Junctions between i-motif tetramers in supramolecular structures.

The symmetry of i-motif tetramers gives to cytidine-rich oligonucleotides the capacity to associate into supramolecular structures (sms). In order to determine how the tetramers are linked together in such structures, we have measured by gel filtration chromatography and NMR the formation and dissociation kinetics of sms built by oligonucleotides containing two short C stretches separated by a non-cytidine-base. We show that a stretch of only two cytidines either at the 3'- or 5'-end is long enough to link the tetramers into sms. The analysis of the properties of sms formed by oligonucleotides differing by the length of the oligo-C stretches, the sequence orientation and the nature of the non-C base provides a model of the junction connecting the tetramers in sms.

Insight into the role of dynamics in the conformational switch of the small GTP-binding protein Arf1.

Activation of the small GTP-binding protein Arf1, a major regulator of cellular traffic, follows an ordered sequence of structural events, which have been pictured by crystallographic snapshots. Combined with biochemical analysis, these data lead to a model of Arf1 activation, in which opening of its N-terminal helix first translocates Arf1-GDP to membranes, where it is then secured by a register shift of the interswitch ß-strands, before GDP is eventually exchanged for GTP. However, how Arf1 rearranges its central ß-sheet, an event that involves the loss and re-formation of H-bonds deep within the protein core, is not explained by available structural data. Here, we used Δ17Arf1, in which the N-terminal helix has been deleted, to address this issue by NMR structural and dynamics analysis. We first completed the assignment of Δ17Arf1 bound to GDP, GTP, and GTPγS and established that NMR data are fully consistent with the crystal structures of Arf1-GDP and Δ17Arf1-GTP. Our assignments allowed us to analyze the kinetics of both protein conformational transitions and nucleotide exchange by real-time NMR. Analysis of the dynamics over a very large range of timescale by (15)N relaxation, CPMG relaxation dispersion and H/D exchange reveals that while Δ17Arf1-GTP and full-length Arf1-GDP dynamics is restricted to localized fast motions, Δ17Arf1-GDP features unique intermediate and slow motions in the interswitch region. Altogether, the NMR data bring insight into how that membrane-bound Arf1-GDP, which is mimicked by the truncation of the N-terminal helix, acquires internal motions that enable the toggle of the interswitch.

[C7GC4]4 association into supra molecular i-motif structures.

The self-associative properties of cytidine-rich oligonucleotides into symmetrical i-motif tetramers give to these oligonucleotides the capacity of forming supramolecular structures (sms) that have potential applications in the nanotechnology domain. In order to facilitate sms formation, oligonucleotides containing two cytidine stretches of unequal length (C(n)XC(m)) separated by a non-cytidine spacer were synthesized. They were designed to associate into a tetramer including an i-motif core built by intercalation of the C.C(+) pairs of the longer C stretch with the two dangling non-intercalated strands of the shorter C stretch at each end. Gel filtration chromatography shows that the non-intercalated C-rich ends give to this structure the capacity of forming extremely stable sms. Using C(7)GC(4) as a model, we find that the sms formation rate varies as the oligonucleotide concentration and increases at high temperature. Competitively with the tetramer involved in sms elongation, C(n)XC(m) oligonucleotides form i-motif dimers that compete with sms elongation. The dimer stability is strongly reduced when the pH is moved away from the cytidine pK. This results in an equilibrium shift towards the tetramer and in the acceleration of the sms formation rate. The chromatograms of the sms formed by C(7)GC(4) indicate a broad distribution. In a 1.5 mM solution incubated at 37 degrees C, the equilibrium distribution is centered on a molecular weight corresponding to the assembly of nine tetramers and the upper limit corresponds to 80 tetramers. The lifetime of this structure is about 4 days at 40 degrees C, pH 4.6.

[5mCCTCTCTCC]4: an i-motif tetramer with intercalated T*T pairs.

The i-motif is a four-stranded structure built by intercalation in a head-to-tail orientation of two parallel duplexes associated by hemiprotonated C(*)C(+) pairs. T*T pairs are nearly isomorphic of C*C(+) pairs; however the structural investigations of i-motif tetramers containing thymidines suggest that the i-motif cannot accommodate T*T pairs in a face-to-face orientation. The tetramer of 5mCCTCTCTCC make an exception. It includes two symmetry related open/closed T3/T7 groups, but the central thymidines form two long-lived T5*T5 pairs that are intercalated in a face-to-face orientation. This observation provides indications of the origin of the conflict that usually hinders T*T intercalation into i-motif structures and more generally of the constraints influencing i-motif formation.

Footprinting analysis of BWYV pseudoknot-ribosome complexes.

Many viruses regulate translation of polycistronic mRNA using a -1 ribosomal frameshift induced by an RNA pseudoknot. When the ribosome encounters the pseudoknot barrier that resists unraveling, transient mRNA-tRNA dissociation at the decoding site, results in a shift of the reading frame. The eukaryotic frameshifting pseudoknot from the beet western yellow virus (BWYV) has been well characterized, both structurally and functionally. Here, we show that in order to obtain eukaryotic levels of frameshifting efficiencies using prokaryotic Escherichia coli ribosomes, which depend upon the structural integrity of the BWYV pseudoknot, it is necessary to shorten the mRNA spacer between the slippery sequence and the pseudoknot by 1 or 2 nucleotides (nt). Shortening of the spacer is likely to re-establish tension and/or ribosomal contacts that were otherwise lost with the smaller E. coli ribosomes. Chemical probing experiments for frameshifting and nonframeshifting BWYV constructs were performed to investigate the structural integrity of the pseudoknot confined locally at the mRNA entry site. These data, obtained in the pretranslocation state, show a compact overall pseudoknot structure, with changes in the conformation of nucleotides (i.e., increase in reactivity to chemical probes) that are first "hit" by the ribosomal helicase center. Interestingly, with the 1-nt shortened spacer, this increase of reactivity extends to a downstream nucleotide in the first base pair (bp) of stem 1, consistent with melting of this base pair. Thus, the 3 bp that will unfold upon translocation are different in both constructs with likely consequences on unfolding kinetics.

The formation pathway of i-motif tetramers.

The i-motif is a four-stranded structure formed by two intercalated parallel duplexes containing hemiprotonated C*C(+) pairs. In order to describe the sequence of reactions by which four C-rich strands associate, we measured the formation and dissociation rates of three [TC(n)](4) tetramers (n = 3, 4 and 5), their dissociation constant and the reaction order for tetramer formation by NMR. We find that TC(n) association results in the formation of several tetramers differing by the number of intercalated C*C(+) pairs. The formation rates of the fully and partially intercalated species are comparable but their lifetimes increase strongly with the number of intercalated C*C(+) pairs, and for this reason the single tetramer detected at equilibrium is that with optimal intercalation. The tetramer half formation times vary as the power -2 of the oligonucleotide concentration indicating that the reaction order for i-motif formation is 3. This observation is inconsistent with a model supposing association of two preformed duplex and suggests that quadruplex formation proceeds via sequential strand association into duplex and triplex intermediate species and that triplex formation is rate limiting.

S1 ribosomal protein functions in translation initiation and ribonuclease RegB activation are mediated by similar RNA-protein interactions: an NMR and SAXS analysis.

The ribosomal protein S1, in Escherichia coli, is necessary for the recognition by the ribosome of the translation initiation codon of most messenger RNAs. It also participates in other functions. In particular, it stimulates the T4 endoribonuclease RegB, which inactivates some of the phage mRNAs, when their translation is no longer required, by cleaving them in the middle of their Shine-Dalgarno sequence. In each function, S1 seems to target very different RNAs, which led to the hypothesis that it possesses different RNA-binding sites. We previously demonstrated that the ability of S1 to activate RegB is carried by a fragment of the protein formed of three consecutive domains (domains D3, D4, and D5). The same fragment plays a central role in all other functions. We analyzed its structural organization and its interactions with three RNAs: two RegB substrates and a translation initiation region. We show that these three RNAs bind the same area of the protein through a set of systematic (common to the three RNAs) and specific (RNA-dependent) interactions. We also show that, in the absence of RNA, the D4 and D5 domains are associated, whereas the D3 and D4 domains are in equilibrium between open (noninteracting) and closed (weakly interacting) forms and that RNA binding induces a structural reorganization of the fragment. All of these results suggest that the ability of S1 to recognize different RNAs results from a high adaptability of both its structure and its binding surface.

The formation pathway of tetramolecular G-quadruplexes.

Oligonucleotides containing guanosine stretches associate into tetrameric structures stabilized by monovalent ions. In order to describe the sequence of reactions leading to association of four identical strands, we measured by NMR the formation and dissociation rates of (TGnT)4 quadruplexes (n = 3-6), their dissociation constants and the reaction orders for quadruplex formation. The quadruplex formation rates increase with the salt concentration but weakly depend on the nature (K+, Na+ or Li+) of the counter ions. The activation energies for quadruplex formation are negative. The quadruplex lifetimes strongly increase with the G-tract length and are much more longer in K+ solution than in Na+ or Li+ solutions. The reaction order for quadruplex formation is 3 in 0.125 M KCl and 4 in LiCl solutions. The kinetics measurements suggest that quadruplex formation proceeds step by step via sequential strand association into duplex and triplex intermediate species. Triplex formation is rate limiting in 0.125 M KCl solution. In LiCl, each step of the association process depends on the strand concentration. Parallel reactions to formation of the fully matched canonical quadruplex may result in kinetically trapped mismatched quadruplexes making the canonical quadruplex practically inaccessible in particular at low temperature in KCl solution.

Management of multicentric lesions of the lower genital tract.

OBJECTIVES: To report management and outcome of multicentric lesions of the lower genital tract. To define risk factors of recurrence. STUDY DESIGN: Retrospective review of multicentric dysplasias treated in our colposcopic clinic between 1996 and 2003. Multicentric dysplasias included CIN with VAIN and/or VIN. After primary treatment, follow-up was colposcopic, cytologic and virologic. RESULTS: Forty-four patients presented multicentric lesions out of 998 patients referred for CIN (4.4%). The average age was 36.8 years. Immunologic disorders were present in 20.4%. Ninety-one percent had cervicovaginal or cervicovulvar lesions, only 9% had three sites of genital dysplasia. 53.3% of lesions were concomitant. 79.5% of CIN were high grade, 62.5% of VAIN low grade and 62.5% of VIN high grade. Therapeutic modalities were as follows: conization for CIN (70.4%), CO2 laser for VAIN (33.3%) and surgery for VIN (41.7%). Forty patients were followed and had at least one post-treatment cytologic control; 55% of them had residual disease. Out of the 23 patients with at least two negative controls after treatment, 43.5% presented recurrence. Risk of recurrence was not statistically bound to such parameters as tabagism, immunologic disorder, high grade lesions, non-surgical treatment, and persistence of HPV infection after treatment. CONCLUSION: Multicentric dysplasias are associated with high rate of residual lesion and recurrence. Management of these lesions require long term follow-up.

Structure, internal motions and association-dissociation kinetics of the i-motif dimer of d(5mCCTCACTCC).

At slightly acidic pH, the association of two d(5mCCTCACTCC) strands results in the formation of an i-motif dimer. Using NMR methods, we investigated the structure of [d(5mCCTCACTCC)]2, the internal motion of the base pairs stacked in the i-motif core, the dimer formation and dissociation kinetics versus pH. The excellent resolution of the 1H and 31P spectra provided the determination of dihedral angles, which together with a large set of distance restraints, improve substantially the definition of the sugar-phosphate backbone by comparison with previous NMR studies of i-motif structures. [d(5mCCTCACTCC)]2 is built by intercalation of two symmetrical hairpins held together by six symmetrical C*C+ pairs and by pair T7*T7. The hairpin loops that are formed by a single residue, A5, cross the narrow grooves on the same side of the i-motif core. The base pair intercalation order is C9*C9+/5mC1*5mC1+/C8*C8+/C2*C2+/T7.T7/C6*C6+/C4*C4+. The T3 bases are flipped out in the wide grooves. The core of the structure includes four long-lived pairs whose lifetimes at 15 degrees C range from 100 s (C8*C8+) to 0.18 s (T7*T7). The formation rate and the lifetime of [d(5mCCTCACTCC)]2 were measured between pH 6.8 and 4.8. The dimer formation rate is three to four magnitude orders slower than that of a B-DNA duplex. It depends on pH, as it must occur for a bimolecular process involving non cooperative association of neutral and protonated residues. In the range of pH investigated, the dimer lifetime, 500 s at 0 degrees C, pH 6.8, varies approximately as 10(-pH).

i-motif solution structure and dynamics of the d(AACCCC) and d(CCCCAA) tetrahymena telomeric repeats.

Using NMR methods, we have resolved the i-motif structures formed by d(AACCCC) and by d(CCCCAA), two versions of the DNA sequence repeated in the telomeric regions of the C-rich strand of tetrahymena chromosomes. Both oligonucleotides form fully symmetrical i-motif tetramers built by intercalation of two hemiprotonated duplexes containing four C*C+ pairs. The structures are extremely stable. In the tetramer of d(AACCCC), the outermost C*C+ pairs are formed by the cytidines of the 5' ends of the cytidine tracts. A2 forms an A2*A2 (H6trans-N7) pair stacked to C3*C3+ and cross-strand stacked to A1. At 0 degrees C, the lifetimes of the hemiprotonated pairs range from 1 ms for the outermost pair to approximately 1 h for the innermost pairs. The tetramer of d(CCCCAA) adopts two distinct intercalation topologies in slow conformational exchange. One, whose outermost C*C+ pairs are built by the cytidines of the 5' end and the other by those of the 3' end. In both topologies, the adenosine bases are fairly well stacked to the adjacent C*C+ pairs. They are not paired but form symmetrical pseudo-pairs with their H6cis amino proton and N1 nitrogen pointing towards each other.

Opening mechanism of G.T/U pairs in DNA and RNA duplexes: a combined study of imino proton exchange and molecular dynamics simulation.

The opening pathway of wobble pairs dG.T and rG.U has been investigated in four DNA and two RNA duplexes. Using NMR spectroscopy, we measured the imino proton exchange of both G(H1) and T/U(H3), catalyzed by ammonia, tris, and OH(-), and we calculated the free energy surface related to G.T/U opening by molecular dynamics simulations. Taken together the experimental and theoretical results, we suggest that wobble pairs open through a coupled rotation of the bases toward the major groove where exchange of both imino protons takes place with the surrounding water.

T.T pair intercalation and duplex interconversion within i-motif tetramers.

The i-motif is a four-stranded structure built by intercalation in head-to-tail orientation of two parallel duplexes associated by hemi-protonated C.C(+) pairs. Using NMR methods, we investigated the structure, the base-pair opening kinetics and the internal motions of three i-motif tetramers: [d(5mCCTCnTCC)](4) (n=1, 2, 3). These tetramers cannot accommodate the intercalation of two T.T pairs in face-to-face orientation. They are built by intercalation of two symmetrical duplexes whose contacting T3/TM thymidine bases (M=5, 6, 7) are either base-paired or unstacked. The arrangement of the unstacked/paired thymidine bases of the two T/T groups results in the formation of two different conformations. One, fully symmetric, whose thymidine bases T3 and TM are unstacked and base-paired respectively. The other is the asymmetric assembly of two duplexes: one where both thymidine bases are unstacked and the other with two T.T pairs. The proportion of the symmetric conformer increases from a value beyond the detection threshold for n=1, to 19% for n=2 and up to more than 95% for n=3. The exchange cross-peaks connecting together the intercalated duplexes of [d(5mCCTCTCC)](4) and [d(5mCCTCCTCC)](4) reveal a structural interconversion induced by the simultaneous opening/closing of the contacting T3/TM thymidine bases. In [d(5mCCTCCTCC)](4) the motion of the T3/T6 groups triggers the interconversion of the symmetric and asymmetric conformations. In [d(5mCCTCTCC)](4) the intercalated duplexes exchange their structures in an apparently concerted motion, suggesting the simultaneous opening/closing of two distant T3/T5* and T5/T3* switching groups. The spectrum of [d(5mCCTCCCTCC)](4) is fully symmetric and, for this reason, its spectrum gives no indication for duplex interconversion. Nevertheless, the imino proton exchange kinetics argues for a switching motion of the T3/T7 group. Duplex interconversion is not detectable in that case, due to the tetramer symmetry. The origin of the structural conflict hindering the intercalation of two T.T pairs into the i-motif is discussed.

[Evaluation of cytological screening for cancers and precancerous lesions of the cervix]. / Evaluation du dépistage cytologique des cancers et lésions précancéreuses du col utérin.

The aim was to study retrospectively different parameters useful for the evaluation of cytological screening for cancers and precancerous lesions of the cervix. The study concerned 23,815 smears obtained consecutively from 18,045 women in 4 years. The proportion of follow up smears was equal to 11.1%. The % of abnormal smears was equal to 10.3% of all smears, 7.7% of screening smears (including 0.85% high grade or invasive lesions) and 30.7% of follow-up smears (including 5.55% high grade lesions). The 20,968 screening smears (from 16,896 women) included 2% unsatisfactory smears, 4.15% Ascus (atypical squamous cells of undetermined significance), 2.6% LSIL (low grade squamous intraepithelial lesion), 0.77% HSIL (high grade SIL), 0.05% squamous carcinomas, 0.10% atypical glandular cells and 0.02% adenocarcinomas. Histological examination of the cervix was obtained in 3.3% of women with negative smears, 17.7% of Ascus, 45.8% of LSIL and 82.9% of HSIL. In 1,082 women with histological examination, an histological diagnosis of high grade or invasive lesion of the cervix was made in 7.2% of women with Ascus, 15.7% of cytological LSIL and 85.1% of cytological HSIL.

Stability of the I-motif structure is related to the interactions between phosphodiester backbones.

The i-motif DNA tetrameric structure is formed of two parallel duplexes intercalated in a head-to-tail orientation, and held together by hemiprotonated cytosine pairs. The four phosphodiester backbones forming the structure define two narrow and wide grooves. The short interphosphate distances across the narrow groove induce a strong repulsion which should destabilize the tetramer. To investigate this point, molecular dynamics simulations were run on the [d(C2)]4 and [d(C4)]4 tetramers in 3'E and 5'E topologies, for which the interaction of the phosphodiester backbones through the narrow groove is different. The analysis of the simulations, using the Molecular Mechanics Generalized Born Solvation Area and Molecular Mechanics Poisson-Boltzmann Solvation Area approaches, shows that it is the van der Waals energy contribution which displays the largest relative difference between the two topologies. The comparison of the solvent-accessible area of each topology reveals that the sugar-sugar interactions account for the greater stability of the 3'E topology. This stresses the importance of the sugar-sugar contacts across the narrow groove which, enforcing the optimal backbone twisting, are essential to the base stacking and the i-motif stability. Tighter interactions between the sugars are observed in the case of N-type sugar puckers.

[Cervical smears: towards an optimal screening for cervical cancer]. / Vers un dépistage optimal des cancers et précancers du col utérin par frottis cervicaux.

THE INTEREST OF CERVICO-UTERINE CYTOLOGY: Cytology is the most appropriate means of screening for cancers and pre-cancerous states of the cervix. It permits the prevention of invasive cancers by identifying the precursor lesions, which exist more than 10 years before the invasion, and their effective excision or destruction. The absence of screening represents the greatest risk factor for invasive cancer and explains its higher frequency in developing countries. The Bethesda system is a language shared by cytologists and clinicians for spotting situations at risk Histology is essential before any therapeutic decision. It is provided by biopsies during colposcopy or by diagnostic coning in ambiguous situations. The cytological diagnosis of a low grade lesion and atypia of undetermined significance sometimes corresponds, histologically, to a high grade lesion. ENHANCING THE RESULTS OF SCREENING: The invasive cervical cancers still observed in France can be attributed to the lack of screening, lack of management and cytological false negatives. Conditions for optimising screening are wide coverage of women, good quality smears, competent cytologists and the appropriate follow-up of abnormal smears.

Unusual DNA conformations: implications for telomeres.

DNA is prone to structural polymorphism: its three-dimensional structure can differ markedly from the classical double helix. Nucleic acid structures composed of more than two strands have also been observed. The guanine-rich sequence of both the telomere and centromere can form a quadruplex based on G-quartets while the complementary cytosine-rich strand can fold into an intercalated tetramer called the i-motif. The G-quartet is a gold mine for structural biologists and the telomere has become a target for anti-cancer drug design since it was observed that deregulation of telomerase favors proliferation of certain tumors. Other DNA sequences may adopt unusual conformations. Polypurine-polypyrimidine sequences capable of forming a triple-stranded structure called H-DNA are found abundantly in the eukaryotic genome and may play a significant role in DNA metabolism, transcription and replication. Triplex-forming oligonucleotides are currently being developed as "anti-gene" agents. Unusual DNA structures may therefore be implicated in fundamental processes such as gene expression and represent unique targets for both structural-specific and sequence-specific agents. In this review, we present work characterizing some of these unusual conformations in terms of structure, stability and formation kinetics and discuss their biological implications.