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.

A new chemical inhibitor of angiogenesis and tumorigenesis that targets the VEGF signaling pathway upstream of Ras.

The efficacy of anti-angiogenic therapies on cancer patients is limited by the emergence of drug resistance, urging the search for second-generation drugs. In this study, we screened an academic chemical library (DCM, University of Grenoble-Alpes) and identified a leader molecule, COB223, that inhibits endothelial cell migration and proliferation. It inhibits also Lewis lung carcinoma (LLC/2) cell proliferation whereas it does not affect fibroblast proliferation. The anti-angiogenic activity of COB223 was confirmed using several in vitro and in vivo assays. In a mouse LLC/2 tumor model, ip administration of doses as low as 4 mg/kg COB223 efficiently reduced the tumor growth rate. We observed that COB223 inhibits endothelial cell ERK1/2 phosphorylation induced by VEGF, FGF-2 or serum and that it acts downstream of PKC and upstream of Ras. This molecule represents a novel anti-angiogenic and anti-tumorigenic agent with an original mechanism of action that deserves further development as an anti-cancer drug.

Synthetic access to the chemical diversity of DNA and RNA 5'-aldehyde lesions.

Hydrogen atom abstraction from the C5'-position of nucleotides in DNA results in direct strand scission by generating alkali-labile fragments from the oxidized nucleotide. The major damage consists in a terminus containing a 5'-aldehyde as part of an otherwise undamaged nucleotide. Moreover it is considered as a polymorphic DNA strand break lesion since it can be borne by any of the four nucleosides encountered in DNA. Here we propose an expeditious synthesis of oligonucleotides (ON) ending with this 5'-aldehyde group (5'-AODN). This straightforward and cheap strategy relies on Pfitzner­Moffatt oxidation performed on solid support followed by a transient protection of the resulting aldehyde function. This method is irrespective of the 5'-terminal nucleobase and most interestingly can be directly extended to RNA to produce the corresponding 5'-AORN. We also report preliminary results on recognition of 5'-AODN by base excision repair (BER) enzymes.

Structure and dynamics of DNA duplexes containing a cluster of mutagenic 8-oxoguanine and abasic site lesions.

Clustered DNA damage sites are caused by ionizing radiation. They are much more difficult to repair than are isolated single lesions, and their biological outcomes in terms of mutagenesis and repair inhibition are strongly dependent on the type, relative position and orientation of the lesions present in the cluster. To determine whether these effects on repair mechanism could be due to local structural properties within DNA, we used (1)H NMR spectroscopy and restrained molecular dynamics simulation to elucidate the structures of three DNA duplexes containing bistranded clusters of lesions. Each DNA sequence contained an abasic site in the middle of one strand and differed by the relative position of the 8-oxoguanine, staggered on either the 3' or the 5' side of the complementary strand. Their repair by base excision repair protein Fpg was either complete or inhibited. All the studied damaged DNA duplexes adopt an overall B-form conformation and the damaged residues remain intrahelical. No striking deformations of the DNA chain have been observed as a result of close proximity of the lesions. These results rule out the possibility that differential recognition of clustered DNA lesions by the Fpg protein could be due to changes in the DNA's structural features induced by those lesions and provide new insight into the Fpg recognition process.

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.

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.

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.

Photoreactive threading agent that specifically binds to abasic sites in DNA.

We report the synthesis and study of a photoreactive nitrobenzamide containing acridine that specifically interacts at abasic site in DNA by threading intercalation and introduces under irradiation a lesion on the opposite strand at the unpaired pyrimidine.

Action of multiple base excision repair enzymes on the 2'-deoxyribonolactone.

Free radical attack on the sugar-phosphate backbone generates oxidized apurinic/apyrimidinic (AP) residues in DNA. 2'-deoxyribonolactone (dL) is a C1'-oxidized AP site damage generated by UV and gamma-irradiation, and certain anticancer drugs. If not repaired dL produces G-->A transitions in Escherichia coli. In the base excision repair (BER) pathway, AP endonucleases are the major enzymes responsible for 5'-incision of the regular AP site (dR) and dL. DNA glycosylases with associated AP lyase activity can also efficiently cleave regular AP sites. Here, we report that dL is a substrate for AP endonucleases but not for DNA glycosylases/AP lyases. The kinetic parameters of the dL-incision were similar to those of the dR. DNA glycosylases such as E. coli formamidopyrimidine-DNA glycosylase, mismatch-specific uracil-DNA glycosylase, and human alkylpurine-DNA N-glycosylase bind strongly to dL without cleaving it. We show that dL cross-links with the human proteins 8-oxoguanine-DNA (hOGG1) and thymine glycol-DNA glycosylases (hNth1), and dR cross-links with Nth and hNth1. These results suggest that dL and dR induced genotoxicity might be strengthened by BER pathway in vivo.

Photoreaction of [Ru(hat)2phen]2+ with guanosine-5'-monophosphate and DNA: formation of new types of photoadducts.

[Ru(hat)2phen]2+ (HAT=1,4,5,8,9,12-hexaazatriphenylene, phen=1,10-phenanthroline) interacts with a good affinity with polynucleotides and DNA by intercalation, despite the presence of a second voluminous ancillary HAT ligand. It photoreacts with guanosine-5'-monophosphate (GMP). From HPLC, ESMS and NMR analyses, it can be concluded that this complex forms photoadducts with GMP. In contrast to the photoadducts isolated with Ru-TAP complexes (TAP=1,4,5,8-tetraazaphenanthrene), the photoadducts with [Ru(hat)2phen]2+ contain a covalent link between the oxygen atom of the guanine unit and a HAT ligand. Formation of oxidised photoadducts and compounds resulting from the addition of two GMP entities to the complex are also detected as side products. In the presence of oligo- and polynucleotides, [Ru(hat)2phen]2+ yields photoadducts when guanine bases are present.

2'-deoxyribonolactone lesion produces G->A transitions in Escherichia coli.

2'-deoxyribonolactone (dL) is a C1'-oxidized abasic site damage generated by a radical attack on DNA. Numerous genotoxic agents have been shown to produce dL including UV and gamma-irradiation, ene-dye antibiotics etc. At present the biological consequences of dL present in DNA have been poorly documented, mainly due to the lack of method for introducing the lesion in oligonucleotides. We have recently designed a synthesis of dL which allowed investigation of the mutagenicity of dL in Escherichia coli by using a genetic reversion assay. The lesion was site-specifically incorporated in a double-stranded bacteriophage vector M13G*1, which detects single-base-pair substitutions at position 141 of the lacZalpha gene by a change in plaque color. In E.coli JM105 the dL-induced reversion frequency was 4.7 x 10(-5), similar to that of the classic abasic site 2'-deoxyribose (dR). Here we report that a dL residue in a duplex DNA codes mainly for thymidine. The processing of dL in vivo was investigated by measuring lesion-induced mutation frequencies in DNA repair deficient E.coli strains. We showed a 32-fold increase in dL-induced reversion rate in AP endonuclease deficient (xth nfo) mutant compared with wild-type strain, indicating that the Xth and Nfo AP endonucleases participate in dL repair in vivo.

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)(2)dip](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.