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J F Constant

Publications and source records attributed to J F Constant.

15 recordsLinked to original sources
PubMed2001

Translesional synthesis on DNA templates containing the 2'-deoxyribonolactone lesion.

A site-specifically modified oligonucleotide containing a single 2'-deoxyribonolactone lesion was used as a template for primer extension reactions catalyzed by M-MuLV reverse transcriptase (RT) and by the Klenow fragments of Escherichia coli DNA polymerase proficient (KF exo(+)) or deficient (KF exo(-)) in exonuclease activity. Analysis of the extension products in the presence of the four dNTPs or of a single dNTP showed that the M-MuLV RT was completely blocked and did not incorporate any dNMP opposite 2'-deoxyribonolactone. KF exo(-) preferentially incorporated nucleotides opposite the lesion following the frequency order dAMP > dGMP >> dTMP approximately dCMP and thus appeared to obey the 'A rule' for preferential incorporation as has been shown previously for the 2'-deoxyribose abasic site. In the sequence context examined, the primer extension by KF exo(-) appeared to be less efficient when dAMP was positioned opposite the lesion as compared with dTMP or dGMP. These two nucleotides promoted a more efficient polymerization accompanied by nucleotide deletion through misalignment incorporations. We therefore predict that the sequence context may strongly influence the translesional synthesis by KF exo(-) and thus the miscoding and mutational potential of the 2'-deoxyribonolactone in E.coli.

Base Sequence↗
PubMed2000

Synthesis and study of a new adenine-acridine tandem, inhibitor of exonuclease III.

A new heterodimer adenine-chain-acridine containing a mixed amido-guanidinium linker chain was synthesized. To achieve the synthesis a new method of introduction of aminoalkyl chain at position 9 of adenine was designed. The heterodimer interacts specifically with the abasic sites in DNA and inhibits the major base excision repair enzyme in Escherichia coli, Exonuclease III.

Acridines↗
PubMed1999

Abasic site recognition in DNA as a new strategy to potentiate the action of anticancer alkylating drugs?

Inhibition of abasic site repair in the cell seems an attractive strategy to potentiate the action of antitumor DNA alkylating drugs. Molecules that bind specifically and strongly to the abasic site are possible candidates to achieve such inhibition. We explored this strategy by preparing molecule 4 that incorporates (1) an aminoacridine intercalator for DNA binding, (2) an adenine moiety for abasic site recognition, and (3) a linker containing two guanidinium functions to increase binding to DNA without inducing cleavage at the base-sensitive abasic site. Compound 4 was compared to analogues containing secondary amines, i.e., 1. We report on synthesis of the new heterodimer 4. We show by physicochemical studies-including determination of association constants with calf-thymus DNA, T(m) measurements, and high-field NMR examination of the complexes formed with abasic DNA duplexes-that 4 binds specifically and more strongly to the abasic site than the analogues. Compound 4 does not cleave abasic plasmid DNA. Compound 4 shows apparent synergy with the anticancer bischloroethylnitrosourea (BCNU) in L1210 and A549 cell lines in vitro. It potentiates BCNU in the in vivo tests. The results favor the pertinence of the strategy.

Animals↗
PubMed1999

Abasic DNA structure, reactivity, and recognition.

Loss of a base in DNA, i.e., creation of an abasic site leaving a deoxyribose residue in the strand, is a frequent lesion that may occur spontaneously, or under the action of radiations and alkylating agents, or enzymatically as an intermediate in the repair of modified or abnormal bases. The abasic site lesion is mutagenic or lethal if not repaired. From a chemical point of view,the abasic site is an alkali-labile residue that leads to strand breakage through beta- and delta- elimination. Progress in the understanding of the chemistry and enzymology of abasic DNA largely relies upon the study of synthetic abasic duplexes. Several efficient synthetic methods have thus been developed to introduce the lesion (or a stable analogue) at defined position in the sequence. Physicochemical and spectroscopic examination of such duplexes, including calorimetry, melting temperature, high-field nmr and molecular modeling indicate that the lesion strongly destabilizes the duplex, although remaining in the canonical B-form with structural modifications strictly located at the site of the lesion. Probes have been developed to titrate the damage in DNA in vitro. Series of molecules have been devised to recognize specifically the abasic site, exhibiting a cleavage activity and mimicking the AP nucleases. Others have been prepared that bind strongly to the abasic site and show promise in potentiating the cytotoxic and antitumor activity of the clinically used nitrosourea (bis-chloroethylnitrosurea).

Antineoplastic Agents↗
PubMed1999

A simple and sensitive method for in vitro quantitation of abasic sites in DNA.

A novel method for the quantitation of abasic sites (AP sites) in DNA is described. As abasic sites can be generated by controlled thermal treatment of base-modified DNA, this method can be used for estimation of the extent of DNA damage resulting from exposure to genotoxic agents. The method involves use of probe molecules 1 and 2 that contain a fluorescent label linked to an aminooxy group which reacts specifically with the aldehydic function of the ring-opened form of abasic sites. The two fluorescent probes 1 and 2 were found to react with 2-deoxyribose, a model substrate, at the optimum of pH 4.0. As spontaneous depurination occurs at low pH, the reactions with abasic DNA were carried out at neutral pH with an excess concentration of the probes. Studies with alkylated, depurinated calf thymus DNA showed that the method is selective and quantitative. Good correlations were found between the level of 7-methylguanine (7-MeGua), generated in vitro in DNA by the methylating agent dimethyl sulfate, and the amount of AP sites as determined by the method presented here. In addition, similar correlations were found when the assay was used to detect abasic sites in DNA isolated from rats treated with carcinogenic alkylating agents. In each case, the level of abasic sites, as expected, is slightly higher than the level of 7-MeGua which is known to represent about 70% of the total modifications of DNA following exposure to the methylating agent. This method may be useful not only in experimental settings but also in studies of DNA damage in humans resulting from chemotherapy or exposure to environmental agents.

Animals↗
PubMed1997

Search for DNA repair inhibitors: selective binding of nucleic bases-acridine conjugates to a DNA duplex containing an abasic site.

The abasic site is one of the most frequent DNA lesions generated by spontaneous or enzymatic cleavage of the N-glycosidic bond. The abasic site is also an intermediate in the nucleotide and base excision DNA repair. We examined molecules which recognize and cleave DNA at the abasic site with high efficiency. These molecules incorporate in their structure a nucleic base for abasic site recognition, an intercalator for DNA binding, and a polyamino linker for ionic interaction and DNA cleavage. Such compounds, by interfering with abasic sites in DNA, are also inhibitors of DNA repair. In order to better understand the parameters of the interaction, we carried out a UV thermal denaturation study of synthetic oligonucleotides containing the lesion both in the absence and in the presence of the drugs. A similar study was also carried out using the corresponding nonmodified oligonucleotide. The results indicate selective binding of the base-chain-intercalator conjugates to the abasic site containing oligonucleotides.

Acridines↗
PubMed1997

NMR and molecular modeling studies of the interaction of artificial AP lyases with a DNA duplex containing an apurinic abasic site model.

Tailor-made molecules, DTAc and ATAc, that incorporate a nucleic base (adenine or 2,6-diaminopurine) linked by a diamino chain to an intercalator (9-amino-6-chloro-2-methoxyacridine) selectively recognize and efficiently cleave abasic sites in DNA via a beta-elimination reaction. The three-dimensional structure of the complexes of DTAc and ATAc bound to a DNA undecamer, the 5'd(C1G2C3A4C5X6C7A8C9G10C11)3' x 3'd(G22C21G20T19G18T17G16T15G14C13G12)5' duplex in which the X residue is a stable abasic site [3-hydroxy-2-(hydroxymethyl)tetrahydrofuran], has been studied by combined NMR-energy minimization methods. Analysis of the NMR spectra reveals that DTAc and ATAc interact with a very similar fashion and form two different complexes with DNA, present in a ratio of 70/30 (+/-10). In both complexes, the acridine ring intercalates exclusively between the C3 x G20 and A4 x T19 base pairs, the linker is located in the minor groove, and the base moiety docks in the abasic site. The principal difference between the major and the minor complexes consists of a 180 degrees rotation of the acridine ring around the Acr-C-N bond within the same intercalation site. Molecular modeling studies with few intermolecular ligand-DNA restraints were used to investigate the geometry of the base pair formed between the diaminopurine of DTAc and the T17 ring. The most energetically favored complex has the 2,6-diaminopurine of DTAc base paired with the T17 ring in a Hoogsteen conformation. The models DTAc and ATAc are also discussed as nuclease mimics and cleaving agents at abasic sites.

DNA↗
PubMed1994

Design of molecules that specifically recognize and cleave apurinic sites in DNA.

We have prepared a series of tailor-made molecules that recognize and cleave DNA at apurinic sites in vitro. These molecules incorporate in their structure different units designed for specific function: an intercalator for DNA binding, a nucleic base for abasic site recognition and a linking chain of variable length and nature (including amino and/or amido functions). The cleavage efficiency of the molecules can be modulated by varying successively the nature of the intercalating agent, the nucleic base and the chain. All molecules bind to native calf thymus DNA with binding constants ranging from 10(4) to 10(6) M-1. Their cleavage activity was determined on plasmid DNA (pBR 322) containing 1.8 AP-sites per DNA-molecule. The minimum requirements for cleavage are the presence of the three units, the intercalator, the nucleic base and at least one amino function in the chain. The most efficient molecules cleave plasmid DNA at nanomolar concentrations. Enzymatic experiments on the termini generated after cleavage of AP-DNA suggest a strand break induced by a beta-elimination reaction. In order to get insight into the mode of action (efficiency, selectivity, interaction), we have used synthetic oligonucleotides containing either a true abasic site at a determined position to analyse the cleavage parameters of the synthetic molecules by HPLC or a chemically stable analog (tetrahydrofuran) of the abasic site for high field 1H NMR spectrometry and footprinting experiments. All results are consistent with a beta-elimination mechanism in which each constituent of the molecule exerts a specific function as indicated in the scheme: DNA targeting, abasic site recognition, phosphate binding and beta-elimination catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Aminothiols linked to quinoline and acridine chromophores efficiently decrease 7,8-dihydro-8-oxo-2'-deoxyguanosine formation in gamma-irradiated DNA.

In a search for more active radioprotective compounds, we have prepared and examined a series of model molecules in which the radioprotective beta-aminothiol unit (free or derivatized as acetate or phosphorothioate) is tethered to the DNA-binding chromophores quinoline and acridine through links of variable length. The modifying activity of these 'hybrid' molecules was estimated by measuring the formation of 8-oxo-2'-deoxyguanosine (8-oxodGuo) in double-strand DNA upon exposure to gamma-rays in oxygen-free solution in the presence of the drugs. We show that all hybrid molecules protect the guanine moiety from oxidation more efficiently than the parent beta-aminothiol units. The degree of protection is the highest for the molecules in which the thiol is linked to the strong binding intercalator acridine through a long polyaminochain.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1988

Heterodimeric molecules including nucleic acid bases and 9-aminoacridine. Spectroscopic studies, conformations, and interactions with DNA.

Heterodimeric molecules have been examined in which the 9-amino-6-chloro-2-methoxyacridine ring is linked to the nucleic acid bases adenine, thymine, and guanine by polymethylenic chains (CH2)n of varying length (n = 3, 5, 6). A detailed analysis has been performed, including hypochromism measurement in the UV, chemical shift variations in Fourier transform proton magnetic resonance, and fluorescence emission. All these techniques show that all molecules exist mainly under folded conformations in water in the temperature range 0-90 degrees C, with the acridine and the base rings being stacked one on top of the other. The thermodynamic parameters for the folded in equilibrium unfolded conformational equilibrium were estimated. The geometry of the intramolecular complexes could be determined. (1) All these data give information on the strength and nature of the base-acridine interactions as a function of different parameters such as solvent, temperature, etc. (2) The molecules under study constitute "spectroscopic models" for the drug-base complexes as they occur in DNA. In particular, we show the dramatic influence of the relative orientations of the two stacked chromophores in the complex upon the magnitude of % H, the percent hypochromism. The quenching and enhancement of acridine fluorescence emission induced respectively by guanine and adenine is evidenced and quantitatively estimated. (3) These base-acridine heterodimers bind to DNA to an extent that is inversely proportional to their degree of intramolecular stacking.

Aminoacridines↗
PubMed1988

9-[(10-(aden-9-yl)-4,8-diazadecyl)amino]-6-chloro-2-methoxy-acridine incises DNA at apurinic sites.

The incision of DNA at apurinic/apyrimidinic sites (AP-sites) by chloro-6-methoxy-2 [(adenyl-9)-11)-4,8 diazadecyl]amino-9 acridine (Ade-Z-Acr), a 9-aminoacridine linked to an adenine, at nanomolar concentrations is described. Moreover, this drug, Ade-Z-Acr, is one of the most efficient drugs which cleaves DNA at AP-sites. The high activity is the result of the composition of the drug, since the individual components have no incising activity in the concentration range studied. The termini left by the Ade-Z-Acr molecule are a 3'deoxyribose and a 5'nucleotide. The termini and the inability of the Ade-Z-Acr to incise DNA with reduced AP-sites suggest that the mechanism of cleavage is beta-elimination.

Acridines↗
PubMed1981

Influence of pH and ionic strength on the lysis of Micrococcus luteus cells by hen lysozyme at low (20 degree C) and high (physiological, 40 degree C) temperature.

From isoactivity curves (showing activity as a function of Ph and ionic strength) it was found that in the pH domain 6.7-8.6 frequently used in experiments involving hen lysozyme, the pH optimum of lysis of Micrococcus luteus cells at low ionic strength (0.02-0.05) by the high-temperature form (40 degree C, physiological temperature) was one to two pH units lower than that by the low-temperature form (20 degree C).

Animals↗
PubMedDate not supplied

Nuclease activity of 1,10-phenanthroline-copper. New conjugates with low molecular weight targeting ligands.

The chemical nuclease activity of 1,10-phenanthroline-copper depends on DNA sequence because the coordination complex has affinity for DNA. In order to target this efficient nucleolytic activity, it is essential to override its inherent specificity. The minimal size of ligands capable of redirecting the specificity has been investigated. A conjugate (HOP) prepared by alkylating Hoechst dye 33258 with 5-(iodoacetamido)-1,10-phenanthroline has a greater preference for A-T-rich regions than the unsubstituted 1,10-phenanthroline-copper complex, reflecting the specificity of this A-T-specific minor-groove binder. However, since quaternizing the dye with 5-(iodoacetamido)-1,10-phenanthroline increases its affinity for DNA, the specificity of cleavage by the conjugate is less than the binding selectivity of the dye. Linking 1,10-phenanthroline with the peptide of the helix-turn-helix domain of the Trp repressor specificity results in a conjugate with greater reactivity for the operator sequence than the unsubstituted complex. The intrinsic affinity of the 1,10-phenanthroline-Cu can only be partially overridden by the conformationally unstable peptide. Attachment of 1,10-phenanthroline to a deoxyoligonucleotide complementary to a single-stranded loop of RNA successfully targets the scission of the chemical nuclease. Cleavage sites are observed not only contiguous to the site of hybridization but also at nonadjacent sequence positions. The latter set of sites must be close in space to the 5' end of the hybridized deoxyoligonucleotide.

Alkylation↗