Analysis of altered DNA structures: cruciform DNA.
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DNA, Cruciform↗
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Effect of anti-cruciform DNA monoclonal antibodies on DNA replication.
To study the possible involvement of DNA cruciforms in the initiation of DNA replication, we used two monoclonal antibodies, 2D3 and 4B4, with anti-cruciform DNA specificity. Synchronized CV-1 cells were released into S phase for hourly intervals up to 6 h and permeabilized in the presence of monoclonal antibodies, under conditions that allow limited DNA replication. Exposure of the permeabilized cells to 2D3 or 4B4 resulted in a 2- to 6-fold enhancement of incorporation of labeled precursor nucleotide over the 6 h period. Approximately 50% of the enhanced synthesis was sensitive to aphidicolin, and the enhancing effect of 2D3 was abolished by absorption with immunobead anti-mouse immunoglobulin. Dot-blot hybridization analyses of DNA isolated from anti-cruciform antibody treatment groups showed a similar 2- to 11-fold increase in the relative copy number of low copy probes. In contrast, exposure of the permeabilized cells to a monoclonal antibody directed against Z-DNA and B-DNA had no significant effect on DNA synthesis. The results suggest that cruciforms are present in replicating DNA and that they are recognized and stabilized by the monoclonal antibodies.
Animals↗
A novel type of interaction between cruciform DNA and a cruciform binding protein from HeLa cells.
We recently identified and enriched a protein (CBP) from HeLa cells with binding specificity for cruciform-containing DNA. We have now studied the interaction of CBP with stable cruciform DNA molecules containing the 27 bp palindrome of SV40 on one strand and an unrelated 26 bp palindrome on the other strand by hydroxyl radical footprinting. The CBP-DNA interaction is localized to the four-way junction at the base of the cruciforms. CBP appears to interact with the elbows of the junctions in an asymmetric fashion. Upon CBP binding, structural distortions were observed in the cruciform stems and in a DNA region adjacent to the junction. These features distinguish CBP from other cruciform binding proteins, which bind symmetrically and display exclusively either contacts with the DNA backbone or structural alterations in the DNA.
Base Sequence↗
Action of RecBCD enzyme on cruciform DNA.
We tested the hypothesis that RecBCD enzyme of Escherichia coli resolves pre-existing Holliday recombination intermediates by examining the action of the purified enzyme on an open-ended DNA cruciform with limited ability to branch migrate. The enzyme cleaved two strands of the cruciform near its base to produce "recombinant" products, with a marked bias in the direction of cleavage. The two nicks necessary to cleave the cruciform were made separately. Cruciforms whose four termini were blocked by synthetic hairpin-shaped oligonucleotides were not detectably nicked by the enzyme. With one terminus open the enzyme made a nick at the base of the cruciform but not a double-strand cut. With two or more termini open the enzyme made double-strand cuts. We infer that RecBCD enzyme molecules must enter the termini of duplex DNA and approach the cruciform from more than one direction in order to cleave it into recombinant products. Previous results on RecBCD-mediated recombination between phage lambda and lambda dv imply that intracellular RecBCD enzyme can approach pre-existing Holliday junctions from only one direction. We infer that intracellular RecBCD enzyme cannot cleave pre-existing Holliday junctions into recombinants and suggest that the enzyme may cleave Holliday junctions in whose formation it participates.
Base Sequence↗
Anti-cruciform monoclonal antibody and cruciform DNA interaction.
Cruciform DNA structure, as a structural feature, has been associated with regulation of transcription, recombination and replication. Previously used to successfully modify DNA replication and affinity-purify origins and autonomously replicating sequences. Using enzyme protection assays, their binding activity has been localized to the base (elbow) of the cruciform stem. We report here the hydroxyl radical footprinting of 2D3 (kappa IgG1) anti-cruciform monoclonal antibody on a stable cruciform structure created by heteroduplexing fragments from two plasmids, identical except for two centrally located palindromes of different sequence. The footprinting was performed at near-physiological salt concentrations, conditions favouring the stacked X-structure of the cruciform. Our data show that binding by the antibody occurs at the four-way junction (elbows) of the stable cruciform. The binding of the antibody seems also to cause associated structural distortions in the heteroduplex, which generally result in greater sensitivity to hydroxyl radicals at the tips of the cruciforms. The data are consistent to hydroxyl radicals at the tips of the cruciforms. The data are consistent with the binding of a single antibody to an antigen-combining site. The results of this study compare favourably with the hydroxyl radical footprinting studies reported recently for a human cruciform binding protein (CBP), which binds at the base of the stem-loop structure and causes similar distortions of the stable cruciform structure. These studies indicate that the four-way junction of the cruciform possesses certain unique structural qualities that are antigenic; the association of this structural determinant with DNA replication and the existence of a novel cellular protein, CBP, of similar binding specificity as the antibody specificity support a role for cruciforms as important regulatory recognition signals in replication.
Antibodies, Monoclonal↗
The ribosomal S16 protein of Escherichia coli displaying a DNA-nicking activity binds to cruciform DNA.
We have recently shown that the ribosomal S16 protein of Escherichia coli is a magnesium-dependent DNase which introduces nicks into supercoiled DNA molecules [Oberto, J., Bonnefoy, E., Mouray, E., Pellegrini, O., Wikstrom, P. M. & Rouvière-Yaniv, J. (1996) Mol. Microbiol. 19, 1319-1330]. In this work we analysed the DNA-binding and DNA-nicking properties of S16 using two different approaches. Gel-retardation assays showed that S16 is a structure-specific DNA-binding protein displaying a preferential binding for cruciform DNA structures. This specific binding to cruciform DNA was further investigated using a supercoiled plasmid carrying the origin of replication of E. coli (oriC) which is an (A+T)-rich DNA region with abundant palindromic sequences susceptible of forming cruciform-like structures in vivo. We show that the nicks introduced by S16 in oriC are not randomly positioned but are precisely localised near such palindromic sequences. In addition, the nicking activity of S16 appeared to be sequence dependent since the cuts introduced by S16 occurred next to an adenine, in most cases an unpaired adenine, usually followed by a GTT sequence. Overall these experiments indicate that S16 requires a cruciform-like DNA structure to bind DNA and the presence of a particular sequence in order to introduce specific single-stranded cuts into a DNA molecule.
Base Sequence↗
Structure and dynamics of supercoil-stabilized DNA cruciforms.
Understanding DNA function requires knowledge of the structure of local, sequence-dependent conformations that can be dramatically different from the B-form helix. One alternative DNA conformation is the cruciform, which has been shown to have a critical role in the initiation of DNA replication and the regulation of transcription in certain systems. In addition, cruciforms provide a model system for structural studies of Holliday junctions, intermediates in homologous DNA recombination. Cruciforms are not thermodynamically stable in linear DNA due to branch point migration, which makes their study using many biophysical techniques problematic. Atomic Force Microscopy (AFM) was applied to visualize cruciforms in negatively supercoiled plasmid DNA. Cruciforms are seen as clear-cut extrusions on the DNA filament with the lengths of the arms consistent with the size of the hairpins expected from a 106 bp inverted repeat. The cruciform exists in two different conformations, an extended one with the angle of ca. 180 degrees between the hairpin arms and a compact, X-type conformation, with acute angles between the hairpin arms and the main DNA strands. The ratio of molecules with the different conformations of cruciforms depends on ionic conditions. In the presence of high salt or Mg cations, a compact, X-type conformation is highly preferable. Remarkably, the X-conformation was highly mobile allowing the cruciform arms to adopt a parallel orientation. The structure observed is consistent with a model of the Holliday junction with a parallel orientation of the exchanging strands.
DNA, Superhelical↗
Diethyl pyrocarbonate: a chemical probe for DNA cruciforms.
Two palindromic DNA sequences were analyzed with respect to their chemical reactivities with diethyl pyrocarbonate. In negatively supercoiled plasmid templates enhanced N7 carbethoxylation was found with individual purines located in presumptive single-stranded loops of DNA cruciform structures. No enhanced reactivity at these positions was observed in linear, relaxed or low superhelical density plasmids. Hyperreactivity was found over a narrow region only, indicating that stable cruciforms contain loops of minimal size. No enhanced chemical reactivity was found with the four-way junction at the base of cruciforms. Diethyl pyrocarbonate has proved a sensitive structural probe for the analysis, with single nucleotide resolution, of DNA cruciform structures.
Base Sequence↗
14-3-3sigma is a cruciform DNA binding protein and associates in vivo with origins of DNA replication.
A human cruciform binding protein (CBP) was previously shown to bind to cruciform DNA in a structure-specific manner and be a member of the 14-3-3 protein family. CBP had been found to contain the 14-3-3 isoforms beta, gamma, epsilon, and zeta. Here, we show by Western blot analysis that the CBP-cruciform DNA complex eluted from band-shift polyacrylamide gels also contains the 14-3-3sigma isoform, which is present in HeLa cell nuclear extracts. An antibody specific for the 14-3-3sigma isoform was able to interfere with the formation of the CBP-cruciform DNA complex. The effect of the same anti-14-3-3sigma antibody in the in vitro replication of p186, a plasmid containing the minimal replication origin of the monkey origin ors8, was also analyzed. Pre-incubation of total HeLa cell extracts with this antibody decreased p186 in vitro replication to approximately 30% of control levels, while non-specific antibodies had no effect. 14-3-3sigma was found to associate in vivo with the monkey origins of DNA replication ors8 and ors12 in a cell cycle-dependent manner, as assayed by a chromatin immunoprecipitation (ChIP) assay that involved formaldehyde cross-linking, followed by immunoprecipitation with anti-14-3-3sigma antibody and quantitative PCR. The association of 14-3-3sigma with the replication origins was maximal at the G(1)/S phase. The results indicate that 14-3-3sigma is an origin binding protein involved in the regulation of DNA replication via cruciform DNA binding.
14-3-3 Proteins↗
Specific recognition of cruciform DNA by nuclear protein HMG1.
Cruciform DNA, a non-double helix form of DNA, can be generated as an intermediate in genetic recombination as well as from palindromic sequences under the effect of supercoiling. Eukaryotic cells are equipped with a DNA-binding protein that selectively recognizes cruciform DNA. Biochemical and immunological data showed that this protein is HMG1, an evolutionarily conserved, essential, and abundant component of the nucleus. The interaction with a ubiquitous protein points to a critical role for cruciform DNA conformations.
Animals↗
DNA-binding parameters of the HU protein of Escherichia coli to cruciform DNA.
We have previously studied the binding characteristics of the HU protein of Escherichia coli to different linear DNAs. In this work, using gel-retardation and footprint analysis, we studied the specific binding of HU protein to a synthetic cruciform DNA. We have quantified our results in order to precisely define the binding and cooperativity constants of HU protein towards cruciform DNA and compare them to those obtained with linear DNA. We used stringent high-salt conditions versus non-stringent low-salt conditions in order to differentiate the non-specific-protein HU-DNA complexes from the specific, high-salt-resistant complexes. We observe that HU-protein dimers bind specifically to the cruciform DNA with a binding constant K = 2.0 x 10(8) M-1 and a value for the cooperativity constant omega = 1 corresponding to a non-cooperative phenomenon. For the first time we observe a footprint pattern of HU protein bound to DNA using the hydroxyl-radical-footprinting technique on HU-protein-cruciform-DNA complexes. The residues protected by HU protein are localized at and near the junction point but interestingly they are mainly present in two of the four oligonucleotides which constitute the cruciform DNA. These two oligonucleotides are unpaired and opposite each other. These results support a model where two HU-protein dimers specifically bind to two equivalent angles present opposite each other in the four-way-junction-DNA structure with almost no dimer-dimer interactions.
Bacterial Proteins↗
Interaction of a protein from rat liver nuclei with cruciform DNA.
We constructed a synthetic cruciform DNA which closely resembles Holliday junctions, a DNA structure formed during recombination or following the transition from interstrand to intrastrand base pairing in palindromic DNA sequences. We identified and partially purified a protein from rat liver that specifically binds to this cruciform DNA molecule and does not bind to single-stranded or double-stranded DNAs of the same sequence. This protein also binds to the cruciform structure formed by a 70 bp palindromic sequence cloned in plasmid pUC18. No detectable nucleolytic activity is associated with the rat liver cruciform-binding protein, in contrast to all cruciform-recognizing proteins known so far.
Animals↗
Cruciform DNA binding protein in HeLa cell extracts.
We have analyzed by band-shift assays HeLa cell protein-DNA interactions on a stable cruciform DNA molecule. The stable cruciform was formed by heteroduplexing the HindIII-SphI fragment of SV40 virus DNA that contains the origin of replication with a derivative mutant containing a heterologous substitution at the central inverted repeat. We have identified a novel binding activity in HeLa cell extracts with specificity for the cruciform-containing DNA and no apparent sequence specificity. The activity is protein-dependent, void of detectable nuclease activity, and distinct from that reported for HMG1. A cruciform binding protein (CBP) with an apparent molecular weight of 66 kDa was enriched from HeLa cell extracts. In addition to the CBP, we have detected sequence-specific binding activities to sites proximal to the cruciform. Binding to one such site is increased in the cruciform-containing heteroduplex DNA by comparison to its linear homoduplex counterpart, suggesting transmission of structural effects by the stem-loops to their local environment.
Base Sequence↗
Multiple DNA secondary structures in perfect inverted repeat inserts in plasmids. Right-handed B-DNA, cruciforms, and left-handed Z-DNA.
The capabilities of five recombinant plasmids, containing relatively long (approximately 60-100 base pairs) perfect inverted repeat (IR) inserts, to support supercoil stabilized non-B-DNA structures were studied in vitro. The IRs were also alternating purine-pyrimidine sequences, thus, each could form either left-handed Z-DNA or cruciforms. Single-strand specific endonucleases, restriction endonucleases and methylases, and OsO4 modifications were used to characterize the DNA structures. Two-dimensional gel electrophoretic studies indicated that three of the five IRs formed both cruciforms and Z-DNA. (C-G) containing inserts preferred to form Z-DNA, whereas (T-G) sequences favored cruciforms. In vivo supercoil relaxation experiments demonstrated the existence of cruciforms in Escherichia coli. The physiological significance of these structures is discussed.
Base Sequence↗
Identification of amino acids of endonuclease VII essential for binding and cleavage of cruciform DNA.
Endonuclease VII is a Holliday-structure-resolving enzyme of bacteriophage T4. The active protein is a homodimer with 157 amino acids/monomer. An amber mutation (amE727 in codon 151) inactivates the nuclease completely, indicating the importance of the seven C-terminal amino acids for nucleolytic activity. The influence of these amino acids on cruciform-DNA binding and cleavage was investigated through functional analysis of C-terminal-truncated proteins derived from deletion constructs. It was found that the three C-terminal amino acids are not necessary for binding and cleavage. A transition from active to inactive protein occurs gradually with truncations of the next four amino acids. Reduction of DNA-binding ability, as measured by electrophoretic mobility shift assays, was determined to be the primary defect in the cleavage-deficient proteins. This was further concluded by the finding that EVII-(1-150)-peptide(amber), a protein with fairly low affinity to cruciform DNA, contributes cleavage activity to reactions of wild-type EVII with cruciform DNA. [Asp62]EVII-(1-156)-peptide lacking one C-terminal amino acid, contains a point mutation in codon 62 that eliminates the nucleolytic activity of the protein while retaining its DNA-binding proficiency. By mixing binding-deficient and cleavage-deficient mutants in the same assay, cleavage of cruciform DNA resumed. Evidence is presented that complementation occurs by heterodimer formation. Our results show that the zinc-binding motif of EVII is not sufficient for cruciform-DNA binding.
Amino Acids↗
Does cruciform DNA provide a recognition signal for DNA-topoisomerase II?
Topoisomerase II displays higher affinity for supercoiled DNA compared to the same relaxed DNA. Moreover, cruciform structures are formed in topologically constrained DNA. Here we report that, using S1 nuclease experiments on supercoiled DNA, hairpin structures are located close to numerous topoisomerase II cleavage sites on the BPV I genome. Therefore, DNA secondary structure may play a role in the recognition mechanism of DNA by topoisomerase II.
Base Sequence↗
Interarm interaction of DNA cruciform forming at a short inverted repeat sequence.
A novel interarm interaction of DNA cruciform forming at inverted repeat sequence was characterized using an S1 nuclease digestion, permanganate oxidation, and microscopic imaging. An inverted repeat consisting of 17 bp complementary sequences was isolated from the bluegill sunfish Lepomis macrochirus (Perciformes) and subcloned into the pUC19 plasmid, after which the supercoiled recombinant plasmid was subjected to enzymatic and chemical modification. In high salt conditions (200 mM NaCl, or 100-200 mM KCl), S1 nuclease cut supercoiled DNA at the center of palindromic symmetry, suggesting the formation of DNA cruciform. On the other hand, S1 nuclease in the presence of 150 mM NaCl or less cleaved mainly the 3'-half of the repeat, thereby forming an unusual structure in which the 3'-half of the inverted repeat, but not the 5'-half, was retained as an unpaired strand. Permanganate oxidation profiles also supported the presence of single-stranded part in the 3'-half of the inverted repeat in addition to the center of the symmetry. Both electron microscopy and atomic force microscopy have detected a thick protrusion on the supercoiled DNA harboring the inverted repeat. We hypothesize that the cruciform hairpins at conditions favoring triplex formation adopt a parallel side-by-side orientation of the arms allowing the interaction between them supposedly stabilized by hydrogen bonding of base triads.
Animals↗
Anti-cruciform DNA affinity purification of active mammalian origins of replication.
A novel approach that employs anti-cruciform DNA monoclonal antibodies was used to isolate segments of cruciform-containing DNA from genomic DNA, in an effort to obtain fragments containing active origins of replication. High molecular weight DNA (greater than 50 kb) was extracted from log phase CV-1 cells and 6 micrograms incubated with approximately 2.5 micrograms of a monoclonal antibody, 2D3, specific for cruciform-containing DNA. The 2D3-bound DNA was digested with EcoRI and antibody-bound fragments were recovered using rabbit anti-mouse immunobeads. The beads were washed free of nonspecifically bound DNA and the 2D3-bound DNA was eluted with 2% sodium dodecyl sulphate (SDS). The yield of DNA recovered by 2D3 was 2000-fold less than the initial amount and was 17-20-fold more than that recovered nonspecifically using the control mAb, P3. The 2D3-bound DNA ranged from 0.15- greater than 23 kb with a major peak at approximately 12 kb. Specific enrichment of origin-containing DNA by 2D3 over P3 was suggested by a 10-100-fold greater recovery of a 9 kb fragment hybridizable to a low-copy monkey autonomously replicating sequence, ors 8. 20 ng of affinity-purified DNA was cloned into lambda Zap II and excised into Bluescript phagemids in vivo. Of nine randomly-selected clones between 0.15 and 3.2 kb, four were able to replicate autonomously when transfected into HeLa cells. Two of the nine clones contained sequences hybridizable to both monkey alpha-satellite and human Alu DNA, and two others to Alu alone. The present work provides further evidence for the involvement of cruciforms at active mammalian origins of DNA replication.
Animals↗