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PubMed1991

Sequence organization and RNA structural motifs directing the mouse primary rRNA-processing event.

The first processing step in the maturation of mouse precursor rRNA involves cleavage at nucleotide ca. +650, at the 5' border of a 200-nucleotide region that is conserved across mammals and contains the sequences that direct the processing. To identify the relevant sequence elements, we used rRNAs with small internal mutations and short pre-rRNA substrates. Much of the region can be mutated without appreciable effect, but nucleotides +655 to +666 appear to be absolutely required and short segments surrounding +750 and +810 markedly stimulate processing. The minimal processing signal corresponds to rRNA nucleotides +645 to +672. Formation of a ribonucleoprotein complex of retarded electrophoretic mobility is evidently necessary but not sufficient for processing. Computer-assisted analysis suggested a phylogenetic- and mutant-supported secondary structure in which the minimal processing signal forms a stem with the +655 region in the loop, and there is a separate branched duplex containing the downstream stimulatory sequences. Use of antisense RNA, in trans and in cis, to sequester the +655 region in a duplex supported the hypothesis that this critical region was needed in a single-stranded conformation for processing and for specific complex formation.

Animals↗
PubMed1992

Oligonucleotide-mediated triple helix formation using an N3-protonated deoxycytidine analog exhibiting pH-independent binding within the physiological range.

Triple helix formation with pyrimidine deoxyoligonucleotides for the sequence-specific recognition of DNA duplex targets suffers from a decrease in affinity as the pH of the medium increases to that of physiological fluids. A solution to this problem has been identified and entails the substitution of N6-methyl-8-oxo-2'-deoxyadenosine (M) for the 5-methyl-deoxycytosine base residues. The triple helix forming ability of an oligonucleotide consisting of thymidine and M residues is pH independent in the physiological range. Furthermore, M has been found to be superior to the previously used 5-methyldeoxycytidine and deoxyguanosine in conferring increased affinity for duplex DNA under physiological salt conditions.

Base Sequence↗
PubMed1998

Asp34 of PvuII endonuclease is directly involved in DNA minor groove recognition and indirectly involved in catalysis.

The PvuII restriction endonuclease is a homodimer that recognizes and cleaves the DNA sequence 5'-CAGCTG-3' in double-stranded DNA, and the structure of this enzyme has been reported. In the wild-type enzyme, Asp34 interacts with the internal guanine of the recognition sequence on the minor groove side. The Asp34 codon was altered to specify Gly (D34G), and in vitro studies have revealed that the D34G protein has lost binding specificity for the central G.C base-pairs, and that it cuts the canonical sequence with 10(-4)-fold reduced activity as compared to the wild-type enzyme. We have now determined the structure at 1.59 A resolution of the D34G PvuII endonuclease complexed with a 12 bp duplex deoxyoligonucleotide containing the cognate sequence. The D34G alteration results in several structural changes relative to wild-type protein/DNA complexes. First, the sugar moiety of the internal guanine changes from a C2'-endo to C3'-endo pucker while that of the 3' guanine changes from C3'-endo to C2'-endo pucker. Second, the axial rise between the internal G.C base-pairs is reduced while that between the G.C and flanking base-pairs is expanded. Third, two distinct monomeric active sites are observed that we refer to as being "primed" and "unprimed" for phosphodiester bond cleavage. The primed and unprimed sites differ in the conformation of the Asp58 side-chain, and in the absence from unprimed sites of four networked water molecules. These water molecules, present in the primed site, have been implicated in the catalytic mechanism of this and other endonucleases; some of them can be replaced by the Mg2+ necessary for cleavage. Taken together, these structural changes imply that the Asp34 side-chains from the two subunits maintain a distinct conformation of its DNA substrate, properly situating the target backbone phosphates and indirectly manipulating the active sites. This provides some insight into how recognition of the specific DNA sequence is linked to catalysis by the highly specific restriction endonucleases, and reveals one way in which the structural conformation of the DNA is modulated coordinately with that of the PvuII protein.

Aspartic Acid↗
PubMed1989

Trypanosoma brucei: conserved sequence organization 3' to telomeric variant surface glycoprotein genes.

We have previously postulated that telomeric variant surface glycoprotein (VSG) genes in Trypanosoma brucei serve more frequently than intrachromosomal VSG genes as basic copies for gene conversion. To examine this further we determined the sequence for approximately 1200 nucleotides 3' to the telomeric IsTat 1 VSG gene, expressed in early variant antigenic types, and compared this sequence with those 3' to other VSG genes. We found that about 200 nucleotides immediately 3' to the 1 VSG gene are homologous to sequences immediately 3' to other telomeric VSG genes. These sequences may function in extended duplex formation 3' to telomeric VSG genes and partially explain their more frequent gene conversion. In addition, further 3' is a highly conserved 49 bp direct repeat, which is not transcribed into stable RNA. These sequences appear to be conserved in various T. brucei stocks, and we have therefore proposed a model which is a modification of one previously proposed (E. H. Blackburn and P. B. Challoner, 1984, Cell, 36, 447-457; L. H. T. Van der Ploeg, A. Y. C. Liu, and P. Borst, 1984, Cell, 36, 459-468) for the sequence organization of a trypanosome telomeric region.

Amino Acid Sequence↗
PubMed2002

Crystal structure of d(GCGAAAGCT) containing parallel-stranded duplex with homo base pairs and anti-parallel duplex with Watson-Crick base pairs.

A DNA fragment d(GCGAAAGCT), known to adopt a stable mini-hairpin structure in solution, has been crystallized under a slightly acidic condition, and its crystal structure has been determined at 2.5A resolution. In the first half of the oligomer, the sequence CGAA forms a parallel duplex with another symmetry-related half through the homo base pairs, C2:C2+ (semi-protonated between the Watson-Crick sites), G3:G3 (between the minor groove sites), A4:A4 (between the major groove sites) and A5:A5 (between the Watson-Crick sites). The second halves of the parallel duplex are split away in the different direction, and extended to form an anti-parallel B-form duplex with another second half.

Base Pairing↗
PubMed1991

1H-NMR investigation of the interaction of the amino terminal domain of the LexA repressor with a synthetic half-operator.

A synthetic half-operator DNA-duplex, d(GCTACTGTATGT), containing a portion of the proposed recognition sequence (CTGT) of several "SOS" genes, has been synthesized. The dodecamer has been characterized through 1H-NMR spectroscopy. Complete assignment of exchangeable hydrogen bonded imino protons has been achieved by applying 1D NOE techniques and an analysis of the temperature dependence of the chemical shifts. In order to determine the specific role of the CTGT consensus sequence in the overall recognition process, the oligonucleotide duplex has been titrated with the amino terminal DNA binding domain of the LexA repressor. The observation of substantial changes of 1H-NMR chemical shifts in the imino proton region upon interaction with the protein strongly suggests that the protein binds specifically to the operator DNA. The largest deviations of 1H-NMR chemical shifts upon protein binding have been observed for protons assigned to the CTGT segment, thus strongly suggesting a direct involvement of this sequence in the binding process. At high potassium chloride concentrations the 1H-NMR chemical shift deviations are reverted which is consistent with the known drop in the affinity constant of LexA for operator DNA at high salt concentrations.

Bacterial Proteins↗
PubMed1998

Continuum solvent studies of the stability of RNA hairpin loops and helices.

We apply continuum solvent models to investigate the relative stability of various conformational forms for two RNA sequences, GGAC(UUCG)GUCC and GGUG(UGAA)CACC. In the first part, we compare alternate hairpin conformations to explore the reliability of these models to discriminate between different local conformations. A second part looks at the hairpin-duplex conversion for the UUCG sequence, identifying major contributors to the thermodynamics of a much large scale transition. Structures were taken as snapshots from multi-nanosecond molecular dynamics simulations computed in a consistent fashion using explicit solvent and with long-range electrostatics accounted for using the Particle-Mesh Ewald procedure. The electrostatic contribution to solvation energies were computed using both a finite-difference Poisson-Boltzmann (PB) model and a pairwise Generalized Born model; non-electrostatic contributions were estimated with a surface-area dependent term. To these solvation free energies were added the mean solute internal energies (determined from a molecular mechanics potential) and estimates of the solute entropy (from a harmonic analysis). Consistent with experiment and with earlier solvated molecular dynamics simulations, the UUCG hairpin was found to prefer conformers close to a recent NMR structure determination in preference to those from an earlier NMR study. Similarly, results for the UGAA hairpin favored an NMR-derived structure over that to be expected for a generic GNRA hairpin loop. Experimental free energies are not known for the hairpin/duplex conversion, but must be close to zero since hairpins are seen in solution and duplexes in crystals; out calculations find a value near zero and illustrate the expected interplay of solvation, salt effects and entropy in affecting this equilibrium.

Computer Simulation↗
PubMed1995

An A-DNA triplet code: thermodynamic rules for predicting A- and B-DNA.

The ability to predict macromolecular conformations from sequence and thermodynamic principles has long been coveted but generally has not been achieved. We show that differences in the hydration of DNA surfaces can be used to distinguish between sequences that form A- and B-DNA. From this, a "triplet code" of A-DNA propensities was derived as energetic rules for predicting A-DNA formation. This code correctly predicted > 90% of A- and B-DNA sequences in crystals and correlates with A-DNA formation in solution. Thus, with our previous studies on Z-DNA, we now have a single method to predict the relative stability of sequences in the three standard DNA duplex conformations.

Base Sequence↗
PubMed1989

Facile interconversion of duplex structures formed by copolymers of d(CG).

Correlations between DNA sequence and reactivity have often been drawn with an implicit or explicit connection to duplex structure. An in vitro model using oligonucleotides of defined sequences has been developed to characterize a potential source of the hypersensitivity that naturally occurring regions of redundant sequence exhibit with many nucleases. S-1 nuclease was used here to diagnose the unusual hybridization of copolymeric DNA, d(CG)6, and related oligomers, through product and kinetic analysis. Fully complementary but redundant sequences reacted with this enzyme almost an order of magnitude faster than did heterogeneous fragments of DNA. Hydrolysis products of the copolymers indicated that conformations with unpaired termini were the sole substrates under these studies, and only a facile equilibrium between aligned and extended structures was required to explain the heightened reactivity of this DNA. For example, d(CG)6 was converted to d(CG)5 and d(CG)4 whereas d(CG)4C was initially processed to an octamer and then only later to a hexamer. Catalysis by S-1 exhibited no other substrate or product specificity; even the disordered bases in the loop region of a hairpin structure, d(CG)3T4(CG)3, did not provide sites of enhanced enzyme action. The rate of DNA consumption under standard conditions was proportional to the expected concentration of overhanging sequences rather than the absolute amount of DNA present. All initial attempts to saturate enzyme activity failed, and therefore, the rate of substrate formation through strand slippage was always faster than the catalytic depletion of unpaired bases. Only a low-energy transition state(s) must then separate the various hybridized species since this structural equilibration proceeded readily under conditions of 10 mM potassium phosphate, pH 7, 100 mM NaCl, and 22 degrees C.

Base Sequence↗
PubMed1991

Specificity and stringency in DNA triplex formation.

Triple-helix formation can in principle serve as a general method for sequence-specific recognition and physical separation of duplex DNA molecules. Realization of this goal depends on how much the triplex is destabilized by mismatches and other defects (specificity) and on finding conditions in which perfect complexes are stable and defect complexes are not (stringency). We have addressed the question of specificity by determining the difference in free energy between perfect and defect complexes by using UV melting curves and equilibrium competition experiments. We find that third strands that bind with either single-base bulges or single mismatches are destabilized relative to the perfect triplex by 2.5-2.9 and 3.2-4.0 kcal/mol (1 cal = 4.184 J), respectively, essentially equivalent to the corresponding values determined for duplex DNA and RNA. Also, we present a method, referred to as stringency clamping, which maintains specific binding under conditions far from normal stringency. To do this, we provide for the formation of a competing structure involving the third strand with stability between that of the perfect and imperfect complexes; the competitive interaction effectively prevents triplex formation at imperfect sites even far below their melting temperature. We illustrate the phenomenon with three different stringency clamps, two of which compete for the all-pyrimidine third strand through Watson-Crick pairing and one that competes through all-pyrimidine pairing at acidic pH. We demonstrate physical separation of two duplex DNA molecules differing by a single base pair in their target sequence for triple-helix formation.

Base Sequence↗
PubMed2005

Spectroscopic characterization of interstrand carbinolamine cross-links formed in the 5'-CpG-3' sequence by the acrolein-derived gamma-OH-1,N2-propano-2'-deoxyguanosine DNA adduct.

The interstrand N2,N2-dG DNA cross-linking chemistry of the acrolein-derived gamma-OH-1,N2-propanodeoxyguanosine (gamma-OH-PdG) adduct in the 5'-CpG-3' sequence was monitored within a dodecamer duplex by NMR spectroscopy, in situ, using a series of site-specific 13C- and 15N-edited experiments. At equilibrium 40% of the DNA was cross-linked, with the carbinolamine form of the cross-link predominating. The cross-link existed in equilibrium with the non-crosslinked N2-(3-oxo-propyl)-dG aldehyde and its geminal diol hydrate. The ratio of aldehyde/diol increased at higher temperatures. The 1,N2-dG cyclic adduct was not detected. Molecular modeling suggested that the carbinolamine linkage should be capable of maintaining Watson-Crick hydrogen bonding at both of the tandem C x G base pairs. In contrast, dehydration of the carbinolamine cross-link to an imine (Schiff base) cross-link, or cyclization of the latter to form a pyrimidopurinone cross-link, was predicted to require disruption of Watson-Crick hydrogen bonding at one or both of the tandem cross-linked C x G base pairs. When the gamma-OH-PdG adduct contained within the 5'-CpG-3' sequence was instead annealed into duplex DNA opposite T, a mixture of the 1,N2-dG cyclic adduct, the aldehyde, and the diol, but no cross-link, was observed. With this mismatched duplex, reaction with the tetrapeptide KWKK formed DNA-peptide cross-links efficiently. When annealed opposite dA, gamma-OH-PdG remained as the 1,N2-dG cyclic adduct although transient epimerization was detected by trapping with the peptide KWKK. The results provide a rationale for the stability of interstrand cross-links formed by acrolein and perhaps other alpha,beta-unsaturated aldehydes. These sequence-specific carbinolamine cross-links are anticipated to interfere with DNA replication and contribute to acrolein-mediated genotoxicity.

Acrolein↗
PubMed1993

Hydrogen-bonding contacts in the major groove are required for human immunodeficiency virus type-1 tat protein recognition of TAR RNA.

The binding site for tat on TAR RNA was analysed by preparing a series of model RNA substrates carrying site-specific functional group modifications. The test RNAs were prepared by annealing two short synthetic oligoribonucleotides to form a duplex structure with a U-rich bulge and flanking sequences identical to TAR RNA. Tat binds these duplex RNAs with approximately half the affinity for wild-type TAR RNA. Substitution at positions U23 or U25 by the base analogue, O4-methyl-dT, which is deficient in its ability to hydrogen-bond at the N3 position reduces tat affinity more than 20-fold. Modifications to purines in the stem of TAR RNA that affect hydrogen-bonding ability in either the major or the minor groove of duplex RNA were also tested. Removal of the nitrogen atom at either the N7 position of G26 or at the N7 position of A27 reduces tat affinity 10- to 20-fold. By contrast removal of the exocyclic amino group in the minor groove at position G26, by substitution with inosine, does not affect tat binding significantly. A single methylphosphonate substitution at the phosphate bond between A22 and U23 also leads to a significant loss of tat binding ability, whereas all other methylphosphonate substitutions in the U-rich bulge are not harmful to tat binding. We conclude that tat forms multiple specific hydrogen bonds to a series of dispersed sites displayed in the major groove of the TAR RNA molecule. These include the N3-H of U23, the N7 of G26, the N7 of A26 and the phosphate between A22 and U23.

Base Sequence↗
PubMed1998

Interaction of methyl green with an oligonucleotide in intramolecular duplex and triplex conformations. Circular dichroism studies.

Interaction of methyl green with the oligonucleotide 5-dGGAAAAGG-[T4]-GGAAAAGG-[T4]-CCTTTTCC (where [T4] is a nucleotide sequence of four thymines) in hairpin duplex and in intramolecular triplex structures has been studied by circular dichroism. We found that methyl green binding to the duplex form shows a complex pattern, exhibiting an exciton contribution when the number of bound molecules increases. Differences between this pattern and previously published results on other DNAs reveals the presence of different types of complexes. In contrast to previous findings with the triple helix poly(dA).2poly(dT) we show that the methyl green is not toallly excluded from this triplex structure made of Pur:Pur:Pyr triplets.

Base Sequence↗
PubMed1995

Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes.

The thermodynamic parameters (delta H degree, delta S degree, and delta G degree 37) for 16 nearest-neighbor sets and one initiation factor are presented here in order to predict stability of RNA/DNA hybrid duplexes. To determine the nearest-neighbor parameters, thermodynamics for 68 different hybrid sequences (136 single-stranded oligonucleotides) with 5-13 nucleotide length including several duplexes with identical nearest-neighbors were measured by UV melting procedure. These sequences were selected to have many different combinations of nearest-neighbor pairs, and so that the number of the 16 nearest-neighbor sequences in the oligomers were as equal as possible. The structures of the hybrids were also investigated by measuring circular dichroism spectra. Comparing delta G degree 37 values of the hybrids with DNA/DNA and RNA/RNA parameters reported previously (Breslauer, K.J., Frank, R., Blöcker, H., & Marky, L.A. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3746-3750; Freier, S.M., Kierzek, R., Jaeger, J.A., Sugimoto, N., Caruthers, M.H., Neilson, T., & Turner, D.H. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9373-9377), RNA/RNA double helix is the most stable of the three kinds of helixes with the same nearest-neighbor sequences. Which is more stable between DNA/DNA and RNA/DNA hybrid duplexes depends on its sequence. Calculated thermodynamic values of hybrid formation with the present parameters reproduce the experimental values within reasonable errors.

Base Sequence↗
PubMed1996

Mercury(II) Site-Selective Binding to a DNA Hairpin. Relationship of Sequence-Dependent Intra- and Interstrand Cross-Linking to the Hairpin-Duplex Conformational Transition.

Hg(II) interacted site selectively with only one of three deoxyribooligonucleotides examined; these "oligos" each had a different number of unmatched T residues. Thus, Hg(II) formed an intrastrand T-Hg-T cross-link between the first and fourth T residues of the hairpin, d(GCGCTTTTGCGC) (T4). The DNA strand formed a loop around the Hg, as if the Hg atom had been lassoed. The interactions of Hg(II) with two other oligos, d(ATGGGTTCCCAT) (T2) and d(GCGCTTTGCGC) (T3), were less specific. Previously, we found that at high DNA and salt concentrations, T2 was a mixture of hairpin and duplex forms while T3 and T4 had the hairpin form; modeling studies showed that in the free T4 hairpin the two T's at the ends of the (T)(4) loop form a T.T wobble base pair. Only in T4 are the T residues positioned to form an intrastrand cross-link readily. The Hg(II)-oligo adducts formed as a function of added Hg(II) were investigated by titrations monitored by UV, CD, and (1)H NMR spectroscopy. The appearance of a new set of (1)H signals with the concomitant decay of the free oligo (1)H signals indicated that 1:1 Hg(II):T2, 1.5:1 Hg(II):T3, and 1:1 Hg(II):T4 adducts were formed with Hg(NO(3))(2). In H(2)O, these adducts all had spectra with very downfield signals for the exchangeable TN(3)H and GN(1)H groups, a characteristic of base-paired regions. All upfield N(3)H signals from the (T)(2) and (T)(3) sequences of the free oligo disappeared in the spectra of the 1:1 Hg(II):T2 and 1.5:1 Hg(II):T3 adducts. The disappearance of the NH signals, the UV spectral changes, and the stoichiometries (1:1 Hg(II):T2 and 1.5:1 Hg(II):T3) indicate that these adducts are duplexes containing two and three T-Hg-T interstrand cross-links for T2 and T3, respectively. The (1)H and (13)C signals of the 1:1 Hg(II):T4 adduct in D(2)O were nearly completely assigned by 2D NMR spectroscopy. The spectrum of the adduct in H(2)O had only two of the four original TN(3)H signals from the (T)(4) sequence present in the spectrum of T4; this result is consistent with the presence of a TN3-Hg-TN3 cross-link. The (13)C chemical shift changes upon Hg(II) binding indicated that the TN3-Hg-TN3 cross-link was between the T's at each end of the (T)(4) loop. The NOESY, CD, and UV spectra were all consistent with a hairpin conformation for the 1:1 Hg(II):T4 adduct. A hairpin conformation also appeared reasonable from molecular modeling calculations. In conclusion, the length of the central (T)(n)() sequence influenced the type of T-Hg-T cross-link formed and, in turn, the conformation of the adducts. For (T)(2) and (T)(3), interstrand T-Hg-T cross-linking favored the duplex form. In contrast, for (T)(4), intrastrand T-Hg-T cross-linking stabilized the hairpin form.

Journal Article↗
PubMed1999

Effect of distance between homologous sequences and 3' homology on the frequency of retroviral reverse transcriptase template switching.

Deletion of direct repeats in retroviral genomes provides an in vivo system for analysis of reverse transcriptase (RT) template switching. The effect of distance between direct repeats on the rate of deletion was determined for 16 murine leukemia virus (MLV)-based vectors containing a 701-bp direct repeat of overlapping fragments of the herpes simplex virus thymidine kinase gene (HTK). The direct repeats were separated by spacer fragments of various lengths (0.1 to 3.5 kb). Southern analysis of infected cells after one replication cycle indicated that all vectors in which the distance between homologous sequences was >1,500 bp deleted at very high rates (>90%). In contrast, vectors containing <1,500 bp between homologous sequences exhibited lower frequencies of deletion (37 to 82%). To analyze the pattern of locations at which RT switched templates, restriction site markers were introduced to divide the downstream direct repeat into five regions. RT switched templates within all five regions of the 701-bp direct repeat and the frequency of template switching was greater within the 5' regions in comparison to the 3' regions. The probability of RT switching templates within the 5' regions doubled when the MLV packaging sequence (Psi) was placed between the 701-bp direct repeats. However, Psi did not increase the rate of template switching for shorter direct repeats. These results indicate that linear distance between homologous sequences increases the rate of template switching and suggest that duplex formation between nascent DNA and homologous template sequences 3' of RT promote template switching.

Animals↗
PubMed1998

Topotecan lactone selectively binds to double- and single-stranded DNA in the absence of topoisomerase I.

We report the first experimental observation that a clinically important camptothecin [CPT; topotecan (TPT), a water-soluble CPT] binds directly and noncovalently to double-stranded DNA and single-stranded DNA structures in the absence of topoisomerase I, but only in the lactone form. We observed clear DNA sequence specificity of the TPT lactone binding to duplex DNA, which was comprised of alternating purine-pyrimidine sequences that contained dT. These structural studies of direct TPT lactone-DNA binding support several important considerations involving possible mechanism(s) of anticancer activity of CPT-type drugs containing a 20(S) lactone moiety.

Antineoplastic Agents↗