Jean-François Constant

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).

Synthesis of fluorescent probes for the detection of abasic sites in DNA

Abstract The three luminescent molecules 1–3 were prepared as highly sensitive and specific probes for the quantification of a major DNA lesion, the abasic site. These molecules incorporate in their structure the reactive oxyamino function linked to the Dansyl or Lissamine-Rhodamine fluorophores through amido or polyether chains.

Determination of DNA guanine sites forming photo-adducts with Ru(II)-labeled oligonucleotides; DNA polymerase inhibition by the resulting photo-crosslinking.

The influence of the distance between the anchoring site of the tethered [Ru(TAP)2dip]2+ complex (TAP=1,4,5,8-tetraazaphenanthrene; dip=4,7-diphenyl-1,10-phenanthroline) on a probe sequence and the guanines of the complementary target strand was studied by (1) the luminescence quenching of the complex (by electron transfer) and (2) the oligodeoxyribonucleotide adduct (ODN adduct) formation which results in photo-crosslinking of the two strands. Moving the guanine moieties away from the complex induces an important decrease of the efficiency of both processes, but clearly affects the ODN adduct formation more specifically than the quenching process. From these results, we determined the positions of the guanine bases in the duplex ODN that are able to form a photo-adduct with the tethered complex. We also examined the possible competition between a long-range hole migration in the duplex ODN and the formation of a photo-adduct by using a sequence labeled with the complex at the 5′-phosphate end. Such a hole migration appears to be inefficient as compared to the ODN adduct formation. Finally, we studied the influence of the photo-crosslinking on the function of two different DNA polymerases. A 17-mer Ru(II)-labeled ODN was hybridized to its complementary sequence located on the 5′-side of a 40-mer matrix. After illumination, the elongation of a 13-mer DNA primer hybridized to the 3′-extremity of the same matrix was stopped at a position corresponding to the formation of the ODN adduct.

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.

Synthesis and bioevaluation of 22-hydroxyacuminatine analogs

A series of 22-hydroxyacuminatine analogs was prepared by using different Friedländer condensations. Several of the new compounds were tested for antiproliferative activity on cancer cell lines and for topoisomerase I inhibitory activity.

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.

DNA repair inhibitors

DNA repair is essential for cell survival by preventing the formation of mutations which can be lethal and in some cases at the origin of tumours. The DNA repair may occur directly by enzymatic removal of damage from the nucleobase or indirectly, step by step (recognition of the damage, excision, DNA resynthesis). During chemotherapeutic treatment of cancer, the action of DNA repair proteins may lead to tumour cell resistance. This resistance might be overcome by the use of DNA repair inhibitors. The better characterised repair proteins are O6-alkylguanine alkyltransferases (AGT) and poly(ADP-ribose) polymerase (PARP). AGT removes alkylgroups from guanine O6 position via a one step suicide mechanism. The inhibition of AGT activity results from alkylation of the enzyme with a reactive O6modified guanine analogue. Encouraging results were obtained in combination with mono-alkylating agents on cell cultures. O6-benzylguanine (O6-BG) has been used in clinical trials. PARP is implicated in single strand break ...

Antibiotic drugs aminoglycosides cleave DNA at abasic sites: shedding new light on their toxicity?

Abasic sites are probably the most common lesions in DNA resulting from the hydrolytic cleavage of glycosidic bonds that can occur spontaneously and through DNA alkylation by anticancer agents, by radiotherapy, and during the repair processes of damaged nucleic bases. If not repaired, the abasic site can be mutagenic or lethal. Thus, compounds able to specifically bind and react at abasic sites have attracted much attention for therapeutic and diagnostic purposes. Here, we report on the efficient cleavage activity of characteristic antibiotic drugs of the major aminoglycosides (AG) family at abasic sites introduced either by depurination in a plasmidic DNA or site specifically in a synthetic oligonucleotide. Among the antibiotic AG drugs selected for this study, neomycin B is the most efficient (a 0.1 μM concentration induces 50% cleavage of an abasic site containing DNA). This cleavage activity could be related to aminoglycoside toxicity but also find medicinal applications through potentiation of cancer radiotherapy and chemotherapy with alkylating drugs. In the search for antibiotic and antiviral agents, we have previously described the synthesis of derivatives of the small aminoglycoside neamine, which corresponds to rings I and II of neomycin B constituted of four rings. The cleavage activity at abasic sites of four of these neamine derivatives is also reported in the present study. One of them appeared to be much more active than the parent compound neamine with cleavage efficiency close to that of neomycin.

Nuclear Magnetic Resonance Solution Structure of DNA Featuring Clustered 2′-Deoxyribonolactone and 8-Oxoguanine Lesions

Ionizing radiation, free radicals, and reactive oxygen species produce hundreds of different DNA lesions. Clustered lesions are typical for ionizing radiation. They compromise the efficiency of the base excision repair (BER) pathway, and as a consequence, they are much more toxic and mutagenic than isolated lesions. Despite their biological relevance, e.g., in cancer radiotherapy and accidental exposure, they are not very well studied from a structural point of view, and while insights provided by structural studies contribute to the understanding of the repair process, only three nuclear magnetic resonance (NMR) studies of DNA containing clusters of lesions were reported. Herein, we report the first NMR solution structure of two DNAs containing a bistranded cluster with the 2-deoxyribonolactone and 8-oxoguanine lesions. Both DNA duplexes feature a 2-deoxyribonolactone site in the middle of the sequence of one strand and differ by the relative position of the 8-oxoguanine, staggered 3 or 5 side on the complementary strand at a three-nucleotide distance. Depending on its relative position, the repair of the 8-oxoguanine lesion by the base excision repair protein Fpg is either almost complete or inhibited. We found that the structures of the two DNAs containing a bistranded cluster of two lesions are similar and do not deviate very much from the standard B-form. As no obvious structural deformations were observed between the two duplexes, we concluded that the differences in Fpg activity are not due to differences in their global conformation.

Oligonucleotide-selenide conjugate: Synthesis and its inducible sequence-specific alkylation of DNA

Oligonucleotide-selenium conjugate was designed and synthesized and its sequence-specific cross-linking ability was investigated. The selenide derivatives can generate covalent interstrand cross-linking with its complementary strand through the formation of o-QM intermediate induced by periodate oxidation. A cross-linking reaction yield of up to 50% was obtained. Hydroxyl radical footprinting experiment revealed that the quinone appendage specifically alkylated the cytosine base extending the duplex formed between the conjugate and the target strand.