Triple helix-specific ligands.

A triple helix is formed upon binding of an oligodeoxynucleotide to the major groove of duplex DNA. A benzo[e]pyridoindole derivative (BePI) strongly stabilized this structure and showed preferential binding to a triplex rather than to a duplex. Energy transfer experiments suggest that BePI intercalates within the triple helix. Sequence-specific inhibition of transcription initiation of a specific gene by Escherichia coli RNA polymerase by a triplex-forming oligodeoxynucleotide is strongly enhanced when the triplex is stabilized by BePI. Upon irradiation with ultraviolet light, BePI induces covalent modifications of the target within the triple helix structure.

The transpososome: control of transposition at the level of catalysis.

Studies of several transposable genetic elements have pinpointed the importance of the transpososome, a nucleoprotein complex involving the transposon ends and a transposon-encoded enzyme--the transposase--as a key in regulating transposition. Transpososomes provide a precise architecture within which the chemical reactions involved in transposon displacement occur. Data are accumulating that suggest they are dynamic and undergo staged conformational changes to accommodate different steps in the transposition pathway. This has been underpinned by recent results obtained particularly with Tn5, Tn10 and bacteriophage Mu.

The terminal inverted repeats of IS911: requirements for synaptic complex assembly and activity.

The bacterial insertion sequence IS911 transposes via a covalently closed circular intermediate. Circle formation involves transposase-mediated pairing of both insertion sequence ends. While full-length transposase, OrfAB, binds poorly in vitro to IS911 DNA fragments carrying a copy of the IS911 end, truncated protein derivatives carrying the first 135 (OrfAB[1-135]) or 149 (OrfAB[1-149]) amino acid residues bind efficiently. They generate a paired-end complex containing two such fragments which resembles that expected for the first synaptic complex. Shorter protein derivatives lacking a region involved in multimerisation do not form these complexes but modify the binding of OrfAB[1-135] and OrfAB[1-149]. DNaseI footprinting demonstrated that OrfAB[1-149] protects a sub-terminal (internal) region of the inverted repeats which includes two blocks of sequence (beta and gamma) conserved between the left (IRL) and right (IRR) ends. DNA binding assays in vitro and measurement of recombination activity in vivo of sequential deletion derivatives of the two inverted repeats suggested a model in which the N-terminal region of OrfAB binds the conserved boxes beta and gamma in a sequence-specific manner and anchors the two insertion sequence ends into a paired-end complex. The external region of the inverted repeat is proposed to contact the C-terminal transposase domain carrying the catalytic site.

Triple helix-directed psoralen crosslinks are recognized by Uvr(A)BC excinuclease.

Pyrimidine oligonucleotides bind to the major groove of an oligopyrimidine-oligopurine DNA sequence by triple helix formation. A 14-mer oligopyrimidine 3'-psoralen-conjugate (P) and a doubly modified 5'-acridine/3'-psoralen-oligonucleotide (PA) were photo-crosslinked to their target site. The crosslinked complexes were tested regarding their sensitivity to Uvr(A)BC excinuclease/DNA complex formation and excision, and compared to free psoralen crosslinked to the same site (M). An electrophoretic mobility-shift assay showed that the crosslinked triple-helix did not hamper formation of the (A)2B complex under conditions where the third strand was bound to its target. In vitro excision experiments performed on damaged DNA fragments containing crosslinked 5-methoxypsoralen (M-target) confirmed that the psoralen photoadduct was recognized by Uvr(A)BC and that excision occurred at the crosslinked site. The major cleavage reaction took place on the 5'-side of oligopurine strand. The excision was less efficient on the 5'-side of the pyrimidine strand. The 3'-side incision either on the purine or pyrimidine strand was even weaker. With optimal Uvr(A) concentrations, it was observed that the incision reaction on (P)- and (PA)-modified targets was clearly inhibited compared to the (M)-modified target, reflecting an effect of the oligonucleotide on the recognition/excision process. These results demonstrate that a triple helix is efficient in promoting inhibition of Uvr(A)BC excision nuclease activity. These results could account for divergent findings concerning the effects of triple helix-forming oligonucleotides on repair systems and open new perspectives to study DNA repair processes through the use of bi-substituted triple helix-forming oligonucleotides.

Triple-helix specific ligands stabilize H-DNA conformation.

Under superhelical stress, oligopurine-oligopyrimidine mirror-repeat sequences are able to adopt H-DNA conformations where a triple-helical and a single-stranded structure co-exist. We have previously shown that a benzo[e]pyridoindole derivative (BePI), an antitumor drug interacting more tightly with triplex than with duplex DNA, strongly stabilizes intermolecular triple helices formed upon binding of homopyrimidine oligonucleotides to the major groove of double-stranded DNA at oligopurine-oligopyrimidine sequences. Here we show that an intramolecular triple helix is also strongly stabilized by this ligand. In vitro elongation performed by different DNA polymerases (bacteriophage T7, Escherichia coli or Taq polymerase) could be irreversibly inhibited by the H-DNA structure in the presence of BePI. A mirror-repeat polypurine-polypyrimidine sequence inserted between the E. coli beta-lactamase gene (conferring ampicillin resistance) and its bla promoter strongly inhibited transcription of the beta-lactamase gene in vivo. In the absence of supercoiling, transition to the H-conformation did not occur, but BePI stabilized the H-DNA structure induced by supercoiling as shown by chemical probes (chloroacetaldehyde). The results presented here open a new field of investigation for antitumor agents targeted to a novel class of genetic structures able to regulate gene expression.

Far upstream sequences of the bla promoter from TN3 are involved in complexation with E. coli RNA-polymerase.

The structure of the final initiation complex between E. coli RNA polymerase (RNAP) and the bla promoter from the transposon TN3 has been probed by footprinting experiments and base accessibility to dimethyl sulfate at 37 degrees C. At RNAP/promoter molar ratios "standard" for these experiments (greater than or equal to 10), the contacts on bla extend from -100 to +20, i.e. a length exceeding twice the dimension of the RNAP major axis [33]. Since footprinting at about equimolar amounts of RNAP and bla extends to the usual (-55 to +20) promoter domain, it is very likely that at least two RNAP's participate in the complex observed at tenfold higher RNAP/bla ratios. Under the latter conditions, the extended footprint (-100 to +20) is observed above 30 degrees C, whereas at 15 degrees C, only the -55 to +20 promoter area is contacted. Furthermore, gel retardation experiments show the presence of two complexes of different migration rates. We have reported earlier [21] that at the "standard" RNAP/bla ratio, transcription initiation from the bla promoter is inhibited. The correlation of this inhibition with the postulated two RNAP/bla complex suggests a regulation of bla gene expression by RNAP availability controlled for instance by growth rate. These results can be correlated with those reported in [14, 15] for the tyrT promoter. Interestingly, both promoter share significant sequence homologies.

How Escherichia coli RNA polymerase can negatively regulate transcription from a constitutive promoter.

We previously described the structures and functions of specific complexes between the bla promoter from Tn3 (present in pBR322) and RNA polymerase (RNAP), showing that, at excess RNAP, complexes can form in which one or two RNAPs bind to the same promoter (1:1 and 2:1 complexes) (Duval-Valentin and Ehrlich, 1988). We report here that the 2:1 complex cannot be detected below 25 degrees C; above that temperature, a 1:1 complex forms at a rate one order of magnitude faster than that of the 2:1 complex, and above 30 degrees C, the amounts of both species become equal for RNAP/promoter ratio r30 less than or equal to r less than or equal to 70. The 2:1 complex decays back to a 1:1 complex losing the last RNAP at a rate about three times that of the 1:1 complex decay. Functional assays of the complexes formed at excess RNAP show that both 1:1 and 2:1 complexes are immediately and permanently inhibited, even when the promoters are pre-incubated with ribonucleotide selections potentially enabling entrance into abortive cycling or formation of a stressed complex. We conclude that the inhibition step probably takes place in the complex formation pathway between RPi and RPo, at a novel stable intermediate isomer, RPj, formed above 25 degrees C. A possible mechanism of formation of the 2:1 complex is outlined. In vivo studies, in which r was modified by varying the bacterial growth rate, show a reduction of bla expression as r values are upshifted, specific to the bla promoter from Tn3.

Dynamic and structural characterisation of multiple steps during complex formation between E. coli RNA polymerase and the tetR promoter from pSC101.

Kinetic, functional and structural studies of the recognition of the tetR promoter from pSC101 by E. coli RNA polymerase allowed the characterization of several steps in the specific complex formation and transcription initiation process. First, enzyme and DNA enter in a short life-time complex. An isomerization will convert this unstable complex into a closed stable one where RNA polymerase is tightly attached without establishing stable chemical contacts with the bases. In the next step, stable close contacts appear between both macromolecules involving mainly the downstream part of the promoter. A further isomerization will lead to an open complex where DNA is locally melted and the system is able to initiate transcription. This latter process is accompanied by changes in the upstream part of the promoter. Finally, in vitro transcription assays showed that the position of the major transcription start sites depends on temperature. From the reported results, it appears that the recognition event is a sequential process where different structural elements of the promoter, that can be located apart in the sequence, are involved in a concerted manner in each stage.

Interaction between E. coli RNA polymerase and the tetR promoter from pSC101: homologies and differences with other E. coli promoter systems from close contact point studies.

The interaction between E. coli RNA polymerase and the tetR promoter from pSC101, was studied by protection and premodification experiments, using dimethyl sulfate, methylation of single stranded cytosines, and DNAase I footprinting. Whereas qualitative and quantitative results from the chemical approach conform to patterns already displayed by other promoter systems, hypersensitive sites to DNAase I attack differ from those of other promoters. Distribution and nature of the contacts suggest that regions of the promoter sequence participates differently in complex formation. The involvement of major and minor grooves of the double helix in the complex with the enzyme, differs along the promoter. After a comparison of the results from seven different promoters, a pattern of conserved contacts seem to appear. Comparison of temperature dependence of local unwinding around the transcription start site (detected by the appearance of single stranded cytosines), and DNAase I footprinting, reveals that the process leading to stable complex formation can be achieved without disruption of base-pairing.

A second RNA-polymerase can bind specifically to the bla promoter of Tn3, repressing transcription initiation.

We showed earlier that the region of the bla promoter of Tn3 protected by the RNA-polymerase (RNAP), has the normal size (about 60bp) at RNAP/promoter molar ratio r less than or equal to 2, but rises to about twice this extent as r increases. We confirm here that the species corresponding to normal and extended footprint distinguish by their electrophoretic mobilities. Furthermore, inspection of the complexes by electron microscopy confirms that at r greater than 2, the bla promoter can bind specifically a second RNAP particle, as compared to the 1:1 complex observed at r less than or equal to 2. At r greater than 2, the ability of the bla promoter to initiate transcription in vitro is repressed when compared to the complex 1:1 obtained at r less than or equal to 2. The unexpected decrease in initiation efficiency as the concentration of RNAP particles is increased, together with the striking sequence homology of the bla promoter with promoters of stable RNA, suggest that in vivo, this promoter could be regulated by growth rate.

Specific inhibition of transcription by triple helix-forming oligonucleotides.

Homopyrimidine oligonucleotides bind to the major groove of a complementary homopyrimidine.homopurine stretch by triple helix formation. The bla gene from transposon Tn3 contains a homopyrimidine.homopurine sequence of 13 base pairs located just downstream of the RNA polymerase binding site. A 13-mer homopyrimidine oligonucleotide targeted to this sequence was tested for its effect on transcription of the bla gene in vitro. We show that the consequence of triple helix formation in front of the Escherichia coli RNA polymerase-promoter complex is to block the holoenzyme at its start site during a period that is dependent on temperature. The temperature dependence of transcription inhibition shows a direct correlation between this effect and the stabilization of the triple helix. Substitution of 5-methylcytosine to cytosine in the 13-mer oligonucleotide enhances triplex stability and transcription inhibition. Transcription inhibition by this synthetic repressor was also confirmed by footprinting studies demonstrating its specificity of action. The 13-mer oligonucleotide containing a psoralen derivative covalently linked to its 5' end shows an irreversible and specific inhibition of transcription initiation after exposure to light of wavelength greater than 310 nm.

Requirement of IS911 replication before integration defines a new bacterial transposition pathway.

Movement of transposable elements is often accompanied by replication to ensure their proliferation. Replication is associated with both major classes of transposition mechanisms: cut-and-paste and cointegrate formation (paste-and-copy). Cut-and-paste transposition is often activated by replication of the transposon, while in cointegrate formation replication completes integration. We describe a novel transposition mechanism used by insertion sequence IS911, which we call copy-and-paste. IS911 transposes using a circular intermediate (circle), which then integrates into a target. We demonstrate that this is derived from a branched intermediate (figure-eight) in which both ends are joined by a single-strand bridge after a first-strand transfer. In vivo labelling experiments show that the process of circle formation is replicative. The results indicate that the replication pathway not only produces circles from figure-eight but also regenerates the transposon donor plasmid. To confirm the replicative mechanism, we have also used the Escherichia coli terminators (terC) which, when bound by the Tus protein, inhibit replication forks in a polarised manner. Finally, we demonstrate that the primase DnaG is essential, implicating a host-specific replication pathway.

Excision of IS492 requires flanking target sequences and results in circle formation in Pseudoalteromonas atlantica.

The gram-negative marine bacterium Pseudoalteromonas atlantica produces extracellular polysaccharide (EPS) that is important in biofilm formation by this bacterium. Insertion and precise excision of IS492 at a locus essential for extracellular polysaccharide production (eps) controls phase variation of EPS production in P. atlantica. Examination of IS492 transposition in P. atlantica by using a PCR-based assay revealed a circular form of IS492 that may be an intermediate in transposition or a terminal product of excision. The DNA sequence of the IS492 circle junction indicates that the ends of the element are juxtaposed with a 5-bp spacer sequence. This spacer sequence corresponds to the 5-bp duplication of the chromosomal target sequence found at all IS492 insertion sites on the P. atlantica chromosome that we identified by using inverse PCR. IS492 circle formation correlated with precise excision of IS492 from the P. atlantica eps target sequence when introduced into Escherichia coli on a plasmid. Deletion analyses of the flanking host sequences at the eps insertion site for IS492 demonstrated that the 5-bp duplicated target sequence is essential for precise excision of IS492 and circle formation in E. coli. Excision of IS492 in E. coli also depends on the level of expression of the putative transposase, MooV. A regulatory role for the circular form of IS492 is suggested by the creation of a new strong promoter for expression of mooV by the joining of the ends of the insertion sequence element at the circle junction.

Extension of the range of recognition sequences for triple helix formation by oligonucleotides containing guanines and thymines.

Oligodeoxynucleotides containing G and T can bind to homopurine.homopyrimidine sequences on double-stranded DNA by forming C.G x G and T.A x T base triplets. The orientation of the third strand in such triple helices depends on the number of GpT and TpG steps. Therefore a single oligonucleotide can be designed to bind to two consecutive homopurine.homopyrimidine sequences where the two homopurine stretches alternate on the two strands of DNA. The oligonucleotide switches from one homopurine strand to the other at the junction between the two sequences. This result shows that it is possible to extend the range of DNA sequences that can be recognized by a single oligonucleotide.

Inhibition of viral growth by an alpha-oligonucleotide directed to the splice junction of herpes simplex virus type-1 immediate-early pre-mRNA species 22 and 47.

A 13-residue alpha-anomeric oligonucleotide [alpha-5'-d(GGGCGTCCTCCTT)3'], 5'-substituted with a psoralen derivative, Pso-alpha-13 psoralen linked to the 5' end of an alpha-anomeric n-residue oligonucleotide, was targeted to the acceptor splice junction of Herpes simplex virus type-1 immediate-early pre-mRNA species 22 and 47. Inhibition of viral growth was observed upon irradiation of Vero cells infected with Herpes simplex virus type-1 and treatment with Pso-alpha-13. The virus titer was decreased by 80% at an oligonucleotide concentration of 0.5 microM and at a multiplicity of infection of 0.1 plaque-forming units/cell. The 13-residue oligonucleotide did not induce any cytotoxic effect after irradiation and the inhibition of viral growth was clearly sequence specific. A non-specific 5' Pso-alpha-15 did not inhibit Herpes simplex virus type-1 growth. The 5' Pso-alpha-13 targeted to the acceptor splice junction of Herpes simplex virus type 1, contained five mismatches with respect to the corresponding sequence of Herpes simplex virus type 2, and did not exhibit any inhibitory effects on Herpes simplex virus type-2 growth. These results show that alpha-oligonucleotides can exhibit a sequence-specific antiviral effect and suggest that they may inhibit splicing reactions and be useful in targeting specific nucleic acid sequences within the cell nucleus.

Transient promoter formation: a new feedback mechanism for regulation of IS911 transposition.

IS911 transposition involves a free circular transposon intermediate where the terminal inverted repeat sequences are connected. Transposase synthesis is usually driven by a weak promoter, p(IRL), in the left end (IRL). Circle junction formation creates a strong promoter, p(junc), with a -35 sequence located in the right end and the -10 sequence in the left. p(junc) assembly would permit an increase in synthesis of transposase from the transposon circle, which would be expected to stimulate integration. Insertion results in p(junc) disassembly and a return to the low p(IRL)- driven transposase levels. We demonstrate that p(junc) plays an important role in regulating IS911 transposition. Inactivation of p(junc) strongly decreased IS911 transposition when transposase was produced in its natural configuration. This novel feedback mechanism permits transient and controlled activation of integration only in the presence of the correct (circular) intermediate. We have also investigated other members of the IS3 and other IS families. Several, but not all, IS3 family members possess p(junc) equivalents, underlining that the regulatory mechanisms adopted to fine-tune transposition may be different.

Alternate strand recognition of double-helical DNA by (T,G)-containing oligonucleotides in the presence of a triple helix-specific ligand.

Triple helix formation requires a polypurine- polypyrimidine sequence in the target DNA. Recent works have shown that this constraint can be circumvented by using alternate strand triplex-forming oligonucleotides. We have previously demonstrated that (T,G)-containing triplex- forming oligonucleotides may adopt a parallel or an antiparallel orientation with respect to an oligopurine target, depending upon the sequence and, in particular, upon the number of 5'-GpT-3' and 5'-TpG-3' steps [Sun et al. (1991) C.R. Acad. Sci. Paris Ser III, 313, 585-590]. A single (T,G)-containing oligonucleotide can therefore interact with two oligopurine stretches which alternate on the two strands of the target DNA. The (T,G) switch oligonucleotide contains a 5'-part targeted to one of the oligopurine sequences in a parallel orientation followed by a 3'-part that adopts an antiparallel orientation with respect to the second oligopurine sequence. We show that a limitation to the stability of such a triplex may arise from the instability of the antiparallel part, composed of reverse-Hoogsteen C.GxG and T.AxT base triplets. Using DNase I footprinting and ultraviolet absorption experiments, we report that a benzo[e]pyridoindole derivative [(3-methoxy- 7H-8-methyl-11-[(3'-amino-propyl) amino] benzo[e]pyrido [4,3-b]indole (BePI)], a drug interacting more tightly with a triplex than with a duplex DNA, strongly stabilizes triplexes with reverse-Hoogsteen C.GxG and T.AxT triplets thus allowing a stabilization of the triplex-forming switch (T,G) oligonucleotide on alternating oligopurine- oligopyrimidine 5'-(Pu)14(Py)14-3' duplex sequences. These results lead to an extension of the range of oligonucleotide sequences for alternate strand recognition of duplex DNA.