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PubMed1995

Structural basis for DNA bending by the architectural transcription factor LEF-1.

Lymphoid enhancer-binding factor (LEF-1) and the closely related T-cell factor 1 (TCF-1) are sequence-specific and cell-type-specific DNA-binding proteins that play important regulatory roles in organogenesis and thymocyte differentiation. LEF-1 participates in regulation of the enhancer associated with the T cell receptor (TCR)-alpha gene by inducing a sharp bend in the DNA and facilitating interactions between Ets-1, PEBP2-alpha, and ATF/CREB, transcription factors bound at sites flanking the LEF-1 site. It seems that LEF-1 plays an architectural role in the assembly and function of this regulatory nucleoprotein complex. LEF-1 recognizes a specific nucleotide sequence through a high-mobility-group (HMG) domain. Proteins containing HMG domains bind DNA in the minor groove, bend the double helix, and recognize four-way junctions and other irregular DNA structures. Here we report the solution structure of a complex of the LEF-1 HMG domain and adjacent basic region with its cognate DNA. The structure reveals the HMG domain bound in the widened minor groove of a markedly distorted and bent double helix. The basic region binds across the narrowed major groove and contributes to DNA recognition.

Amino Acid Sequence↗
PubMedDate not supplied

[Defects in the secondary structure of DNA recognizable by nuclease S1 and their possible role in the aging of mammalian somatic cells].

The amount of defects in DNA secondary structure recognized by nuclease S1 and their localization by sequences of various nucleotide composition were studied in relation to the age factor. It was established that a significant twofold increase in defects of DNA secondary structure was observed in hepatic cells of very old (30 months and older) intact mice only, as well as in radiation-induced acceleration of aging at the age 19 months. The prevailing localization of the defects of DNA secondary structure has been demonstrated in those sequences which were enriched with AT pairs by 3% (as compared with the average level). It was concluded that the mentioned defects of DNA secondary structure are not the cause of aging. Nevertheless they can play an essential role in the process of irreversible destruction of genome in the terminal phase of aging.

Aging↗
PubMed1997

The acidic tail of the high mobility group protein HMG-D modulates the structural selectivity of DNA binding.

HMG-D is one of the Drosophila counterparts of the vertebrate HMG1/2 class of abundant chromosomal proteins and contains three domains: an HMG domain followed by a basic region and a short acidic carboxyterminal tail. We show that the HMG domain of HMG-D does not bind to deformed DNA structures such as DNA bulges, cis-platinated DNA or four-way junctions but does bind tightly to DNA microcircles, suggesting that in vivo the natural ligands of this domain are tightly bent DNA loops. The flanking basic region substantially increases the DNA-binding activity of the HMG domain to DNA ligands other than microcircles. We demonstrate that the acidic tail alters the structural selectivity of DNA binding by increasing the affinity for deformed DNA and decreasing the affinity for linear B-DNA. Finally, we show that the acidic tail increases the efficiency of constraining preformed negative supercoils but conversely decreases the efficiency of supercoiling relaxed DNA in the presence of topoisomerase I.

Amino Acid Sequence↗
PubMed1989

Sequence- and structure-dependent DNA base dynamics: synthesis, structure, and dynamics of site and sequence specifically spin-labeled DNA.

A nitroxide spin-labeled analogue of thymidine (1a), in which the methyl group is replaced by an acetylene-tethered nitroxide, was evaluated as a probe for structural and dynamics studies of sequence specifically spin-labeled DNA. Residue 1a was incorporated into synthetic deoxyoligonucleotides by using automated phosphite triester methods. 1H NMR, CD, and thermal denaturation studies indicate that 1a (T*) does not significantly alter the structure of 5'-d(CGCGAATT*CGCG) from that of the native dodecamer. EPR studies on monomer, single-stranded, and duplexed DNA show that 1a readily distinguishes environments of different rigidity. Comparison of the general line-shape features of the observed EPR spectra of several small duplexes (12-mer, 24-mer) with simulated EPR spectra assuming isotropic motion suggests that probe 1a monitors global tumbling of small duplexes. Increasing the length of the DNA oligomers results in significant deviation from isotropic motion, with line-shape features similar to those of calculated spectra of objects with isotropic rotational correlation times of 20-100 ns. EPR spectra of a spin-labeled GT mismatch and a T bulge in long DNAs are distinct from those of spin-labeled Watson-Crick paired DNAs, further demonstrating the value of EPR as a tool in the evaluation of local dynamic and structural features in macromolecules.

Base Sequence↗
PubMed2004

NMR structure of the DNA decamer duplex containing double T*G mismatches of cis-syn cyclobutane pyrimidine dimer: implications for DNA damage recognition by the XPC-hHR23B complex.

The cis-syn cyclobutane pyrimidine dimer (CPD) is a cytotoxic, mutagenic and carcinogenic DNA photoproduct and is repaired by the nucleotide excision repair (NER) pathway in mammalian cells. The XPC-hHR23B complex as the initiator of global genomic NER binds to sites of certain kinds of DNA damage. Although CPDs are rarely recognized by the XPC-hHR23B complex, the presence of mismatched bases opposite a CPD significantly increased the binding affinity of the XPC-hHR23B complex to the CPD. In order to decipher the properties of the DNA structures that determine the binding affinity for XPC-hHR23B to DNA, we carried out structural analyses of the various types of CPDs by NMR spectroscopy. The DNA duplex which contains a single 3' T*G wobble pair in a CPD (CPD/GA duplex) induces little conformational distortion. However, severe distortion of the helical conformation occurs when a CPD contains double T*G wobble pairs (CPD/GG duplex) even though the T residues of the CPD form stable hydrogen bonds with the opposite G residues. The helical bending angle of the CPD/GG duplex was larger than those of the CPD/GA duplex and properly matched CPD/AA duplex. The fluctuation of the backbone conformation and significant changes in the widths of the major and minor grooves at the double T*G wobble paired site were also observed in the CPD/GG duplex. These structural features were also found in a duplex that contains the (6-4) adduct, which is efficiently recognized by the XPC-hHR23B complex. Thus, we suggest that the unique structural features of the DNA double helix (that is, helical bending, flexible backbone conformation, and significant changes of the major and/or minor grooves) might be important factors in determining the binding affinity of the XPC-hHR23B complex to DNA.

Adenine↗
PubMed1995

Atomic model of a pyrimidine dimer excision repair enzyme complexed with a DNA substrate: structural basis for damaged DNA recognition.

T4 endonuclease V is a DNA repair enzyme from bacteriophage T4 that catalyzes the first reaction step of the pyrimidine dimer-specific base excision repair pathway. The crystal structure of this enzyme complexed with a duplex DNA substrate, containing a thymine dimer, has been determined at 2.75 A resolution. The atomic structure of the complex reveals the unique conformation of the DNA duplex, which exhibits a sharp kink with a 60 degree inclination at the central thymine dimer. The adenine base complementary to the 5' side of the thymine dimer is completely flipped out of the DNA duplex and trapped in a cavity on the protein surface. These structural features allow an understanding of the catalytic mechanism and implicate a general mechanism of how other repair enzymes recognize damaged DNA duplexes.

Adenine↗
PubMed2004

Interactive DNA sequence and structure design for DNA nanoapplications.

DNA sequence and structure design is very important for DNA nanoapplications. A computer-aided design tool is needed for exploring DNA sequence and structure of interests before experimental synthesis, which is a time- and labor-consuming process. In this paper, an interactive DNA sequence and structure design software tool called DNA shop is proposed and implemented. The visualization tool can generate DNA structures by specifying, selecting, and moving DNA sequences around and display corresponding structures. Using the tool, DNA sequence and structure can be visually inspected in three-dimensional space before experimental studies.

Algorithms↗
PubMedDate not supplied

[Physical mapping of Streptomyces coelicolor A3(2) actinophage DNA. II. Transposon-like structure in DNA molecules of phiC43 phage DNA, localization of region responsible for establishment of the lysogenic state].

Structural properties of DNA molecules of phages phi C43, phi C43del and mutant phage phi C311yg33 were studied. Actinophages phi C43del and phi C311yg33 have been isolated and shown to have a phenotype characteristic of phages defective in integration, i. e. turbid plaques and inability to establish the lysogenic state. By means of heteroduplexing, deletions were mapped in the genomes of these phages. DNA molecules of phi C43del and phi C11yg33 are devoid of the common fragment, which suggests that the mutant phenotypic character is associated with structural alterations in DNA molecules. The presence of transposon-like structure in phi C43 DNA molecules has been inferred from the analysis of phi C43/phi C43del heteroduplexes and phi C43 homoduplexes. Also, a deletion in phi C43 genome has been detected covering the same region where deletions in phi C43del and phi C11yg33 DNAs were located.

Bacteriophages↗
PubMed1998

Structural analysis of slipped-strand DNA (S-DNA) formed in (CTG)n. (CAG)n repeats from the myotonic dystrophy locus.

The mechanism of disease-associated trinucleotide repeat length variation may involve slippage of the triplet-containing strand at the replication fork, generating a slipped-strand DNA structure. We recently reported formation in vitro of slipped-strand DNA (S-DNA) structures when DNAs containing triplet repeat blocks of myotonic dystrophy or fragile X diseases were melted and allowed to reanneal to form duplexes. Here additional evidence is presented that is consistent with the existence of S-DNA structures. We demonstrate that S-DNA structures can form between two complementary strands containing equal numbers of repeats. In addition, we show that both the propensity for S-DNA formation and the structural complexity of S-DNAs formed increase with increasing repeat length. S-DNA structures were also analyzed by electron microscopy, confirming that the two strands are slipped out of register with respect to each other and confirming the structural polymorphism expected within long tracts of trinucleotide repeats. For (CTG)50.(CAG)50 two distinct populations of slipped structures have been identified: those involving </=10 repeats per slippage, which appear as bent/kinked DNA molecules, and those involving >10 repeats, which have multiple loops or hairpins indicative of complex alternative DNA secondary structures.

DNA↗
PubMed1994

A single 2'-hydroxyl group converts B-DNA to A-DNA. Crystal structure of the DNA-RNA chimeric decamer duplex d(CCGGC)r(G)d(CCGG) with a novel intermolecular G-C base-paired quadruplet.

We have found that the introduction of a single 2'-hydroxyl group on the sugar-phosphate backbone of the B-DNA decamer d(CCGGCGCCGG) transforms it to A-DNA. Thus, for the first time the X-ray structures of the same sequence have been observed in both the A and B-DNA conformations, permitting a comparison. Crystals of the DNA-RNA chimeric decamer d(CCGGC)r(G)d(CCGG) belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 25.63 A, b = 45.24 A and c = 47.99 A, and one decamer duplex in the asymmetric unit. The structure was solved by a rigid body search using the coordinates of the isomorphous structure d(CCCGGCCGGG) and refined to an R value of 0.136 using 2753 unique reflections at 1.9 A resolution. The final model contains 406 nucleotide atoms and 61 water molecules. The chimeric duplex exhibits typical A-DNA geometry, with all the sugars in the C(3')-endo puckering and the base-pairs inclined and displaced from the helix axis. The 2'-hydroxyl groups on rG6 and rG16 protrude into the minor groove surface and form different types of hydrogen bonds; that on strand 1 forms an intermolecular hydrogen bond with the furanose ring O(4') of a symmetry-related C1 residue, while that on strand 2 is involved in two water bridges. Crystal packing forces the G4-G17 base-pair in the top half of the duplex to slide significantly into the minor groove compared to the corresponding G7-G14 base-pair in the bottom half, resulting in these base-pairs exhibiting different base stacking and intermolecular interactions. The base G4 of the G4-G17 base-pair forms an unorthodox base "triple", G4*(G10-C11), hydrogen-bonding through its minor groove sites N(2) and N(3) to the minor groove atoms N(2) and O(2) of both bases of the G10-C11 base-pair of a symmetry-related molecule. The base G10 of this triple in turn forms a second similar unorthodox base triple, G10*(G3*C18), with the adjacent base-pair G3-C18 of the duplex, thus G10 is involved in a double triple. On the other hand, in the bottom half of the duplex, the C7-G14 base-pair is involved only in a single similar unorthodox base triple with G20, (C7-G14)*G20, while the adjacent base-pair rG6-C15 is involved in a novel quadruple with C1-G20, (rG6-C15) *(C1-G20), where the latter base-pairs are hydrogen-bonded to each other via the minor groove sites G(N(2))...C(O(2)).(ABSTRACT TRUNCATED AT 400 WORDS)

Base Composition↗
PubMed1987

Alteration of DNA tertiary structure by physical and chemical carcinogens: involvement in DNA repair processes.

Parameters defining the topological state of DNA seem extremely important for describing the reactive state of the same DNA molecules. We have shown that physical and chemical DNA modifying agents alter the tertiary structure of DNA molecules. Variations in the tertiary structure of DNA were studied by one dimensional electrophoresis on an agarose gel of partially relaxed plasmid DNA topoisomers, a technique allowing the measurement of alterations in the degree of supercoiling equivalent to fractions of superhelical turns. Unwinding angles of -10.1 degrees or -8.7 degrees per pyrimidine or thymine dimer respectively, of -12 degrees per apurinic site, and of -3.4 degrees per methylated purine were obtained by titrating the number of damaged sites necessary to reduce the number of superhelical turns by one in each topoisomer. On the contrary, enzymatic methylation of the C-5 position of cytosine (a modified base present in prokaryotic and eukaryotic DNAs) did not alter the DNA tertiary structure. We have also shown that local alterations in DNA structure caused by UV-irradiation inhibit bacterial DNA topoisomerase I and DNA methylase, and that the topological state of DNA substrate influences the mode of methylation of Hpa II DNA methylase. These findings suggest that the natural topological state of DNA substrate (linear, relaxed, or covalently closed duplex DNA with varying degrees of supercoiling) influences the mode of action of enzymes possibly involved in DNA repair processes, while DNA structural alterations caused by DNA modifying agents might influence DNA repair processes in two ways: either by driving the interaction between repair enzymes and the modified sites of DNA, or by inhibiting or changing the mode of action of enzymes normally acting on unmodified DNA.

Animals↗
PubMed1991

Effects of the antitumor antibiotic mithramycin on the structure of repetitive DNA regions adjacent to its GC-rich binding site.

Regions of An.Tn, (GA)n.(TC)n, and (GT)n.(AC)n have been cloned into the SmaI (CCC/GGG) site of plasmid pUC19. HindIII-EcoRI restriction fragments containing these inserts have been used as substrates for footprinting experiments using DNase I, DNase II, and micrococcal nuclease as probes. These present good mithramycin binding sites (GGG) flanking repetitive regions to which the drug does not bind. In each case, mithramycin footprints are observed at the CCC/GGG sites, which are not affected by the nature of the surrounding sequences. Some weaker binding is detected at TCGA and ACCA sites and at regions of alternating GA. No binding is found to regions of alternating GT. An.Tn inserts (n = 23 or 69) are normally resistant to cleavage by all these probes; in the presence of mithramycin, a dramatic increase in DNase I cleavage is observed throughout the entire insert and is indicative of an alteration in DNA structure. Similar changes are seen with DNase II and micrococcal nuclease. These changes cannot be explained by invoking changes in the ratio of free substrate to cleavage agent. In contrast, cleavage of (GA)n.(CT)n and (GT)n.(AC)n inserts is not affected by drug binding. The results are consistent with a model in which mithramycin causes dramatic changes in the width of the DNA minor groove, generating a structure which has some properties of A-DNA, and suggest that this can be propagated into surrounding DNA regions in a sequence-dependent manner. The structural alterations with An.Tn are highly cooperative and can be transmitted over at least three turns of the DNA helix.

Base Composition↗
PubMed2001

Structure of NaeI-DNA complex reveals dual-mode DNA recognition and complete dimer rearrangement.

NaeI, a novel DNA endonuclease, shows topoisomerase and recombinase activities when a Lys residue is substituted for Leu 43. The NaeI-DNA structure demonstrates that each of the two domains of NaeI recognizes one molecule of DNA duplex. DNA recognition induces dramatic rearrangements: narrowing the binding site of the Topo domain 16 A to grip DNA, widening that of the Endo domain 8 A to encircle and bend DNA 45 degrees for cleavage, and completely rebuilding the homodimer interface. The NaeI-DNA structure presents the first example of novel recognition of two copies of one DNA sequence by two different amino acid sequences and two different structural motifs in one polypeptide.

Amino Acid Motifs↗
PubMed2000

Solution structure of the MEF2A-DNA complex: structural basis for the modulation of DNA bending and specificity by MADS-box transcription factors.

The solution structure of the 33 kDa complex between the dimeric DNA-binding core domain of the transcription factor MEF2A (residues 1-85) and a 20mer DNA oligonucleotide comprising the consensus sequence CTA(A/T)(4)TAG has been solved by NMR. The protein comprises two domains: a MADS-box (residues 1-58) and a MEF2S domain (residues 59-73). Recognition and specificity are achieved by interactions between the MADS-box and both the major and minor grooves of the DNA. A number of critical differences in protein-DNA contacts observed in the MEF2A-DNA complex and the DNA complexes of the related MADS-box transcription factors SRF and MCM1 provide a molecular explanation for modulation of sequence specificity and extent of DNA bending ( approximately 15 versus approximately 70 degrees ). The structure of the MEF2S domain is entirely different from that of the equivalent SAM domain in SRF and MCM1, accounting for the absence of cross-reactivity with other proteins that interact with these transcription factors.

Amino Acid Sequence↗
PubMed1995

Targeting peptide nucleic acid-protein conjugates to structural features within duplex DNA.

A convenient small scale synthesis has been developed for obtaining peptide nucleic acid oligomers (PNAs). PNAs have been conjugated to a protein, staphylococcal nuclease, through disulfide exchange between a cysteine at the 3'-(carboxy) end of the PNA and an introduced cysteine on the surface of the nuclease. Site specific DNA cleavage by the attached nuclease has been used to examine the Watson-Crick hybridization of the PNAs to duplex DNA. Substantial affinity cleavage occurred when target sites contained inverted repeats which have the potential to form non B-DNA structures such as cruciforms. No affinity cleavage was observed at a site lacking apparent potential for non B-DNA structures. These results indicate that the Watson-Crick hybridization of PNAs to duplex DNA by strand displacement is favored by the presence of potential alternative secondary structures within the target sequence.

Base Sequence↗
PubMed1987

Mapping structurally perturbed sites in DNA by replication arrest and run-off replication.

We describe a technique for rapid fine mapping of sites of torsion-induced perturbations of DNA structure. The technique involves strand scission or chemical base modification at structurally perturbed sites, replication arrest in a double-strand DNA sequencing reaction, and size analysis of replication products by electrophoresis on sequencing gels. Besides being less complicated and faster than site identification by conventional end-labeling methods, the technique assures high sequence specificity through the use of oligomeric sequencing primers. This property should be useful for in vivo mapping of DNA structural perturbations with known sequence within complex genomes.

Acetaldehyde↗
PubMed2001

Activation and autoregulation of DNA-PK from structured single-stranded DNA and coding end hairpins.

DNA-dependent protein kinase (DNA-PK) acts through an essential relationship with DNA to participate in the regulation of multiple cellular processes. Yet the role of DNA as a cofactor in kinase activity remains to be completely elucidated. For example, although DNA-PK activity appears to be required for the resolution of hairpin coding ends in variable diversity joining recombination, kinase activity remains to be demonstrated from hairpin ends or other DNA structures. In the present study we report that DNA-PK is strongly activated from hairpin ends and structured single-stranded DNA, but that the phosphorylation of many heterologous substrates is blocked efficiently by inactivation of the kinase through autophosphorylation. However, substrates that bound efficiently to single-stranded DNA such as p53 and replication protein A were efficiently phosphorylated by DNA-PK from structured DNA. DNA-PK also was found to be active toward heterologous substrates from hairpin ends on double-stranded DNA under conditions where autophosphorylation was minimized. These results suggest that the role of DNA-PK in resolving coding end hairpins is likely to be enzymatic rather than structural, expand understanding of how DNA-PK binding to structured DNA relates to enzyme activity, and suggest a mechanism for autoregulatory control of its kinase activity in the cell.

Base Sequence↗
PubMed1983

Structure of T-DNA in roots transformed by Agrobacterium rhizogenes.

DNA isolated from hairy roots incited on Daucus carota by Agrobacterium strains harboring the Ri plasmid was probed with Ri plasmid fragments to identify plasmid sequences transferred and integrated into the plant genome. One hairy root line was incited by an A. rhizogenes natural isolate strain harboring two large plasmids in addition to the Ri plasmid. DNA from hairy roots incited by this strain contained a total of five nonidentical copies of T-DNA which together comprised a T-DNA complement representing a 34-42 kb segment of the Ri plasmid. A second hairy root line was incited by a Ti plasmidless A. tumefaciens strain into which the Ri plasmid had been conjugated. Hairy root DNA incited by this strain contained four identical T-DNA copies 17-18 kb in length and one truncated copy. T-DNA borders occurred at preferential sites of the Ri plasmid. T-DNA structure in these hairy root lines is similar to T-DNA structure in tumors incited by Ti plasmids of A. tumefaciens.

DNA↗