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At least 541 records · Page 30Linked to original sources
PubMed1996

A DNA structure is required for geminivirus replication origin function.

The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two single-stranded circular DNAs, A and B, that replicate through a rolling-circle mechanism in nuclei of infected plant cells. The TGMV origin of replication is located in a conserved 5' intergenic region and includes at least two functional elements: the origin recognition site of the essential viral replication protein, AL1, and a sequence motif with the potential to form a hairpin or cruciform structure. To address the role of the hairpin motif during TGMV replication, we constructed a series of B-component mutants that resolved sequence changes from structural alterations of the motif. Only those mutant B DNAs that retained the capacity to form the hairpin structure replicated to wild-type levels in tobacco protoplasts when the viral replication proteins were provided in trans from a plant expression cassette. In contrast, the same B DNAs replicated to significantly lower levels in transient assays that included replicating, wild-type TGMV A DNA. These data established that the hairpin structure is essential for TGMV replication, whereas its sequence affects the efficiency of replication. We also showed that TGMV AL1 functions as a site-specific endonuclease in vitro and mapped the cleavage site to the loop of the hairpin. In vitro cleavage analysis of two TGMV B mutants with different replication phenotypes indicated that there is a correlation between the two assays for origin activity. These results suggest that the in vivo replication results may reflect structural and sequence requirements for DNA cleavage during initiation of rolling-circle replication.

Base Sequence↗
PubMed1986

Stable stem-loop and cruciform DNA structures: isolation of mutants with rearrangements of the palindromic sequence at the simian virus 40 replication origin.

With the objective of generating DNA molecules that form stable stem-loop structures or cruciform structures in solution, we have altered the palindromic sequence at the Simian Virus 40 (SV40) replication origin. These alterations include: deletion of 18 of the 27 base pairs (bp) in the 13-bp inverted repeat; deletion of 26 of the 27 bp; substitution of the entire 27 bp with a totally different 26-bp sequence containing a 13-bp inverted repeat; and substitution of the 27 bp with an 8-bp sequence containing a 4-bp inverted repeat. The DNA from these mutants was purified. Mutant DNAs were hybridized to wild-type SV40 DNA or to each other, and the heteroduplexes were purified. The predicted structures were verified by S1 and restriction endonuclease digestion. The mutants - the heteroduplexes have not been tested - are capable of complementing tsA mutants and/or transforming mouse cells.

Autoradiography↗
PubMed1997

Models of DNA structure achieve almost perfect discrimination between normal prostate, benign prostatic hyperplasia (BPH), and adenocarcinoma and have a high potential for predicting BPH and prostate cancer.

In our previous studies of DNA, wavenumber-absorbance relationships of infrared spectra analyzed by principal components analysis (PCA) were expressed as points in space. Each point represented a highly discriminating measure of structural modifications that altered vibrational and rotational motion, thus changing the spatial orientation of the points. PCA/Fourier transform-infrared technology has now provided a virtually perfect separation of clusters of points representing DNA from normal prostate tissue, BPH, and adenocarcinoma. The findings suggest that the progression of normal prostate tissue to BPH and to prostate cancer involves structural alterations in DNA that are distinctly different. The hydroxyl radical is likely a major contributor to these structural alterations, which is consistent with previous studies of breast cancer. Models based on logistic regression of infrared spectral data were used to calculate the probability of a tissue being BPH or adenocarcinoma. The models had a sensitivity and specificity of 100% for classifying normal vs. cancer and normal vs. BPH, and close to 100% for BPH vs. cancer. Thus, the PCA/Fourier transform-infrared technology was shown to be a powerful means for discriminating between normal prostate tissue, BPH and prostate cancer and has considerable promise for risk prediction and clinical application.

Adenocarcinoma↗
PubMed1993

Genomic DNA structure of a gene encoding cytosolic ascorbate peroxidase from Arabidopsis thaliana.

A genomic DNA clone encoding cytosolic ascorbate peroxidase was isolated from a genomic library of Arabidopsis thaliana, using a cDNA for the enzyme as a probe. Nucleotide sequence and primer extension analyses of this gene (APX1) revealed nine exons split by eight introns, one of which is inserted in the 5'-untranslated region. The exon/intron organization of the APX1 gene differs from that of the guaiacol peroxidase genes.

Amino Acid Sequence↗
PubMed2001

Conformations of an adenine bulge in a DNA octamer and its influence on DNA structure from molecular dynamics simulations.

Molecular dynamics simulations have been applied to the DNA octamer d(GCGCA-GAAC). d(GTTCGCGC), which has an adenine bulge at the center to determine the pathway for interconversion between the stacked and extended forms. These forms are known to be important in the molecular recognition of bulges. From a total of ~35 ns of simulation time with the most recent CHARMM27 force field a variety of distinct conformations and subconformations are found. Stacked and fully looped-out forms are in excellent agreement with experimental data from NMR and x-ray crystallography. Furthermore, in a number of conformations the bulge base associates with the minor groove to varying degrees. Transitions between many of the conformations are observed in the simulations and used to propose a complete transition pathway between the stacked and fully extended conformations. The effect on the surrounding DNA sequence is investigated and biological implications of the accessible conformational space and the suggested transition pathway are discussed, in particular for the interaction of the MS2 replicase operator RNA with its coat protein.

Adenine↗
PubMed1991

Probing of DNA structure with osmium tetroxide,2,2'-bipyridine. Adduct-specific antibodies.

Antibodies against DNA modified with a single-strand selective probe, OsO4 in complex with 2,2'-bipyridine (Os,bipy), were raised in rabbits. These antibodies were fractionated using affinity column chromatography and fractions S89-II and S89-III characterized as highly specific for DNA-Os,bipy adduct with no cross reactivity to at least 1000-fold excess of unmodified DNA, RNA and Os,bipy-modified and unmodified proteins. Cross-reactivity to Os,bipy-modified RNA was very small. S89-II showed no cross-reactivity to DNA modified with OsO4 complexed with tetramethylethylenediamine or with bathophenanthroline disulphonic acid and to DNA oxidized with KMnO4. It cross-reacted, however, with DNA modified with OsO4,1,10-phenanthroline complex. The limit of detection of immunodot-blot analysis of extensively Os,bipy-modified DNA was below 0.5 pg. Small extent of Os,bipy-modification of supercoiled and linearized plasmids can be detected by DNA gel retardation and immunoblotting techniques. E. coli cells contain DNA regions in which bases are accessible to the single-strand selective probe.

2,2'-Dipyridyl↗
PubMed2006

DinI and RecX modulate RecA-DNA structures in Escherichia coli K-12.

RecA plays a central role in recombination, DNA repair and SOS induction through forming a RecA-DNA helical filament. Biochemical observations show that at low ratios to RecA, DinI and RecX stabilize and destabilize RecA-DNA filaments, respectively, and that the C-terminal 17 residues of RecA are important for RecX function. RecA-DNA filament formation was assayed in vivo using RecA-GFP foci formation in log-phase and UV-irradiated cells. In log-phase cells, dinI mutants have fewer foci than wild type and that recX mutants have more foci than wild type. A recADelta17::gfp mutant had more foci like a recX mutant. dinI recX double mutants have the same number of foci as dinI mutants alone, suggesting that dinI is epistatic to recX. After UV treatment, the dinI, recX and dinI recX mutants differed in their ability to form foci. All three mutants had fewer foci than wild type. The dinI mutant's foci persisted longer than wild-type foci. Roles of DinI and RecX after UV treatment differed from those during log-phase growth and may reflect the different DNA substrates, population of proteins or amounts during the SOS response. These experiments give new insight into the roles of these proteins.

Bacterial Proteins↗
PubMed1976

Effects of alkylation by dimethyl sulfate, nitrogen mustard, and mitomycin C on DNA structure as studied by the ethidium binding assay.

The extent of alkylation of DNA by dimethyl sulfate, nitrogen mustard, and the antibiotic mitomycin C is related to the resulting decrease in the fluorescence of intercalated ethidium. The fluorescence losses due to the first two types of reagents show a marked pH dependence, with greater losses of fluorescence being observed at alkaline pH values. At pH 11.6 the fluorescence shows a slow recovery, so that with low levels of methylation (4% deoxyguanosine residues modified) one observes complete return of fluorescence. We postulate that these phenomena are due to conversion of 7-methyldeoxyguanosine to the zwitterionic form, and partial denaturation of the DNA duplex with loss of ethidium binding sites. Hydroxide-ion-catalyzed imidazole ring opening, and the removal of the positive charge permits reannealing with concomitant return of the ethidium intercalation sites. This conclusion is substantiated by enzymatic hydrolysis of 14C-labelled methylated DNA and identifiions of the ethidium assay. The distinctly different behavior of mitomycin C confirms previous conclusions that its alkylation, preferentially on guanine, does not take part at the N-7 position.

Alkylating Agents↗
PubMedDate not supplied

Restriction fragment length polymorphism (RFLP) heterogeneity of HLA-DQ beta genes associated with DNA fragment identical to the DR1-beta DNA structure.

Restriction fragment length polymorphism (RFLP) analyses of DR1 positive peripheral blood leukocytes DNA was carried out. The Taq I digested DNA was hybridized with cDNA probes for HLA-DR and -DQ beta genes. The DR probe detected fragments commonly observed in the DR1 specificity, whereas a new DQ-beta fragment was detected in some DR1 haplotypes when the DQ-beta probe was used. This fragment had an RFLP pattern identical to the DQ-beta fragment typically associated with most DR2 and some DRw6 specificities.

Alleles↗
PubMed1985

[DNA structure in the particles of 5 bacteriophages from the data of circular dichroism].

DNA optical activities in situ were studied in the particles of five medium sized bacteriophages (SB1, F15; IRA, SD and T7). Delta epsilon in the CD spectrum of intraphage DNA is not shown to correlate with the sizes of phage heads or with the light scattering characteristics of phage suspension. Bacteriophage SB1, studied for the first time, has the amplitude of CD spectrum in the 260-300 nm region higher, than the CD spectrum of its free phage DNA. CD magnitude in the 260-300 nm region is different for varying phages while the red shift of the positive band in the CD spectrum takes place for all phages studied. The dense packing of DNA is suggested to be a common factor for the red shift being observed for all phages. The different delta epsilon in the 260-300 nm region might reflect the different nature in changes of helical DNA geometry inside phage particles as compared with the changes in solutions. The increase in melting temperatures for intraphage DNA as compared with the temperature for free DNA was not shown to correlate with the CD spectrum difference of intraphage DNAs. This property of intraphage DNAs is supposed to be connected with the dense packing of DNA in bacteriophage deoxyribonucleoprotein.

Circular Dichroism↗
PubMed1990

Effect of monovalent cation-induced telomeric DNA structure on the binding of Oxytricha telomeric protein.

Oligonucleotides bearing 4 repeats of telomeric deoxyguanosine-rich sequence undergo a monovalent cation-induced transition to a folded conformation with G-G base pairs, modeled as a 'G-quartet' structure. We have now deduced the rates of folding and unfolding of d(TTTTGGGG)4, which has four repeats of the Oxytricha telomeric DNA sequence. The estimated average values of delta G for the folded form at 37 degrees C are -2.2 kcal/mol and -4.7 kcal/mol in 50 mM na+ and K+, respectively. The fully folded DNA is not recognized by the Oxytricha telomere-binding protein; the substrate for protein binding has properties consistent with its being partly or fully unfolded. In confirmation of this conclusion, prevention of DNA folding by methylation enables the protein to bind as rapidly in the presence of monovalent cations as in their absence. The slow unfolding (t1/2 = 4 hr and 18 hr at 37 degrees C in Na+ and K+, respectively) of the DNA suggests that such structures would be long-lived if they formed in vivo, unless they can be actively unfolded. The inability of the telomere-binding protein to bind the stable, folded form of the 4-repeat telomeric sequence is a problem that may be circumvented in vivo by avoiding four single-stranded repeats.

Base Composition↗
PubMed2003

X-ray crystallographic study of the possible binding sites of the monovalent cations in the Z-DNA structure.

Determination of the binding positions of light metal ions in the structure of oligonucleotides remains a challenge owing to their irregular coordination geometries, low electron densities and mobility in the crystals. To study the interactions between monovalent cations and DNA, the heavier alkali metal ion Rb(+) was used to replace Na(+) and K(+) in the Z-DNA crystal and the structure was analyzed using X-ray cryocrystallography. The resolution of the Rb-d(CGCGCG)(2) crystal data set is 1.76 A, with an R(merge) of 0.061. The final residual factor (R factor) for the crystal structure is 0.175. The positions of 16 Rb(+) ions were tentatively assigned, with most of them having partial occupancies. Three rubidium ions with full occupancies coordinated to two DNA double strands were located in the Z-DNA crystal. The results of this work suggest that there are numerous relatively low-energy sites for binding of monovalent cations to Z-DNA and that these sites are cation-water hybrid positions.

Binding Sites↗
PubMed1988

Promoter recognition by Escherichia coli RNA polymerase. Influence of DNA structure in the spacer separating the -10 and -35 regions.

Escherichia coli RNA polymerase contacts promoter DNA at two regions (the -10 and -35 regions) which are separated by a segment of spacer DNA. Previously we showed that base substitutions in the spacer DNA can affect promoter strength both in vitro and in vivo; these results were interpreted to reflect altered structural properties of the substituted DNAs. Here we provide experimental support for this interpretation. The pattern of cleavage of the promoters with Neurospora crassa endonuclease and the reactivity of their guanine residues with dimethyl sulfate (DMS) suggest that the structures of the spacer DNAs in the promoters with altered transcriptional activities are distinct. In addition, the binding of RNA polymerase to the latter promoters induces characteristic enhancements in the extent to which specific guanine residues in the spacer DNAs react with DMS. We propose that for these promoters the substitutions in the spacer DNAs have affected the relative orientation of the -10 and -35 regions. The observed differences in promoter activity then would reflect the requirement for realignment of these regions during the process of open complex formation; we postulate that two such realignments occur.

Autoradiography↗