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PubMed1997

D-2-deoxyribose and D-arabinose, but not D-ribose, stabilize the cytosine tetrad (i-DNA) structure.

Described here are studies exploring the effect of the sugar-phosphate backbone on the stability of i-tetrads in solution [K. Gehring et al. Nature 363, 561-565 (1993)]. In the accompanying paper, branched oligonucleotides are shown to be effective probes for organizing oligodeoxycytidine strands into I-motif structures (C-tetrads). Specifically, the joining of a pair of parallel deoxycytidylate strands with a riboadenosine "linker" leads to marked enhancement in stability of the tetrad structure. To further characterize the nature of the sugar-sugar interactions in this novel structure, branched oligonucleotides containing D-arabinocytidine and D-ribocytidine were synthesized and their association properties examined. The ribo oligomers were prepared in two regioisomeric forms differing only in the connectivities of the deoxycytidine strands, i.e., 3'-to-5' versus 2'-to-5' linked dC5 strands. The branched D-deoxycytidine analogue, rA(2',5'-dC5)3',5'-dC5, which previously has been shown to fold into a bimolecular I-motif, served as model system. It is found that the arabinose substitution leads to hypochromic structures that are characteristic of four-stranded intercalated DNA and has little, if any, effect on the stability of the complex formed. Parallel experiments with the branched ribocytidine analogs gave very weak or no discernible UV transitions, consistent with no strand association in this case [Lacroix et al., Biochemistry 35, 8715-8722 (1996)]. These results are discussed in relation to expected steric interactions of oligocytidine strands within the I-structure. The findings increase our understanding of the impact of the sugar and internucleotide connectivity on the stability of this higher-order nucleic acid structure.

Arabinose↗
PubMed1985

Cleavage of stem-and-loop structure DNA by bleomycin. Reaction on the bacteriophage G4 origin of complementary strand synthesis.

The cleavage by bleomycin-Fe(II) complex in the presence of dithiothreitol of 3'-or 5'-end-labeled DNA from the region of the bacteriophage G4 origin of complementary strand synthesis was investigated by using the DNA-sequencing technique. Bleomycin cleaved a single-stranded DNA substrate preferentially at inverted repeat sequences, which potentially form stem-and-loop structures, while it cleaved double-stranded DNA substrates with different specificity. The results support the formation of three adjoining stem-and-loop structures in the region of the phage G4 origin of complementary strand synthesis under the low-salt conditions used and suggest a difference in the form of the double helix between the stem and the double-stranded DNA fragment. Bleomycin appears to be a useful reagent for searching stem-and-loop structures. The results may also contribute to the understanding of the mode of action of bleomycin as an antitumor antibiotic.

Bacteriophages↗
PubMed1994

Secondary structure and interaction of phage D108 Ner repressor with a 61-base-pair operator: evidence for altered protein and DNA structures in the complex.

Ner repressors of the transposable phages Mu and D108 play a central role in regulating the expression of the early (transposase) operon and in ensuring that phage growth proceeds along a lytic pathway. The latter function is analogous to that performed by the Cro protein of phage lambda. Unlike lambda Cro, however, the structural basis of operator recognition is not known for the Ner repressors. In order to elucidate the structural features underlying operator recognition by Ner repressors, we have employed Raman spectroscopy as a probe of the solution secondary structures of both D108 Ner and Mu Ner. Additionally, we have obtained Raman spectra of the D108 Ner repressor when bound to a 61-base-pair oligodeoxynucleotide containing the 55-base-pair D108 ner binding site. Conformation-sensitive Raman bands show that both D108 and Mu Ner contain similar, highly alpha-helical (approximately 45%) secondary structures. The Raman markers also show that the substantial nonhelical secondary structure of both D108 Ner and Mu Ner is largely beta-stranded. The protein-free 61-bp D108 ner operator exhibits Raman marker bands diagnostic of an uninterrupted B DNA duplex. In the D108 Ner:DNA complex, we find the following: (i) B DNA stereochemistry is fully conserved, although with significant perturbations to the B form backbone geometry, particularly in AT-rich regions of the bound operator. (ii) The specific interactions that occur between Ner repressor and operator involve B DNA major groove sites. (iii) A small (8 +/- 3%) increase in alpha-helix content of the Ner repressor is detected upon operator binding. (iv) Finally, the local environments of many aromatic amino acids are substantially altered in the D108 Ner:DNA complex. We propose a molecular model for binding of D108 Ner to its operator that is consistent with both the present spectroscopic findings and the results of recent biochemical studies. Essential features of this model are bending of the DNA double helix and contact of operator sites with repressor domains bearing sequence homologies with the helix-turn-helix (HTH) motifs of other DNA-binding proteins. The Raman fingerprint of the Ner:DNA complex is shown to be clearly distinguishable from that of the lambda cI:DNA complex, even though both gene regulatory complexes are presumed to employ HTH recognition motifs. The unique Raman signatures observed for these repressor complexes suggest that the Raman methodology may be useful in discriminating different modes of operator recognition by the HTH motifs of regulatory proteins.

Amides↗
PubMed1992

A reduced set of coordinates for modeling DNA structures: (I). A B-to-A transition pathway driven by pseudorotational angle.

The A-DNA and the B-DNA are two well characterized polymorphous forms of DNA duplex. By using Metropolis Monte Carlo Simulations in a reduced coordinate space, we have shown that the B in equilibrium with A transitions can be induced by forcing pseudorotational angle (W) to change between C3'-endo and C2'-endo puckerings. The energy barrier for the transition pathway is less than 10 Kcal.mol-1. Base-pair parameters x-displacement (Dx) and roll (rho), which have the largest differences between the two forms of structures, cannot drive the transition. Our results support the view that the bistable states of the DNA duplex are due to the bistable structures of the sugar ring.

Computer Simulation↗
PubMed1999

An A-DNA structure with two independent duplexes in the asymmetric unit.

The crystal and molecular structure of the self-complementary A-DNA decamer sequence d(G4CGC4) was solved at 1.9 A resolution. The decamer crystallizes in space group P21 with two independent duplexes in the asymmetric unit. Duplex 1 has interactions which are distributed symmetrically about its length compared with duplex 2. The two end base pairs of duplex 1 have a similar NH.O hydrogen-bond pattern involving GGC segments of duplex 2 and a symmetry-related neighbour, while the end base pairs of duplex 2 interact with the GCC and GGG segments of its symmetry-related neighbours through NH.O and NH.N hydrogen bonds and a water-mediated hydrogen bond between the carboxyl groups of C40 and C8. In addition to the C4'-C5' torsion angle gamma assuming the trans conformation in certain steps, this angle also adopts the gauche- conformation at C37 as opposed to the preferred gauche+ conformation, with a concomitant change in phosphodiester P-O5' (alpha) in the opposite sense. This facilitates stacking between adjacent bases. The study suggests that the structural alterations in the two molecules in the asymmetric unit originate from an inherent propensity of the d(G4CGC4) base sequence for varied intermolecular interactions and malleability.

Base Sequence↗
PubMed1980

Molecular cloning of avian sarcoma virus closed circular DNA: structural and biological characterization of three recombinant clones.

Unintegrated, circular viral DNA, isolated from Prague A avian sarcoma virus (PrA-ASV)-infected quail cells (QT6), was cloned in the lambda vector lambda gtWES x lambda B. Three independent lambda-ASV recombinants were identified, and each contained a complete copy of the PrA-ASV genome. The arrangement of the ASV sequences within the recombinants was determined by restriction enzyme analysis and hybridization with labeled ASV-specific complementary DNA. One of the recombinants (lambda RPA101) resulted from cloning at the EcoRI site located within the terminally repeated sequence and therefore was virtually co-linear with PrA-ASV virion RNA. The other two recombinants (lambda RPA102 and 103) resulted from cloning at the EcoRI site located within the viral env gene. By restriction enzyme analysis and by measurement of R-loops formed between lambda RPA101 and PrA-ASV virion 35S RNA, the viral genome was estimated to be 9,100 bases in length. Genome length viral DNA purified from clones lambda RPA102 and 103 was biologically active. Transfection of chicken embryo cells with viral DNA, in the form of either circles or linear dimers, produced foci of transformed cells within 8 to 10 days. Linear DNA was much less efficient at inducing transformation. Viral DNA from the clone lambda RPA101 was unable to cause transformation; the basis for this defect is unknown.

Alpharetrovirus↗
PubMed1990

A gene for cytochrome c oxidase subunit III (COXIII) in broad bean mitochondrial DNA: structural features and sequence evolution.

A nucleotide sequence of broad bean mitochondrial DNA (mtDNA) that contains the coxIII gene is presented, and compared to corresponding sequences of Oenothera and corn mtDNAs. Upstream from the broad bean coxIII gene are three potential secondary structures: a single stem and loop (hairpin) that is conserved in the Oenothera and corn sequences; a second single stem and loop; and a double stem and loop. The rate of evolution of the coxIII gene has been slower in plants than in mammals. Constraints on the fixation of at least some kinds of mutations in silent (synonymous) third position nucleotides, as well as of mutations that cause amino acid replacements, seem to have contributed to this slower rate.

Amino Acid Sequence↗
PubMed1982

Restriction endonuclease and nucleotide sequence analyses of molecularly cloned unintegrated avian tumor virus DNA: structure of large terminal repeats in circle junctions.

Avian tumor virus supercoiled DNA was isolated from infected quail tumor cells and molecularly cloned in pBR322. Four different recombinant clones denoted pATV-6, pATV-7, pATV-8, and pATV-9 were characterized in detail by restriction endonuclease mapping and by DNA sequencing. The results of these studies indicate that (i) the two large terminal repeats (LTRs) present in PATV-6, are different sizes, (ii) pATV-8 and pATV-9 contain only one LTR, (iii) pATV-7 contains an inversion of 0.6 kilobase in the env gene and a deletion of the U3 region and the src gene, and (iv) the src gene is deleted in pATV-6 and pATV-9. Circle formation from linear molecules was also examined in several of the clones by DNA sequencing through the circle joint. pATV-6 is an example of one class of circular molecules and contains a partially repeated LTR similar to that reported by Ju and Skalka (Cell 22:379-386, 1980). A second class of circles was exemplified by pATV-8 and pATV-9, which contain a single copy of the LTR with no base changes or deletions. This is in contrast to a class of circles containing a complete double LTR structure described by Swanstrom et al. (Proc. Natl. Acad. Sci. U.S.A. 78:124-128, 1981) and suggests that circles containing a single intact LTR may be formed by a homologous recombinational event in which an entire LTR or complementary regions from both LTRs are removed from the linear DNA molecule during circularization.

Avian Sarcoma Viruses↗
PubMed1982

Higher order DNA structure in macronuclear chromatin of the hypotrichous ciliate Oxytricha nova.

On lysis of macronuclei from the ciliated protozoan Oxytricha at 0.5-2 M NaCl, the DNA, which is normally found as discrete molecules ranging from 0.5 to 20 kilobases, appears in high molecular weight aggregates. Various treatments of the macronuclear lysate (i.e., nucleases, proteases, variation of salt, pH, and temperature) indicate that preservation of the aggregate structure depends on both nucleic acid-nucleic acid and nucleic acid-protein interactions. Purification of the DNA-protein complex after lysing the nuclei in 2 M NaCl shows that one major nuclear protein copurifies with the DNA. As shown by DNA-protein binding experiments, this protein has a high affinity for DNA; however, no evidence for sequence specificity of the protein binding was obtained. Chromatin reconstitution experiments suggest that the protein in itself is not sufficient for DNA aggregation in nuclei, but other factors, possibly the native chromatin structure, are required. Electron microscopy of the purified DNA-protein complex showed structures similar to those observed previously with in vitro-aggregated purified macronuclear DNA (14). A model is presented in which the terminal inverted repeat sequences found on all macronuclear DNA molecules interact with each other forming multistranded DNA complexes. The formation of these structures may be accelerated and stabilized by a protein in vivo.

Animals↗
PubMed1998

DNA structure and flexibility in the sequence-specific binding of papillomavirus E2 proteins.

The papillomavirus E2 proteins are transcriptional regulators that bind to a consensus DNA sequence ACCG NNNN CGGT. Multiple copies of this binding site are found in the viral genomes. The affinities of the naturally occurring binding sites for the E2 proteins are predominantly dependent upon the sequence of the NNNN spacer. The hierarchies of binding site affinities among the sites present in the viral genomes result in differential occupancy during the viral life-cycle. In turn, this differential binding regulates transcription from viral promoters, including those for the oncogenes E6 and E7. Structural and biochemical studies have shown that E2 proteins bend the DNA to which they specifically bind. Atomic resolution structures of complexes of the bovine papillomavirus strain 1 (BPV-1) E2 protein and DNA show that the protein does not contact the spacer DNA. A direct comparison of the binding of the DNA-binding domains of the E2 proteins from BPV-1 and human papillomavirus strain 16 (HPV-16) to a series of binding sites as a function of the sequence of their central spacer and/or the presence of a nick or gap in one strand of the spacer DNA is presented in this paper. The BPV-1 E2 DNA-binding domain is only moderately sensitive to the nature of the central spacer; less than several fold differences in affinity were observed when the DNA sequence of the spacer was varied and/or a nick or gap was introduced. In contrast, the HPV-16 E2 DNA-binding domain binds to sites containing A:T-rich central spacers with significantly increased affinity. The introduction of a nick or gap into the spacer of these high affinity sequences is very detrimental to HPV-16 E2 binding while comparable nicks or gaps have only small effects in the low affinity sequences. These results suggest that the HPV-16 E2 protein recognizes the structure of the DNA spacer and that the mechanism of DNA-sequence specific binding of the homologous HPV-16 E2 and BPV-1 E2 proteins is significantly different.

Animals↗
PubMed1995

Poly(dA:dT), a ubiquitous promoter element that stimulates transcription via its intrinsic DNA structure.

Many yeast promoters contain homopolymeric dA:dT sequences that affect nucleosome formation in vitro and are required for wild-type levels of transcription in vivo. Here, we show that poly(dA:dT) is a novel promoter element whose function depends on its intrinsic structure, not its interaction with sequence-specific, DNA-binding proteins. First, poly(dA:dT) stimulates Gcn4-activated transcription in a manner that is length dependent and inversely related to intracellular Gcn4 levels. Second, Datin, the only known poly(dA:dT)-binding protein, behaves as a repressor through poly(dA:dT) sequences. Third, poly(dG:dC), a structurally dissimilar homopolymer that also affects nucleosomes, has transcriptional properties virtually identical to those of poly(dA:dT). Three probes of chromatin structure including HinfI endonuclease cleavage in vivo indicate that poly(dA:dT) increases accessibility of the Gcn4 binding site and adjacent sequences in physiological chromatin. These observations suggest that, by virtue of its intrinsic structure, poly(dA:dT) locally affects nucleosomes and increases the accessibility of transcription factors bound to nearby sequences.

Base Sequence↗
PubMed1998

The mouse Lect2 gene: cloning of cDNA and genomic DNA, structural characterization and chromosomal localization.

We previously purified bovine leukocyte cell-derived chemotaxin 2 (LECT2) as a 16 kDa-secreted protein with a neutrophil chemotactic activity. LECT2 protein is thought to be multifunctional, since it was recently found to be identical to chondromodulin-II, a growth stimulator of chondrocyte cells. We report here the cloning and structural analysis of mouse Lect2 cDNAs and genomic DNA, and chromosomal mapping. Two types of mouse Lect2 cDNAs were cloned: one encoded the mouse counterpart of human and bovine LECT2 proteins, and the other encoded a queer type LECT2 protein whose amino-acid sequence in the carboxy terminus was different from that of the normal type LECT2 protein. The mouse Lect2 gene spanned approx. 8 kb and consisted of five exons and four introns. The genomic organization revealed that two type transcripts arose by an alternative splicing event involving exon 4. A primer extension analysis revealed that several transcription initiation sites occurred within 60-210 nucleotides upstream from the translation initiation codon. The mouse Lect2 gene was mapped to a region adjacent to D13Mit13, D13Mit21 and Il-9 on chromosome 13 by interspecific backcross mapping.

Amino Acid Sequence↗
PubMed1997

Sequence-dependent variations of DNA structure modulate radiation-induced strand breakage.

Using a 80 base pair DNA fragment, the sequence-dependence was compared for: (i) the probability of fast neutrons induced strand breakage, (ii) the accessibility of the H4'- and H5'-atoms to OH. attack, (iii) the width of the minor groove, and (iv) the probability of OH. reactions with H4'- or H5'-atoms. The probability of strand breakage was measured using sequencing gel electrophoresis. The accessibility and the probability of reaction were calculated for the energy-minimized modelled DNA fragment. A Monte-Carlo simulation was used for calculating the probabilities of H-atom abstraction by OH.. It was observed that reduced breakage occurs in sequences exhibiting low accessibility of H4' and H5'2 and low probability of H-atom abstraction by OH., due to a narrow, minor groove. This shows that the breakage probability at a given nucleotide site is not determined by the chemical nature of the nucleotide (A, T, G or C), but mainly by the local sequence-modulated intrinsic structure. Fitting the experimental results with the calculated probabilities of reaction suggests that a C4'-centered radical evolves towards a strand break three times more efficiently than the C5' one, and that half of the breaks occur via the 4'-path and half via the 5'-path.

Base Sequence↗
PubMed1991

Inhibition of telomerase by G-quartet DNA structures.

The ends or telomeres of the linear chromosomes of eukaryotes are composed of tandem repeats of short DNA sequences, one strand being rich in guanine (G strand) and the complementary strand in cytosine. Telomere synthesis involves the addition of telomeric repeats to the G strand by telomere terminal transferase (telomerase). Telomeric G-strand DNAs from a variety of organisms adopt compact structures, the most stable of which is explained by the formation of G-quartets. Here we investigate the capacity of the different folded forms of telomeric DNA to serve as primers for the Oxytricha nova telomerase in vitro. Formation of the K(+)-stabilized G-quartet structure in a primer inhibits its use by telomerase. Furthermore, the octanucleotide T4G4, which does not fold, is a better primer than (T4G4)2, which can form a foldback structure. We conclude that telomerase does not require any folding of its DNA primer. Folding of telomeric DNA into G-quartet structures seems to influence the extent of telomere elongation in vitro and might therefore act as a negative regulator of elongation in vivo.

Animals↗
PubMed1999

Three dimensional DNA structures in computing.

We show that 3-dimensional graph structures can be used for solving computational problems with DNA molecules. Vertex building blocks consisting of k-armed (k = 3 or 4) branched junction molecules are used to form graphs. We present procedures for the 3-SAT and 3-vertex-colorability problems. Construction of one graph structure (in many copies) is sufficient to determine the solution to the problem. In our proposed procedure for 3-SAT, the number of steps required is equal to the number of variables in the formula. For the 3-vertex-colorability problem, the procedure requires a constant number of steps regardless of the size of the graph.

Animals↗
PubMed2004

Influence of DNA structure on the reactivity of the guanine radical cation.

Oxidative damage of DNA via radical cation formation is a common cause of mutagenesis, cancer and of the physiological changes associated with aging. By using state-of-the-art ab initio molecular dynamics simulations, we study the mechanism that guides the first steps of this process. In the mechanism proposed here, guanine, which among the bases has the lowest oxidation potential, and the phosphate backbone play a crucial role. We found that the rate limiting step is the water protolysis. We illuminate the role of the local environment in considerably lowering the barrier. Of particular relevance in this respect is the role of the phosphate backbone.

Cytosine↗
PubMed1983

The reconstitution of higher-order DNA structure after X-irradiation of mammalian cells.

X-ray-induced DNA repair in mouse leukemia (L1210) cells was studied by alkaline elution, which measures the amount of DNA strand breakage, coupled with nucleoid sedimentation, which measures DNA compactness. Two phases of X-ray repair were detected. An initial phase was rapid (t1/2 less than 10 min). During this phase most strand breaks were rejoined and some compaction occurred. After a lag of 1-2 hours, a second phase occurred which exhibited very little or no additional ligation but further compaction of the nucleoid DNA. Both the DNA strand rejoining and initial nucleoid compaction of the first phase were inhibited by 3-ABA2 but not by novobiocin, and the second phase was inhibited by novobiocin but not by 3-ABA. The two phases of reconstitution of nucleoid compactness following X-irradiation are thus distinguishable by their time of occurrence and by their sensitivity to inhibitors of DNA-related enzymes. A coordinated process of ligation followed by compaction may be intrinsic to DNA repair following X-irradiation.

Animals↗