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PubMed1989

Transcription regulation in vitro by an E. coli promoter containing a DNA cruciform in the '-35' region.

A promoter with the potential to adopt a 50 basepair (bp) cruciform spanning from -19 to -69 has been constructed in the plasmid pBR322 tetracycline resistance gene (tet) by forming an inverted repeat from '-35' sequences. Compared to a control promoter, the sequence of this cruciform promoter differs only by a 22 bp insertion between -48 and -69, upstream from the usual location of promoter sequences. The cruciform is extruded in a supercoil-dependent manner, and transcription from this promoter in vitro by RNA polymerase decreases as the negative supercoil density of the plasmid DNA increases. In contrast, transcription from the control promoter increases with negative supercoiling. Thus, DNA secondary structure in the '-35' region can affect promoter-polymerase interaction. The tet promoter cruciform also influences expression of the pBR322 beta-lactamase gene (bla). This apparently results when extrusion of the cruciform reduces the superhelicity of the plasmid molecule to a level that is below the optimum for expression from the bla promoter, illustrating one mechanism for how DNA secondary structure may effect action-at-a-distance. Transcription from both promoters in vivo does not differ from controls, suggesting that this cruciform is not generated to a significant extent intracellularly, most probably as a result of the slow kinetics of extrusion.

Base Composition↗
PubMed1990

The interaction of adenosine diphosphoribosyl transferase (ADPRT) with a cruciform DNA.

Adenosine Diphosphoribosyl Transferase is a eucaryotic nuclear protein that catalyses the transfer of ADP-ribose moiety of NAD+ to itself and other cellular proteins. DNA is required for this post-translational modification process. We present novel evidence that Adenosine Diphosphoribosyl Transferase interacts with the base of plasmid pUC7 cruciform. We speculate that, unlike a sequence-specific DNA binding protein, the transferase may recognize an unusual DNA conformation.

Animals↗
PubMed1992

T4 endonuclease VII resolves cruciform DNA with nick and counter-nick and its activity is directed by local nucleotide sequence.

Endonuclease VII of phage T4 resolves Holliday structures in vitro by nicking pairs of strands across the junction. We report here analyses of this reaction between endonuclease VII and a Holliday structure analogue, made in vitro from synthetic oligonucleotides. The enzyme cleaves the structure in a non-concerted way and nicks each strand independently. Combinations of nicks with counter-nicks in strands across the junction resolve the construct. The specificity of the enzyme for DNA secondary structures was tested with a series of branched molecules made from oligonucleotides with the same nucleotide sequence in one strand. Results show that the number, location and relative cleavage efficiencies depend largely on the local nucleotide sequence, rather than on the branch type. In particular, endonuclease VII cleaves a complete four-armed cruciform as efficiently as a three-armed Y-junction or its derivatives, a semi-Y, a fork with two single-strand overhangs, a single-strand overhang, and a nicked DNA. However, exchange or addition of one or more nucleotides within the cleavage area flanking the structural signal for endonuclease VII strongly affects the cleavage pattern as well as their relative efficiency of usage. Examples with a single-stranded overhang are presented and show in summary that the enzyme has a fivefold preference for pyrimidines rather than purines.

Base Sequence↗
PubMed1997

The high-mobility group protein T160 binds to both linear and cruciform DNA and mediates DNA bending as determined by ring closure.

The high-mobility group protein T160 was isolated by screening a phage library from a murine pre-B-cell line L1210. South-Western experiments have previously shown that this protein binds to V-(D)-J recombination signal sequences, suggesting that it may be a sequence-specific DNA-binding protein. However, neither gel-shift nor footprinting analyses have been successfully employed with the T160 protein, despite an extensive effort. In this study, the T160 protein or truncated forms made soluble through denaturing and renaturing cycles in urea were successfully used in gel-shift experiments showing that T160 binds to cruci-form or linear duplex DNA with no apparent sequence specificity. Furthermore, fragments longer than 100 bp efficiently formed covalently closed circular monomers in the presence of T160 and T4 DNA ligase, indicating that the protein is capable of introducing bends into the duplex. Last, tissue distribution by Western blotting analysis showed that the T160 protein is expressed in various murine tissues in addition to those of lymphoid origin. Considering its broad evolutionary conservation (from plants to mammals) also, these results suggest that the functional role of the T160 protein is not limited to V-(D)-J recombination, but might be involved in basic processes such as DNA replication and repairing, where irregular DNA structures are generated and very likely recognized by HMG domain proteins.

Animals↗
PubMed2000

Interaction of the bacteriophage T4 gene 59 helicase loading protein and gene 41 helicase with each other and with fork, flap, and cruciform DNA.

Bacteriophage T4 gene 59 helicase loading protein accelerates the loading of T4 gene 41 DNA helicase and is required for recombination-dependent DNA replication late in T4 phage infection. The crystal structure of 59 protein revealed a two-domain alpha-helical protein, whose N-terminal domain has strong structural similarity to the DNA binding domain of high mobility group family proteins (Mueser, T. C., Jones, C. E., Nossal, N. G., and Hyde, C. C. (2000) J. Mol. Biol. 296, 597-612). We have previously shown that 59 protein binds preferentially to fork DNA. Here we show that 59 protein binds to completely duplex forks but cannot load the helicase unless there is a single-stranded gap of more than 5 nucleotides on the fork arm corresponding to the lagging strand template. Consistent with the roles of these proteins in recombination, we find that 59 protein binds to and stimulates 41 helicase activity on Holliday junction DNA, and on a substrate that resembles a strand invasion structure. 59 protein forms a stable complex with wild type 41 helicase and fork DNA in the presence of adenosine 5'-O-(thiotriphosphate). The unwinding activity of 41 helicase missing 20 C-terminal amino acids is not stimulated by 59 protein, and it does not form a complex with 59 protein on fork DNA.

Base Sequence↗
PubMed1999

The binding motif recognized by HU on both nicked and cruciform DNA.

The heterodimeric HU protein, highly conserved in bacteria and involved in transposition, recombination, DNA repair, etc., shares similarity with histones and HMGs. HU, which binds DNA with low affinity and without sequence specificity, binds strongly and specifically to DNA junctions and DNA containing single-strand breaks. The fine structure of these specific complexes was studied by footprinting and HU chemically converted into nucleases. The positioning of HUalphabeta on nicked DNA is asymmetrical and specifically oriented: the beta-arm binds the area surrounding the break whereas the alpha-arm lies on the 3' DNA branch. This positioning necessitates a pronounced bend in the DNA at the discontinuous point, which was estimated by circular permutation assay to be 65 degrees. At junctions, HU is similarly asymmetrically positioned in an identical orientation: the junction point plays the role of the discontinuous point in the nicked DNA. The HU binding motif present in both structures is a pair of inclined DNA helices.

Base Sequence↗
PubMed1984

Nucleosome "phasing" and cruciform structures in circular supercoiled pBR322 DNA.

Cruciform structures have been detected in pBR322 supercoiled DNA, both in its naked state and when complexed with histone octamer, using S1 endonuclease cleavage and EcoRI restriction. An inspection of the DNA sequence shows that the S1-hypersensitive sites are very near to AT-rich regions of pBR322 genome. A nucleosome "phasing" in these regions, as found on AT-rich regions of SV40 DNA (15), has been shown by restriction enzymes analysis. On the basis of these results it can be proposed that cruciform structures protrude on the nucleosome surface. This model explains the reason why these structures, which need high superhelical density, can exist in supercoiled DNA partially relaxed by nucleosome formation.

Base Sequence↗
PubMed1988

Effect of base composition at the center of inverted repeated DNA sequences on cruciform transitions in DNA.

We have analyzed the effect of base composition at the center of symmetry of inverted repeated DNA sequences on cruciform transitions in supercoiled DNA. For this we have constructed two series of palindromic DNA sequences: one set with differing center and one set with differing center and arm sequences. The F series consists of two 96-base pair perfect inverted repeats which are identical except for the central 10 base pairs which consist of pure AT or GC base pairs. The S series was constructed such that the overall base composition of the inverted repeats was identical but in which the positioning of blocks of AT- and GC-rich sequences varied. The rate of cruciform formation for the inverted repeats in plasmid pUC8 was dramatically influenced by the 8-10 base pairs at the center of the inverted repeat. Inverted repeats with 8-10 AT base pairs in the center were kinetically much more active in cruciform formation than inverted repeats with 8-10 GC base pairs in the center. These experiments show a dominant influence of the center sequences of inverted repeats on the rate of cruciform formation.

Base Sequence↗
PubMed2002

Crp1p, a new cruciform DNA-binding protein in the yeast Saccharomyces cerevisiae.

A synthetic cruciform DNA (X-DNA) was used for screening cellular extracts of Saccharomyces cerevisiae for X-DNA-binding activity. Three X-DNA-binding proteins with apparent molecular mass of 28kDa, 26kDa and 24kDa, estimated by SDS-PAGE, were partially purified. They were identified as N-terminal fragments originating from the same putative protein, encoded by the open reading frame YHR146W, which we named CRP1 (cruciform DNA-recognising protein 1). Expression of CRP1 in Escherichia coli showed that Crp1p is subject to efficient proteolysis at one specific site. Cleavage leads to an N-terminal subpeptide of approximately 160 amino acid residues that is capable of binding specifically X-DNA with an estimated dissociation constant (K(d)) of 800nM, and a C-terminal subpeptide of approximately 305 residues without intrinsic X-DNA-binding activity. The N-terminal subpeptide is of a size similarly to that of the fragments identified in yeast, suggesting that the same cleavage process occurs in the yeast and the E.coli background. This makes the action of a site-specific protease unlikely and favours the possibility of an autoproteolytic activity of Crp1p. The DNA-binding domain of Crp1p was mapped to positions 120-141. This domain can act autonomously as an X-DNA-binding peptide and provides a new, lysine-rich DNA-binding domain different from those of known cruciform DNA-binding proteins (CBPs). As reported earlier for several other CBPs, Crp1p exerts an enhancing effect on the cleavage of X-DNA by endonuclease VII from bacteriophage T4.

Bacteriophage T4↗
PubMed2002

The 14-3-3 protein homologues from Saccharomyces cerevisiae, Bmh1p and Bmh2p, have cruciform DNA-binding activity and associate in vivo with ARS307.

We have previously shown that, in human cells, cruciform DNA-binding activity is due to 14-3-3 proteins (Todd, A., Cossons, N., Aitken, A., Price, G. B., and Zannis-Hadjopoulos, M. (1998) Biochemistry 37, 14317-14325). Here, wild-type and single- and double-knockout nuclear extracts from the 14-3-3 Saccharomyces cerevisiae homologues Bmh1p and Bmh2p were analyzed for similar cruciform-binding activities in relation to these proteins. The Bmh1p-Bmh2p heterodimer, present in the wild-type strain, bound efficiently to cruciform-containing DNA in a structure-specific manner because cruciform DNA efficiently competed with the formation of the complex, whereas linear DNA did not. In contrast, the band-shift ability of the Bmh1p-Bmh1p and Bmh2p-Bmh2p homodimers present in the bmh2(-) and bmh1(-) single-knockout cells, respectively, was reduced by approximately 93 and 82%, respectively. The 14-3-3 plant homologue GF14 was also able to bind to cruciform DNA, suggesting that cruciform-binding activity is a common feature of the family of 14-3-3 proteins across species. Bmh1p and Bmh2p were found to associate in vivo with the yeast autonomous replication sequence ARS307, as assayed by formaldehyde cross-linking, followed by immunoprecipitation with anti-Bmh1p/Bmh2p antibody and conventional PCR. In agreement with the finding of an association of Bmh1p and Bmh2p with ARS307, another immunoprecipitation experiment using 2D3, an anti-cruciform DNA monoclonal antibody, revealed the presence of cruciform-containing DNA in ARS307.

14-3-3 Proteins↗
PubMed1994

11q23 translocations split the "AT-hook" cruciform DNA-binding region and the transcriptional repression domain from the activation domain of the mixed-lineage leukemia (MLL) gene.

Translocations involving chromosome band 11q23, found in acute lymphoid and myeloid leukemias, disrupt the MLL gene. This gene encodes a putative transcription factor with homology to the zinc fingers and other domains of the Drosophila trithorax gene product and to the "AT-hook" motif of high mobility group proteins. To map potential transcriptional activation or repression domains of the MLL protein, yeast GAL4 DNA-binding domain and MLL hybrid protein-expressing plasmids were cotransfected with chloramphenicol acetyltransferase reporter plasmids in a transient transfection system. We found that MLL contains a strong activation domain and a repression domain. The former, located telomeric (3') to the breakpoint region, activated transcription 18-fold to > 200-fold, depending on the promoter and cell line used for transfection. A repression domain that repressed transcription 4-fold was located centromeric (5') to the breakpoint region of MLL. The MLL AT-hook domain protein was expressed in bacteria and was utilized in a gel mobility shift assay to assess DNA-binding activity. The MLL AT-hook domain could bind cruciform DNA, recognizing structure rather than sequence of the target DNA. In translocations involving MLL, loss of an activation domain with retention of a repression domain and a DNA-binding domain on the der(11) chromosome could alter the expression of downstream target genes, suggesting a potential mechanism of action for MLL in leukemia.

Animals↗
PubMed2004

Influence of global DNA topology on cruciform formation in supercoiled DNA.

DNA supercoiling plays an important role in many genetic processes such as replication, transcription, and recombination. Supercoiling provides energy for helix un-pairing and drives the formation of alternative DNA structural transitions, like cruciforms. Supercoiling also allows distant DNA regions to be brought into close proximity through the formation of inter-wound supercoils. Recently, we showed that the inverted repeat-to-cruciform transition acts as a molecular switch, influencing the global topology of a topological plasmid domain. As alternative DNA structures can affect global topology, a corollary hypothesis might be that the localization of a specific DNA sequence within a topological domain may affect the energetics required for formation of an alternative DNA structure. Here, we test this hypothesis and show that the localization of an inverted repeat to an apical position increases the rate of cruciform formation and reduces the superhelical energy required to drive the transition. For this, we created a series of plasmids containing an inverted repeat and an A-tract bent DNA sequence. The A-tract forms a permanent 180 degrees bend irrespective of DNA topology. The inverted repeat and the bent sequence were placed either at six o'clock or nine o'clock positions with respect to each other. Using 2D agarose gel electrophoresis, we show that the six o'clock construct extrudes the cruciform at a lower superhelical density than a control plasmid without the bend. Atomic force microscopy shows that the nine o'clock construct has the propensity to form branched molecules with the cruciform at the end of one branch. These results demonstrate that the localization of sequences within specific regions of a topological domain can affect the energetics of structural transitions as well as the branching structure of the domain. As structural transitions can be involved in biological processes, localization of alternative conformation-forming sequences to specific locations within a domain provides an additional means for gene regulation.

DNA, Superhelical↗
PubMedDate not supplied

Non-B-DNA structures on the interferon-beta promoter?

The high mobility group (HMG) I protein intervenes as an essential factor during the virus induced expression of the interferon-beta (IFN-beta) gene. It is a non-histone chromatine associated protein that has the dual capacity of binding to a non-B-DNA structure such as cruciform-DNA as well as to AT rich B-DNA sequences. In this work we compare the binding affinity of HMGI for a synthetic cruciform-DNA to its binding affinity for the HMGI-binding-site present in the positive regulatory domain II (PRDII) of the IFN-beta promoter. Using gel retardation experiments, we show that HMGI protein binds with at least ten times more affinity to the synthetic cruciform-DNA structure than to the PRDII B-DNA sequence. DNA hairpin sequences are present in both the human and the murine PRDII-DNAs. We discuss in this work the presence of, yet putative, non-B-DNA structures in the IFN-beta promoter.

Base Sequence↗
PubMed1994

Unusual DNA structures, chromatin and transcription.

Extensive studies of DNA secondary structure during the past decade have shown that DNA is a dynamic molecule, whose structure depends on the underlying nucleotide sequence and is influenced by the environment and the overall DNA topology. Three major non-B-DNA structures have been described (Z-DNA, triplex DNA and cruciform DNA) which are stabilized by unconstrained negative supercoiling and can be formed under physiological conditions. In this essay we summarize the DNA primary structure features that are pertinent to the formation of these conformers and present data concerning the occurrence of these sequences in the eukaryotic genome. The evidence in favor of the existence of these unusual DNA structures in vivo is discussed. The effect of alternative non-B-DNA structures on the way DNA is organized in chromatin is considered, and this is followed by evaluation of the data relating these structures to eukaryotic transcription. Some possible mechanisms by which the effect of non-B structures on transcription might be exerted are proposed.

Animals↗
PubMed2000

Single and multiple topologically driven structural transitions in DNA.

We derive some exact general results concerning the behavior of topological absorbers (i.e., sequences undergoing topologically driven structural transitions) in closed circular DNA molecules. Starting from the formal physical framework that covers all known structural transitions, like those from standard B-DNA to nonstandard conformers Z-DNA, H-DNA, cruciform-DNA, melt-DNA or others, we develop a reduced state space description that leads to an analytically simplified "black box" view of absorbers. The latter contains only a single state variable-the total sequence unwinding u describing the topological state of the absorber. We show that the statistical mechanics of u is determined by the (one-dimensional) absorption free energy function G(abs) and find explicit expressions for G(abs) and for moments in terms of the standard experimental observable-the absorption function alpha:= . The reduced state space method is then applied to systems consisting of several interacting topologically coupled absorbers and a formula predicting their collective behavior (superposition) in terms of their individual absorptions is derived. Using these results we formulate and discuss solution methods for two basic types of inverse problems that turn out to be fundamental for future absorber construction.

DNA↗
PubMed1986

Unusual DNA structures in the adenovirus genome.

More than 80% (approximately 29 kilobase pairs) of the adenovirus serotype 2 genome was surveyed for the presence of unusual DNA conformations. Seven recombinant DNAs containing the largest HindIII fragments of AD2 DNA were analyzed for the presence of negative supercoil-dependent S1 nuclease-sensitive sites. Four plasmids each contained a specific site of S1 nuclease sensitivity whereas the other three showed no reaction. Further investigation was focused on a plasmid containing one of the positively reacting fragments (fragment C) which contained the major late promoter at coordinate 16.4 on the genome; three serotypes (Ad2, Ad7, Ad12) were studied. Fine mapping studies revealed the S1-sensitive sites to be a small region (approximately 6 base pairs) located at the TATA box of the major late promoter in all three cases. Other determinations (supercoil relaxation, T7 gene 3 product sensitivity, bromoacetaldehyde reactivity, anomalous gel mobility, the influence of negative superhelical density on nuclease sensitivity) led to the conclusion that the B-helix deformation was not due to a previously recognized DNA conformation (left-handed Z-DNA, cruciform, bent DNA), but may be accounted for by the homopurine X homopyrimidine nature of this region.

Acetaldehyde↗
PubMed1983

Slow cruciform transitions in palindromic DNA.

Extrusion of cruciform structures in self-complementary regions of DNA is known to be favored by negative supercoiling of DNA. We show here that, in moderately supercoiled DNA, cruciform extrusion is a very slow process. In plasmid pUC7 DNA, with a 48-base-pair palindrome, the half-time of extrusion at 50 degrees C is typically several hours; rates are even slower at lower temperature. The rates increase significantly with increasing DNA supercoiling but are only slightly faster in DNA species with much longer palindromes. The reabsorption of cruciform arms is also very slow. The equilibrium between cruciform and regular DNA structures is sensitive to changes in the linking number. Measurement of this equilibrium leads to an estimate of 18 kcal/mol (75.3 kJ/mol) for the free energy required to generate a cruciform structure. In bacterial cells, cruciform DNA may be rare, even when it is thermodynamically favored, because of its slow formation.

Animals↗