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At least 487 records · Page 27Linked to original sources

PubMed1984

Oligonucleotide directed mutagenesis: selection of mutants by hemimethylation of GATC-sequences.

We have developed a selection procedure for mutants obtained by oligonucleotide directed mutagenesis based on asymmetrical A-methylation of GATC-sequences in the duplex DNA. The method involves the construction of gapped duplexes of circular single-stranded phage DNA. An oligonucleotide, complementary to part of the gap except for a single mismatch, is hybridized to the gapped duplex DNA and the remaining single stranded regions are filled-in enzymatically. When the template is undermethylated, the yield of mutants is almost solely dependent on the priming efficiency of the oligonucleotide. The approach was used to introduce an AT----CG transversion in the mut L region of phage lambda. Under optimal conditions, about 50-60% of the transformants were of the mutant genotype. Although situated adjacent to a known nut L mutation, the present mutation was phenotypically silent. The possibility of screening for mutants by means of a coupled, easily detectable marker was also investigated.

Bacteriophage lambda↗

PubMed1991

A single-stranded region located downstream of the repeated sequence in the ori region of pSC101 is required for binding of the Rep protein in vitro.

Purified replication initiator protein (Rep) of plasmid pSC101 binds preferentially to two inverted repeats (IR) overlapping the promoter of its own structure gene, rep. However, the protein has much lower binding affinity for directly repeated (DR) sequences in the replication origin (ori) that are similar to the symmetric sequences. Exonuclease III (exo III) promotes in vitro binding of Rep to the origin repeats. In the present studies, DNA containing the DR sequences was degraded unidirectionally by exo III and then formed a complex with Rep. Analyses of DNA from the complex revealed that Rep bound to the DR sequences only when the degradation proceeded from the 3' end proximal to IR to the DR sequences, resulting in conversion of the duplex structure in a specific downstream region of DR into the single-stranded form. The degradation in the opposite direction had no effect on binding of Rep. These results suggest that a localized structural change of DNA adjacent to DR is required for Rep binding to double-stranded DR sequences. By contrast, exo III strikingly inhibited binding of Rep to DNA containing the IR sequences by introducing a single-stranded moiety into duplex IR sequences.

Adenosine Triphosphatases↗

PubMed2002

The bacterial histone-like protein HU specifically recognizes similar structures in all nucleic acids. DNA, RNA, and their hybrids.

HU, a major component of the bacterial nucleoid, shares properties with histones, high mobility group proteins (HMGs), and other eukaryotic proteins. HU, which participates in many major pathways of the bacterial cell, binds without sequence specificity to duplex DNA but recognizes with high affinity DNA repair intermediates. Here we demonstrate that HU binds to double-stranded DNA, double-stranded RNA, and linear DNA-RNA duplexes with a similar low affinity. In contrast to this nonspecific binding to total cellular RNA and to supercoiled DNA, HU specifically recognizes defined structures common to both DNA and RNA. In particular HU binds specifically to nicked or gapped DNA-RNA hybrids and to composite RNA molecules such as DsrA, a small non-coding RNA. HU, which modulates DNA architecture, may play additional key functions in the bacterial machinery via its RNA binding capacity. The simple, straightforward structure of its binding domain with two highly flexible beta-ribbon arms and an alpha-helical platform is an alternative model for the elaborate binding domains of the eukaryotic proteins that display dual DNA- and RNA-specific binding capacities.

Bacterial Proteins↗

PubMed1983

Sequence dependence of hydrogen exchange kinetics in DNA duplexes at the individual base pair level in solution.

The kinetics for hydrogen exchange at individual base pairs in self-complementary deoxydodecanucleotide duplexes have been estimated from NMR saturation recovery measurements on the resolved imino protons as a function of temperature. The imino protons of dA . dT base pairs in the center of the fully alternating d(C-G-C-G-T-A-T-A-C-G-C-G) duplex exchange a factor of 2- to 3-fold faster than the corresponding protons at the same positions in the partially alternating d(C-G-C-G-A-A-T-T-C-G-C-G) duplex. These exchange parameters are a direct measure of the rate constants for transient opening of individual dA . dT base pairs in the dodecanucleotide duplexes and demonstrate faster opening kinetics for the "TATA" box region compared to the related "AATT" segment.

Base Sequence↗

PubMed1994

Sequence dependence of DNA structure in solution.

Sequence-dependent structural variations in DNA can influence its binding by ligands and proteins. However, relatively little is known about sequence-structure relationship for arbitrary DNA sequences. The 1H two-dimensional NOESY data presented here for ten oligonucleotide duplexes show pronounced sequence-dependent changes in at least two types of internucleotide distances commonly used in sequential connectivity assignments, namely the H-6/H-8 to preceding (5') H-1' distance (S1) and the H-6/H-8 to preceding (5') H-2'' distance (S2). On the basis of these two measured distances, all dinucleotide steps can be divided into four different groups: Y-R, R-Y, R-R and Y-Y, where R is purine and Y is pyrimidine. These data suggest some rules for the variation in these distances along DNA duplexes. Correlations between these distances and helical parameters of DNA are discussed in comparison to well-resolved X-ray structures of B-type DNA.

Base Sequence↗

PubMed1993

Thermodynamic characterization of daunomycin-DNA interactions: comparison of complete binding profiles for a series of DNA host duplexes.

Using a combination of spectroscopic and calorimetric techniques, we have determined complete thermodynamic binding profiles (delta G degree, delta H degree, and delta S degree) for the complexation of daunomycin to a series of 10 polymeric DNA duplexes. We find the resulting drug binding data to be sensitive to the base composition and sequence of the host duplex, with the binding free energies ranging from -7.5 to -10.8 kcal/mol of bound drug and the binding enthalpies ranging from +4.11 to -10.76 kcal/mol of bound drug at 25 degrees C. The smaller range in the free energy term reflects the impact of large enthalpy-entropy compensations. We observe that the three synthetic duplexes which exhibit the highest daunomycin binding affinities all contain GC (or IC) base pairs as part of alternating purine/pyrimidine sequence motifs, with these high binding affinities being strongly enthalpy driven at 25 degrees C. Specific comparisons between the binding profiles for daunomycin complexation with select pairs of host duplexes lead to the following observations: (1) The presence or absence of a major-groove methyl group does not alter daunomycin binding thermodynamics. (2) The presence or absence of a minor-groove amino group does alter daunomycin binding thermodynamics. (3) Duplexes with different base compositions but identical minor-groove functionality exhibit similar daunomycin binding thermodynamics. (4) Homopolymeric duplexes composed of either AT or AU base pairs, but not GC base pairs, exhibit large enthalpy-entropy compensations in their daunomycin binding profiles. We propose interpretations of these and other features of our thermodynamic data in terms of specific daunomycin-DNA interactions deduced from available structural data.

Base Composition↗

PubMed1978

Periodic organisation of foldback sequences in Physarum polycephalum nuclear DNA.

Nuclear DNA from the slime mould Physarum polycephalum is shown to contain interspersed inverted repeat sequences, such that denatured fragments of DNA containing pairs of these sequences form intra-chain duplexes under appropriate conditions. The organisation and distribution of the nucleotide sequences responsible for the formation of foldback structures in Physarum DNA have been investigated using the electron microscope. The majority of foldback duplexes have sizes ranging up to 800 base pairs, and about 60-80% of DNA molecules 2.2 X 10(4) bases in length contain interspersed foldback elements. The size of individual foldback duplexes, and also the length of the intervening sequences which separate them, are non-random. The results can best be explained by a model in which separate foldback foci in Physarum DNA are spaced periodically at regular intervals. The regions containing foldback foci are thought to contain smaller, tandemly-arranged sequences of discrete sizes, in some cases related to other nucleotide sequences of a similar nature in the same locality in Physarum DNA.

Base Sequence↗

PubMed1996

Novel Hoogsteen-like bases for configurational recognition of the T-A base pair by DNA triplex formation.

Effective sequence-specific recognition of duplex DNA is possible by triplex formation with natural oligonucleotides via Hoogsteen H-bonding. However, triplex formation is in practice limited to pyrimidine oligonucleotides binding duplex A-T or G-C base-pair DNA sequences specifically at homopurine sites in the major groove as T-A-T and C+.G-C triplets. Here we report the successful modeling of novel unnatural nucleosides that recognize the T-A DNA base pair by Hoogsteen interaction. Since the DNA triplex can be considered to assume an A-type or B-type conformation, these novel Hoogsteen nucleotides are tested within model A-type and B-type conformation triplex structures. A triplet consisting of the T-A base pair and one of the novel Hoogsteen nucleotides replaces the central T.A-T triplet in the triplex using the same deoxyribose-phosphodiester and base-deoxyribose dihedral angle configuration. The entire triplex is energy minimized and the presence of any structural or energetic perturbations due to the central triplet is assessed with respect to the unmodified energy-minimized (T.A-T)11 proposed starting structures. Incorporation of these novel triplets into both A-type and B-type natural tiplex structures provokes minimal change in the configuration of the central and adjacent triplets. The plan is to produce a series of Hoogsteen-like bases that preferentially bind the T-A major groove in either an A-type or B-type conformation. Selective recognition of the T-A major groove with respect to the G-C major groove, which presents similar keto and amine placement, is also assessed with configurational preference. Evaluation of the triplex solution structure by using these unnatural bases as binding conformational probes is a prerequisite to the further design of triplet forming bases.

Adenine↗

PubMed2006

Covalent incorporation of methyl red dyes into double-stranded DNA for their ordered clustering.

An ordered dye cluster of Methyl Reds was formed in double-stranded DNA by hybridizing two complementary DNA-dye conjugates, each involving a Methyl Red moiety on a threoninol linker and a 1,3-propanediol spacer arranged alternately in the middle of the DNA sequence. In the duplex, Methyl Reds from each strand were axially stacked antiparallel to each other, as determined from NMR analysis. This clustering of Methyl Reds induced distinct changes in both UV/Vis and CD spectra. Single-stranded DNA-Methyl Red conjugates on D-threoninol linkers and (1,3-propanediol) spacers exhibited broad absorption spectra with lambda(max) at around 480 nm, and almost no CD was observed at around the absorption maximum of Methyl Red. However, as Methyl Reds were clustered by hybridization, lambda(max) shifted towards shorter wavelengths with respect to its monomeric transition. This hypsochromic shift increased as the number of Methyl Red molecules increased. Furthermore, a positive couplet was also strongly induced here. These dye clusters are H-aggregates, in which molecular excitons are coupled. The positive couplet demonstrates that the clusters on D-threoninol form a right-handed helix. In contrast, the induced CD became much weaker with Methyl Red on L-threoninol, which intrinsically prefers counterclockwise winding. Thus, mutual orientation of the stacked dye molecules was controlled by the chirality of the linker.

Azo Compounds↗

PubMed1996

Capture in the gel: intermoleculare triplex formation during gel electrophoresis.

Analysis of unusual gel mobility patterns formed by certain DNA triplexes has revealed that intermolecular triplex formation can occur during gel electrophoresis when a faster migrating single strand overtakes a slower migrating band containing a duplex of appropriate sequence. Control experiments showed that this capture of the third strand occurs by sequence-specific hybridization rather than some nonspecific retardation. This phenomenon can be used to detect triplexes by a gel-shift assay even if their lifetime is much shorter than the time of gel electrophoresis.

Base Composition↗

PubMed2001

Structural alterations in the DNA ahead of the primer terminus during displacement synthesis by reverse transcriptases.

Unlike most DNA polymerases, reverse transcriptases can initiate DNA synthesis at a single-strand break and displace the downstream non- template strand simultaneously with extension of the primer. This reaction is important for generation of the long terminal repeat sequences in the duplex DNA product of retroviral reverse transcription. Oligonucleotide-based model displacement constructs were used to study the interaction of human immunodeficiency virus type 1 and Moloney murine leukemia virus reverse transcriptases with the DNA. Under conditions where the DNA is saturated with enzyme, there is no protection against DNase I cleavage of the 5' single-stranded extension that would correspond to the already-displaced strand. However, the DNase I footprint on the non-template strand extends from the +1 to the +9 position for the human immunodeficiency virus type 1 enzyme and from +1 to +7 or +8 for the Moloney enzyme. This extent of protection on the non-template strand is similar to what was observed previously for the template strand downstream from the primer terminus. Use of potassium permanganate as a probe for unpaired bases in the region ahead of the primer terminus reveals that the two base-pairs immediately in front of the enzyme are melted by the bound enzyme. These findings are consistent with a displacement mechanism in which the reverse transcriptase plays an active role in unpairing the DNA ahead of the translocating polymerase. The results are interpreted in light of a recent crystal structure showing the nature of the protein-DNA contacts with the template strand ahead of the primer terminus.

DNA Footprinting↗

PubMed1993

Antisense oligonucleotides made of 2'-O-alkylRNA: their properties and applications in RNA biochemistry.

Oligo(2'-O-alkylribonucleotides) have been developed recently as novel oligonucleotide analogues with properties that enhance their use as antisense probes. They possess high chemical stability and are resistant to hydrolysis by DNA- or RNA-specific nucleases. Many forms of oligo(2'-O-alkylribonucleotides) hybridise specifically and efficiently to complementary RNA sequences, forming stable duplexes that are not substrates for cleavage by RNAse H. In combination with prosthetic reporter groups, such as biotin, DNP or fluorophores, oligo(2'-O-alkylribonucleotides) have important applications in a wide range of biochemical studies on RNA function and structure.

Alkylation↗

PubMed1990

Energetics of B-Z junction formation in a sixteen base-pair duplex DNA.

We report analysis of the NaCl-induced B-Z transition in a 16 base-pair duplex DNA with sequence designed such that when NaCl is increased the left half of the molecule undergoes the B-Z transition while the right half remains in the B-form. An equilibrium thermodynamic model based on the body of available published experimental data and the recent theoretical work of Soumpasis, which indicate, in the salt range above approximately 0.9 M-NaCl, the transition free-energy of B-Z conversion in DNA is a linear function of the NaCl concentration, is employed. Analysis of the B-Z transition of the junction-containing molecule indicates the B-Z junction formed in this 16 base-pair DNA is composed of approximately three base-pairs and has a free energy of formation of approximately 4.7 kcal/mol junction. These values for the junction are in excellent agreement with published estimates of B-Z junction size and energy derived from much longer DNA pieces.

Base Sequence↗

PubMed1990

A note on graphing helical parameters of dynamics structure of DNA.

A graphical procedure for analysis of helical parameters in dynamic structure of DNA is described. The performance of the procedure is illustrated by analysis of a 20 ps dynamics simulation of the non-self-complementary ninemer, 5'CAAACAGGA:5'TCCTGTTTG, which is a part of DNA from lacI gene. The dynamics trajectory of the duplex shows sequence-dependent fluctuations of helical parameters.

Base Sequence↗

PubMed1988

Preference of human mitochondrial RNA polymerase for superhelical templates with mitochondrial promoters.

The RNA polymerase of HeLa cell mitochondria has been purified free of endonuclease and DNA topoisomerase activities, permitting evaluation of the effect of template topology on transcription in vitro. On single-stranded DNA templates, transcription is nonspecific and does not require mitochondrial DNA sequences. In contrast, duplex DNA templates are efficiently transcribed only when they (1) carry the mitochondrial D-loop region and (2) are negatively supercoiled. These findings suggest a role for template superhelicity in modulating mitochondrial transcription in vivo.

Chlorides↗

PubMed2000

Energetics of DNA intercalation reactions.

Isothermal titration calorimetry has been used to determine the binding enthalpy and heat capacity change (DeltaC(p)()) for a series of DNA intercalators, including ethidium, propidium, daunorubicin, and adriamycin. Temperature-dependent binding enthalpies were measured directly for the ligands, from which DeltaC(p)() values of -140 to -160 cal mol(-)(1) K(-)(1) were calculated. Published van't Hoff plots were reanalyzed to obtain DeltaC(p)() values of -337 to -423 cal mol(-)(1) K(-)(1) for the binding of actinomycin D to several DNA oligonucleotide duplexes with defined sequences. Heat capacity changes for DNA intercalation were found to correlate with the alterations in solvent-accessible surface area calculated from available high-resolution structural data. Multiple linear regression was used to derive the relationship DeltaC(p)() = 0. 382(+/-0.026)DeltaA(np) - 0.121(+/-0.077)DeltaA(p) cal mol(-)(1) K(-)(1), where DeltaA(np) and DeltaA(p) are the binding-induced changes in nonpolar and polar solvent-accessible surface areas (in square angstroms), respectively. The DeltaC(p)() terms were used to estimate the hydrophobic contribution to intercalative binding free energies, yielding values that ranged from -11.2 (ethidium) to -30 kcal mol(-)(1) (actinomycin D). An attempt was made to parse the observed binding free energies of ethidium and propidium into five underlying contributions. Such analysis showed that the DNA binding behavior of these simple intercalators is driven almost equally by hydrophobic effects and van der Waals contacts within the intercalation site.

Animals↗

PubMed1994

Iron(II) bleomycin-mediated degradation of a DNA-RNA heteroduplex.

The effect of iron(II) bleomycin on a DNA-RNA heteroduplex was investigated using a substrate formed by reverse transcription of Escherichia coli 5S ribosomal RNA. Both strands of the heteroduplex were cleaved by FeII.BLM A2 at comparable concentrations; complete digestion of both strands was observed using 5 microM FeII.BLM A2. The DNA strand of the heteroduplex was cleaved predominantly at 5'-G-pyr-3' sites; the sites of cleavage of the DNA strand were a subset of those observed for the corresponding DNA strand of a DNA duplex of identical sequence. The sites of cleavage of the RNA strand of the heteroduplex involved both purines and pyrimidines and were found to be different than the sites of cleavage of the 5S rRNA alone, demonstrating that cleavage of the former must actually have involved heteroduplex recognition by FeII.BLM A2. Both the DNA and RNA strands of the heteroduplex were cleaved by FeII.BLM A2 in the presence of physiological concentrations of Mg2+, consistent with the possibility that DNA-RNA heteroduplexes may be therapeutically relevant targets for bleomycin.

Base Sequence↗

PubMed1994

Synthesis of oligodeoxynucleotides containing analogs of O6-methylguanine and reaction with O6-alkylguanine-DNA alkyltransferase.

O6-Alkylguanine-DNA alkyltransferase (AGT) repairs the mutagenic O6-methylguanine (O6-mG) lesion by transferring a methyl group from the 6-position of guanine to a cysteine residue on the protein. The simplest possible mechanism is an SN2 process in which the cysteine displaces the methyl group off of the guanine in a concerted reaction. To probe the interactions between the protein and guanine leaving group, oligodeoxynucleotide duplexes containing analogs of O6mG were synthesized and then reacted with AGT. The analogs, which were incorporated into deoxynucleotides include O6-methylhypoxanthine (O6-mH),S6-methyl-6-thioguanine (S6mG),S6-methyl-6-thiohypoxanthine (S6mH),Se6-methyl-6-selenoguanine (Se6mG),Se6-methyl-6-selenohypoxanthine (Se6mH), O6-methyl-1-deazaguanine (O6m1DG), O6-methyl-3-deazaguanine (O6m3DG), and O6-methyl-7-deazaguanine (O6m7DG), differ from O6mG in that the heteroatoms have been replaced so that they are poorer hydrogen bond participants and proton acceptors. AGT was reacted with oligonucleotide duplexes of the sequence 5'-GGC GCT XGA GGC GTG-3' in which X was O6mG or an analog in which X was paired with C. The reactions in 50 mM Tris-HCl and 1 mM EDTA, pH7.6 and 37 degrees C, were followed by anion-exchange HPLC in 10 mM NaOH with a NaCl gradient. All detected reactions were demethylations of the oligodeoxynucleotides except for O6m3DG, which reacted in an unknown manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗