Biomedical subjects

P Laugâa

Publications and source records attributed to P Laugâa.

8 recordsLinked to original sources

PubMed1999

A Y form of hammerhead ribozyme trapped by photo-cross-links retains full cleavage activity.

The conformation in solution of a small bipartite I-III hammerhead ribozyme has been deduced from the photo-crosslinks formed between cleavable ribo-deoxysubstrates appropriately substituted with the probe deoxy-4-thiouridine and ribozyme residues. The ribozyme-substrate complex is able to adopt a Y-like structure with stems I and II in close proximity in the presence of 400 mM Na+ only. Indeed, a cross-link joining stem I (1.6) to loop II (AL2.4) forms in significant amount under these conditions. This cross-linked complex furthermore elicits, upon Mg2+ addition, a catalytic activity similar to that exhibited by the complexes cross-linked at the distal ends of either stem I or stem III or of the non-substituted bipartite complex. This shows that the reaction mechanism is fully compatible with a strong structural constraint between stems I and II and that sodium ions at high concentration (400 mM) are able to promote a proper folding of hammerhead ribozymes. None of the multiple cross-links formed within the ribozyme core (probe in position 16.1 or 1.1) was found catalytically active. The cross-link patterns nevertheless indicate a higher flexibility of the core in Na+ than in Mg2+. While most of the cross-links can be accommodated by the Y solution structure, some of them (16.1 to U4 and 2.1) definitely can not, suggesting that additional alternative inactive conformations exist in solution.

Buffers↗

PubMed1998

Thionucleobases as intrinsic photoaffinity probes of nucleic acid structure and nucleic acid-protein interactions.

In the past few years thionucleobases have been extensively used as intrinsic photolabels to probe the structure in solution of folded RNA molecules and to identify contacts within nucleic acids and/or between nucleic acids and proteins, in complex nucleoprotein assemblies. These thio residues such as 4-thiouracil found in E. coli tRNA and its non-natural congeners 4-thiothymine, 6-thioguanine and 6-mercaptopurine absorb light at wavelengths longer than 320 nm and, thus, can be selectively photoactivated. Synthetic or enzymatic procedures have been established, allowing the random or site-specific incorporation of thionucleotide(s) within a RNA (DNA) chain which, in most cases, retains unaltered structural and biological properties. Owing to the high photoreactivity of their triplet state (intersystem yield close to unity), 4-thiouracil and 4-thiothymine derivatives exhibit a high photocrosslinking ability towards pyrimidines (particularly thymine) but also purines. From the nature of the photoproducts obtained in base or nucleotide mixtures and in dinucleotides, the main photochemical pathway was identified as a (2 + 2) photoaddition of the excited C-S bond onto the 5, 6 double bond of pyrimidines yielding thietane intermediates whose structure could be characterized. Depending on the mutual orientation of these bonds in the thietanes, their subsequent dark rearrangement yielded, respectively, either the 5-4 or 6-4 bipyrimidine photoadduct. A similar mechanism appears to be involved in the formation of the unique photoadduct formed between 4-thiothymidine and adenosine. The higher reactivity of thymine derived acceptors can be explained by an additional pathway which involves hydrogen abstraction from the thymine methyl group, followed by radical recombination, leading to methylene linked bipyrimidines. The high photocrosslinking potential of thionucleosides inserted in nucleic acid chains has been used to probe RNA-RNA contacts within the ribosome permitting, in particular, the elucidation of the path of mRNA throughout the small ribosomal subunit. Functional interactions between the mRNA spliced sites and U RNAs could be detected within the spliceosome. Analysis of the photocrosslinks obtained within small endonucleolytic ribozymes in solution led to a tertiary folded pseudo-knot structure for the HDV ribozyme and allowed the construction of a Y form of a hammerhead ribozyme, which revealed to be in close agreement with the structure observed in crystals. Thionucleosides incorporated in nucleic acids crosslink efficiently amino-acid residues of proteins in contact with them. Despite the fact that little is known about the nature of the photoadducts formed, this approach has been extensively used to identify protein components interacting at a defined nucleic acid site and applied to various systems (replisome, spliceosome, transcription complexes and ribosomes).

Animals↗

PubMed1996

Folding of the HDV antigenomic ribozyme pseudoknot structure deduced from long-range photocrosslinks.

A trans-acting system has been designed in order to explore the three-dimensional structure of the anti-genomic HDV ribozyme. In this system, the substrate (SANT) is associated by base-pairing to the catalytic RNA (RzANT) forming helix H1. RzANT is able to cleave specifically the RNA substrate as well as a deoxysubstrate analogue containing a single ribocytidine at the cleavage site (position -1). This demonstrates that such deoxysubstrate analogues are valuable tools for structural studies of this ribozyme domain. They form however weak complexes with RzANT which is due in part to their ability to fold as stable hairpins unlike the RNA substrate. Using a set of full deoxy or of mixed deoxy-ribo substrate analogues site-specific substituted with the photoaffinity probe deoxy-4-thiouridine, ds4U, at a defined position, we were able to determine a number of long range contacts between the substrate and the ribozyme core. In particular, crosslinks between substrate position -1 and position -2 with residues C15, G19 and C67, thought to be involved in the ribozyme catalytic site, were detected. A three dimensional model of the antigenomic ribozyme system, derived from the structure proposed by Tanner et al. [Current Biol (1994) 4, 488-498] for the genomic system was constructed. Apart from residue deletion or insertion, only minor accommodations were needed to account for all photocrosslinks but one which is attributed to an alternative hybridization of the substrate with the ribozyme. This study therefore further supports the structure proposed by Tanner et al. for the pseudoknot model.

Base Sequence↗

PubMed1996

Interaction of human leukocyte elastase with a N-aryl azetidinone suicide substrate: Conformational analyses based on the mechanism of action of serine proteinases.

The three-dimensional interaction of the enzyme-activated (suicide) inhibitor AA 231-1 [N-(2-chloromethyl)-3, 3-difluoro-azetidin-2-one] with human leukocyte elastase has been studied using computer graphics and molecular mechanics. Systematic conformational analyses and energy minimizations have been performed for the inhibitor AA 231-1 and its presumed complexes formed during the enzymatic process of inactivation, i.e., the Michaelis complex, the acyl-enzyme, and the inactivated enzyme with the covalently bound inhibitor. The beta-lactam ring characteristics of modeled AA 231-1 were in agreement with crystallographic data of related structures. Lowest energy conformations were found when the angle between the planes of the beta-lactam ring and that of its phenyl substituent was about -60 or 60 degrees. To study the interaction with the enzyme, the enzyme-inhibitor complexes were constructed by docking the inhibitor in the active site using enzyme coordinates from an X-ray crystallographic structure. The whole enzyme structure was used for conformational analyses and energy mechanics. Favorable conformations for the Michaelis complex have been obtained in which the carbonyl oxygen of the inhibitor was located in the oxyanion hole and the hydroxyl of Ser195 was in position to interact with the beta-lactam carbonyl carbon on the alpha face of AA 231-1. Simulations of the approach of the benzylic carbon by the nucleophilic amino acid His40 or His57 through an SN2 displacement on the halomethyl group of AA 231-1 were performed. The results agreed with the alkylation of the imidazole nitrogen N epsilon 2 of His57 leading to the inactivated enzyme (bis-adduct form).

Azetidines↗

PubMed1995

Hammerhead ribozyme: a three dimensional model based on photo-crosslinking data.

The hammerhead ribozyme is a small catalytic RNA motif made up of 3 base-paired stems connected by conserved sequences which are essential for catalysis. We have modelled its 3 dimensional structure, taking advantage of proximity data between several substrate and ribozyme residues determined by photo-crosslinking experiments. It is characterized by an Y shape of the 3 stems stabilized in the central core by a network of hydrogen bonds involving in particular 2 non Watson-Crick G:A base-pairs. The 5' conserved sequence CUGA makes a sharp turn, the G residue exchanging hydrogen bonds with a conserved base-pair of stem III. The substrate is stretched at the cleavage site. Overall this structure is consistent with that deduced from X-ray crystallography but differences are observed at the level of the CUGA turn.

Cross-Linking Reagents↗

PubMed1987

DNA bis-intercalators as new anti-tumour agents: modulation of the anti-tumour activity by the linking chain rigidity in the ditercalinium series.

Ditercalinium (2,2'-([4,4'-bipiperidine-1,1'-diyl] di-2,1-ethane diyl) bis (10-methoxy-7H-pyrido[4,3c] carbazolium) tetra(methyl sulphonate--NSC 366241), a DNA bis-intercalating compound presently under clinical trial, elicits an original mechanism of action and thus appears as the first of a new class of anti-tumour drugs. Previous studies have shown that a reduced flexibility of the linking chain of these dimers is essential for their biological activity. In order to analyze their mechanism of action at the molecular level and to obtain structure-activity relationships in this series, new derivatives with additional methylene groups between the two piperidine rings have been synthesized. Whereas the addition of a single methylene group in the chain preserves the anti-tumour activity of the dimers, the addition of a second methylene diminishes it; the addition of three methylenes completely abolishes it. Lengthening of sonicated DNA and unwinding of supercoiled DNA support a bis-intercalation mechanism for these drugs. In addition, analyses of poly d(A-T) melting curves in the presence of the drugs, and competition experiments with ethidium dimer, show that these compounds bind to DNA with high affinity (10(7)-10(8) M-1). N.m.r. studies of the dimers in aqueous medium show that the introduction of a single methylene group in the linker leads to compounds with a conformationally-induced decrease of intermolecular stacking interactions, which might be related to the DNA affinity enhancement observed for these dimers. Different hypotheses concerning structure-activity relationships in the different series are discussed.

Animals↗

PubMed1986

Cytotoxicity and SOS-inducing ability of ethidium and photoactivable analogs on E. coli ethidium-bromide-sensitive (Ebs) strains.

In a recently-characterized ethidium-bromide-sensitive E. coli strain, DNA appears to be much more accessible to DNA-binding agents. This strain therefore appears to be of interest for studying the mutagenic properties of chemicals. For this purpose, a series of ethidium-sensitive E. coli strains (Ebs) with normal and defective DNA-repair capacity was constructed and made lysogenic for lambda (sfiA::lacZ). These strains were used to study the cytotoxicity and SOS-inducing ability of ethidium and its two photoactivable analogs 8-azido- and 3,8-diazido-ethidium. When non-covalent DNA complexes are formed, these dyes elicit only a bacteriostatic effect in the Ebs strains, which is almost independent of the strain's DNA-repair capacity. The SOS system is not induced. When covalent DNA adducts are formed after photoactivation of ethidium azido analogs, the effects are quite different. The formation of about 5 DNA monoadducts per cell induces a lethal hit in the Ebs uvrB recA strain and measurable SOS induction in the Ebs uvrB (lambda (sfiA::lacZ) strain. The formation of more than 1000 DNA adducts in the Ebs strain with normal DNA-repair capacity does not induce any measurable cytotoxic effect.

Azides↗

PubMed1985

DNA tris-intercalation: first acridine trimer with DNA affinity in the range of DNA regulatory proteins. Kinetic studies.

A trimer made up of three acridine chromophores linked by a positively charged poly(aminoalkyl) chain was synthesized as a potential tris-intercalating agent. The length of the linking chain was selected to allow intercalation of each chromophore according to the excluded site model. 1H NMR studies have shown that, at 5 mM sodium, pH 5, the acridine trimer occurred under a folded conformation stabilized by stacking interactions between the three aromatic rings. DNA tris-intercalation of the dye at a low dye/base pair ratio was shown by measurements of both the unwinding of PM2 DNA and the lengthening of sonicated rodlike DNA. The trimer exhibits a high DNA affinity for poly[d(A-T)] (Kapp = 8 X 10(8) M-1, 1 M sodium) as shown by competition experiments with ethidium dimer. Kinetic studies of both the association with poly[d(A-T)] and the exchange between poly[d(A-T)] and sonicated calf thymus DNA have been performed as a function of the ionic strength. In 0.3 M sodium the on-rate constant (k1 = 2.6 X 10(7) M-1 s-1) is similar to that reported for other monoacridines or bis(acridines), whereas the off-rate constant is much smaller (k-1 = 1.2 X 10(-4) s-1), leading to an equilibrium binding constant as large as Kapp = 2.2 X 10(11) M-1. A plot of log (k1/k-1) as a function of log [Na+] yielded a straight line whose slope shows that 5.7 ion pairs (out of 7 potential) are formed upon the interaction with DNA. From this linear relationship a Kapp value of 10(14) M-1 in 0.1 M sodium can be estimated. Such a value reaches and even goes beyond that of some DNA regulatory proteins. This acridine trimer appears to be the first synthetic ligand with such a high DNA affinity.

Acridines↗