protons of adenosine and guanosine) of this 12-mer duplex have been made. The results show that this 12-mer duplex maintains an overall B-form DNA with all anti base orientation throughout in aqueous solution at room temperature. Hydroxyl-radical footprinting experiments on a 21-mer sequence that contains this 12-mer duplex used for NMR studies showed that the minor groove is somewhat narrowed at the 7G-8T and 17A-18C steps, as indicated by the inhibition of cleavage at these locations. Although both high-field NMR and hydroxyl-radical footprinting experiments supported a bent-like structure for this 12-mer duplex, nondenaturing gel electrophoresis on the ligated 21-mer sequence that contains this 12-mer duplex did not show the abnormally slow migration characteristic of a bent DNA duplex. Analysis of the NMR data sets reveals several local structural perturbations similar to those found on an (A)n tract DNA duplex. For example, the existence of a propeller twist was detected within the A.T-rich region for both the 12-mer and the (A)n tract DNA duplexes. The 18CH5 aromatic resonance that is directly adjacent to the 3' side of the 5'TAA segment was significantly shifted upfield with a chemical shift of 5.10 ppm, which is almost within the region normally associated with sugar H3' protons. The sugar geometries for 18C and 7G, which are located to the 3' side of the 5'TAA segment, are proposed to be in the neighborhood of C3'-endo and O1'-endo in equilibrium C3'-endo, respectively. We propose that this unusually upfield-shifted resonance signal for 18CH5 and the average C3'-endo sugar geometry for 18C nucleotide on the 12-mer duplex is connected with the peculiar conformation, possibly a transient kink, within the 5'AC/GT step. The results of the NOESY restrained molecular mechanics and dynamics calculations on the 12-mer sequence reveal two kinks, which are located on either side of the 18C nucleotide that has an average C3'-endo sugar geometry.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗

PubMed1996

Control of telomere growth by interactions of RAP1 with the most distal telomeric repeats.

Telomeres, the specialized DNA-protein structures at the ends of eukaryotic chromosomes, are required for chromosomal stability and integrity. Regulation of the overall length of the telomeric DNA repeat tract is likely to be a key requirement for its various biological roles. We have studied telomere length regulation in the yeast Kluyveromyces lactis, which has long (25 base pairs) homogeneous telomeric repeat units that make it highly suitable for telomere studies. In the related Saccharomyces cerevisiae, the DNA-sequence-specific duplex-binding protein RAP1 is a component of the telomeric complex. Here we show that the phenotypic severity of previously described telomerase RNA (ter1) mutations is directly proportional to the loss of RAP1 binding to mutated telomeric repeats. Using a carboxy-terminal-tail mutant of K. lactis RAP1, we also show that, unexpectedly, RAP1 interaction with the most terminal telomeric repeats is crucial for telomere length control.

Base Sequence↗

PubMed1992

Influence of enzyme-substrate contacts located outside the EcoRI recognition site on cleavage of duplex oligodeoxyribonucleotide substrates by EcoRI endonuclease.

A complete understanding of the sequence-specific interaction between the EcoRI restriction endonuclease and its DNA substrate requires identification of all contacts between the enzyme and substrate, and evaluation of their significance. We have searched for possible contacts adjacent to the recognition site, GAATTC, by using a series of substrates with differing lengths of flanking sequence. Each substrate is a duplex of non-self-complementary oligodeoxyribonucleotides in which the recognition site is flanked by six base pairs on one side and from zero to three base pairs on the other. Steady-state kinetic values were determined for the cleavage of each strand of these duplexes. A series of substrates in which the length of flanking sequence was varied on both sides of the hexamer was also examined. The enzyme cleaved both strands of each of the substrates. Decreasing the flanking sequence to fewer than three base pairs on one side of the recognition site induced an asymmetry in the rates of cleavage of the two strands. The scissile bond nearest the shortening sequence was hydrolyzed with increasing rapidity as base pairs were successively removed. Taken together, the KM and kcat values obtained may be interpreted to indicate the relative importance of several likely enzyme-substrate contacts located outside the canonical hexameric recognition site.

Base Composition↗

PubMed2001

VHL gene alterations in renal cell carcinoma patients: novel hotspot or founder mutations and linkage disequilibrium.

Mutations in the von Hippel-Lindau (VHL) gene are frequently detected in human sporadic renal cell carcinoma (RCC). We analysed 102 Swedish RCCs for VHL mutations by PCR-SSCP and sequencing. In 47 patients (46.1%), 70 different mutations were found, and most of them represented novel variations of the VHL gene. Mutations in the VHL gene were found in 54% of clear cell renal cell carcinomas (CCRCC) and in 18% of chromophilic cancers but in no chromophobe cancers or oncocytomas (P=0.016). Three novel hotspot or founder mutations were detected in our study: four CCRCCs carried a missense mutation (glutamic acid to lysine) at codon 160 which is critical in the stabilization of the H1 helix of the alpha domain and the alpha-beta domain interface in the VHL protein. Five CCRCCs and one chromophilic RCC harbored a 15-nucleotide in-frame deletion (codons 41-45) at a duplex tandem repeat sequence site. Moreover, this deletion was in linkage disequilibrium with a C-->T transition in the promoter region. The frequency of linkage was 17 times more common than chance. Five patients with this linked mutation resided in the same hospital district and at least three of them showed the two sequence variants in the tumor-adjacent tissue. In 5/6 patients the wild-type allele was lost in the tumor samples, suggesting a causal role for the mutations in RCC. These linked mutations might be novel polymorphisms maintained in a relative isolated population. Multiple mutations in VHL were found in 17 tumors out of 47 tumors with the VHL mutation. A higher multiple mutation detected rate (33%) was observed in grade 3 CCRCCs than those in grade 1 (22%) and grade 2 (9%) (P=0.04). This is evidence on the association between VHL mutation and extent of nuclear atypia.

Age of Onset↗

PubMed1993

Molecular dynamics simulations provide a structural basis for the experimentally observed nucleotide preferences for DNA interstrand cross-links induced by aziridinylbenzoquinones.

Two-electron reduction of the structurally related aziridinylbenzoquinones DZQ and MeDZQ to their hydroquinone forms, DZHQ and MeDZHQ, respectively, generates species which interact and cross-link DNA at distinct nucleotide sequences. Within single target site duplex oligonucleotides, DZHQ was found to cross-link DNA at 5'-GC-3' and 5'-GNNC-3' sequences, whereas MeDZHQ was found to cross-link predominantly at 5'-GNC-3' within a 5'-GTCA-3' sequence. In a multitarget site duplex oligonucleotide, which contains either the target sequence 5'-TGCAC-3' or 5'-TGCTC-3', DZHQ was found to cross-link at both a 5'-GC-3' (a 1,2 cross-link) and a 5'-GNNC-3' (a 1,4 cross-link) site with approximately equal efficiency. Molecular dynamics simulations were able to accurately reproduce the experimental results and provide a structural basis for the alkylation preferences. Calculations were performed to determine the mobility of the hydroquinone species following guanine N7 (G) alkylation at 5'-TGCAC-3' and 5'-TGTCA-3' sequences. Conformations consistent with the formation of both 1,2 and 1,4 cross-links were observed when DZHQ was placed within a 5-TGCAC-3' sequence. The 1,2 cross-link orientation was more stable and thermodynamically favored. For MeDZHQ at the same site the ligand was unable to form stable 1,2 or 1,4 cross-linking conformations, primarily due to clashes with thymine methyl groups. In contrast, the MeDZHQ monoadduct with a 5'-TGTCA-3' sequence adopted a very stable conformation consistent with formation of a 1,3 cross-link.

Alkylation↗

PubMed1991

CpG methylation inhibits binding of several sequence-specific DNA-binding proteins from pea, wheat, soybean and cauliflower.

To elucidate how methylation of specific sites in plant DNA might control transcription, we examined the effect of DNA methylation at CpG sequences on the binding of plant nuclear factors to an oligonucleotide duplex containing the consensus sequence for mammalian CREB (cAMP response element binding protein). CREB is part of the ATF (activating transcription factor) family of mammalian proteins specifically binding to 5'-TGACGTCA-3' and related sequences. Proteins recognizing the CREB-specific ligand were identified in nuclear extracts of pea seeds, wheat germ, cauliflower, and soybean leaves using electrophoretic mobility shift assays. Cytosine methylation inhibited binding of this protein in all these extracts, and so this sequence-specific DNA-binding activity is referred to as methylation-inhibited binding protein 1 (MIB-1). Sites somewhat similar to that of the CREB ligand are found in the upstream regions of a wheat histone H3 gene and tomato and pea ribulose 1,5-bisphosphate carboxylase genes. These sites were bound preferentially by distinct proteins that may be related to the previously described plant proteins HBP-1, HSBF, ASF-1, or GBF. Methylation of cytosine residues at these sites and at a site for MIB-1 located upstream of a soybean proline-rich protein gene also reduced specific binding with all the nuclear extracts tested. Similarly, substitution of the central CpG dinucleotide with TpG decreased binding.

Base Sequence↗

PubMed2003

Repairing the Sickle Cell mutation. II. Effect of psoralen linker length on specificity of formation and yield of third strand-directed photoproducts with the mutant target sequence.

Three identical deoxyoligonucleotide third strands with a 3'-terminal psoralen moiety attached by linkers that differ in length (N = 16, 6 and 4 atoms) and structure were examined for their ability to form triplex-directed psoralen photoproducts with both the mutant T residue of the Sickle Cell beta-globin gene and the comparable wild-type sequence in linear duplex targets. Specificity and yield of UVA (365 nm) and visible (419 nm) light-induced photoadducts were studied. The total photoproduct yield varies with the linker and includes both monoadducts and crosslinks at various available pyrimidine sites. The specificity of photoadduct formation at the desired mutant T residue site was greatly improved by shortening the psoralen linker. In particular, using the N-4 linker, psoralen interaction with the residues of the non-coding duplex strand was essentially eliminated, while modification of the Sickle Cell mutant T residue was maximized. At the same time, the proportion of crosslink formation at the mutant T residue upon UV irradiation was much greater for the N-4 linker. The photoproducts formed with the wild-type target were fully consistent with its single base pair difference. The third strand with the N-4 linker was also shown to bind to a supercoiled plasmid containing the Sickle Cell mutation site, giving photoproduct yields comparable with those observed in the linear mutant target.

Anemia, Sickle Cell↗

PubMed2004

Influence of minor groove substituents on the structure of DNA Holliday junctions.

The inosine-containing sequence d(CCIGTACm(5)CGG) is shown to crystallize as a four-stranded DNA junction. This structure is nearly identical to the antiparallel junction formed by the parent d(CCGGTACm(5)()CGG) sequence [Vargason, J. M., and Ho, P. S. (2002) J. Biol. Chem. 277, 21041-21049] in terms of its conformational geometry, and inter- and intramolecular interactions within the DNA and between the DNA and solvent, even though the 2-amino group in the minor groove of the important G(3).m(5)C(8) base pair of the junction core trinucleotide (italicized) has been removed. In contrast, the analogous 2,6-diaminopurine sequence d(CCDGTACTGG) crystallizes as resolved duplex DNAs, just like its parent sequence d(CCAGTACTGG) [Hays, F. A., Vargason, J. M., and Ho, P. S. (2003) Biochemistry 42, 9586-9597]. These results demonstrate that it is not the presence or absence of the 2-amino group in the minor groove of the R(3).Y(8) base pair that specifies whether a sequence forms a junction, but the positions of the extracyclic amino and keto groups in the major groove. Finally, the study shows that the arms of the junction can accommodate perturbations to the B-DNA conformation of the stacked duplex arms associated with the loss of the 2-amino substituent, and that two hydrogen bonding interactions from the C(7) and Y(8) pyrimidine nucleotides to phosphate oxygens of the junction crossover specify the geometry of the Holliday junction.

2-Aminopurine↗

PubMed2000

[Cleavage of RNA in hybrid duplexes by ribonuclease H from E. coli. I. Substrate properties of complexes formed by RNA and tandem of short oligodeoxyribonucleotides].

We studied the E. coli RNase H cleavage of a 5'-labeled RNA fragment within two hybrid duplexes with identical sequences, one of which is formed by RNA and a 20-mer oligodeoxyribonucleotide (RNA/p20), whereas the second, by RNA and a tandem of short oligodeoxyribonucleotides (octanucleotide: (RNA/tandem). It was shown that RNA in the RNA/p20 complex is hydrolyzed from the 3'-end to yield consecutively the 17-, 14-, 11-, 8-, and 5-mer 5'-labeled fragments. On hydrolysis of RNA in complex RNA/tandem, the same products were registered, but their accumulation rates in this case differed. Thus, the initial rates of accumulation of the 17- and 8-mer were close. Moreover, the accumulation of the final 5-mer differed considerably: in the RNA/tandem complex it appeared within first minutes of the reaction, but only after a considerable lag period in complex RNA/p20. These data testify that the tandem is involved not only in the consecutive accumulation of the shortened products (which is characteristic of complexes including extended oligonucleotides) but also in the parallel accumulation. This results from hydrolysis of each duplex segment formed by RNA and the short oligonucleotide of the tandem. Although the order of recognition and cleavage of RNA target by ribonuclease H depends on the type of the hybrid duplex, the destruction of RNA target within complex RNA/tandem and in complex with the full-size oligonucleotide occurs with a close effectiveness.

Electrophoresis, Polyacrylamide Gel↗