Hijacking of the human alkyl-N-purine-DNA glycosylase by 3,N4-ethenocytosine, a lipid peroxidation-induced DNA adduct.

Lipid peroxidation generates aldehydes, which react with DNA bases, forming genotoxic exocyclic etheno(epsilon)-adducts. E-bases have been implicated in vinyl chloride-induced carcinogenesis, and increased levels of these DNA lesions formed by endogenous processes are found in human degenerative disorders. E-adducts are repaired by the base excision repair pathway. Here, we report the efficient biological hijacking of the human alkyl-N-purine-DNA glycosylase (ANPG) by 3,N(4)-ethenocytosine (epsilonC) when present in DNA. Unlike the ethenopurines, ANPG does not excise, but binds to epsilonC when present in either double-stranded or single-stranded DNA. We developed a direct assay, based on the fluorescence quenching mechanism of molecular beacons, to measure a DNA glycosylase activity. Molecular beacons containing modified residues have been used to demonstrate that the epsilonC.ANPG interaction inhibits excision repair both in reconstituted systems and in cultured human cells. Furthermore, we show that the epsilonC.ANPG complex blocks primer extension by the Klenow fragment of DNA polymerase I. These results suggest that epsilonC could be more genotoxic than 1,N(6)-ethenoadenine (epsilonA) residues in vivo. The proposed model of ANPG-mediated genotoxicity of epsilonC provides a new insight in the molecular basis of lipid peroxidation-induced cell death and genome instability in cancer.

Characterisation of new substrate specificities of Escherichia coli and Saccharomyces cerevisiae AP endonucleases.

Despite the progress in understanding the base excision repair (BER) pathway it is still unclear why known mutants deficient in DNA glycosylases that remove oxidised bases are not sensitive to oxidising agents. One of the back-up repair pathways for oxidative DNA damage is the nucleotide incision repair (NIR) pathway initiated by two homologous AP endonucleases: the Nfo protein from Escherichia coli and Apn1 protein from Saccharomyces cerevisiae. These endonucleases nick oxidatively damaged DNA in a DNA glycosylase-independent manner, providing the correct ends for DNA synthesis coupled to repair of the remaining 5'-dangling nucleotide. NIR provides an advantage compared to DNA glycosylase-mediated BER, because AP sites, very toxic DNA glycosylase products, do not form. Here, for the first time, we have characterised the substrate specificity of the Apn1 protein towards 5,6-dihydropyrimidine, 5-hydroxy-2'-deoxyuridine and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine deoxynucleotide. Detailed kinetic comparisons of Nfo, Apn1 and various DNA glycosylases using different DNA substrates were made. The apparent K(m) and kcat/K(m) values of the reactions suggest that in vitro DNA glycosylase/AP lyase is somewhat more efficient than the AP endonuclease. However, in vivo, using cell-free extracts from paraquat-induced E.coli and from S.cerevisiae, we show that NIR is one of the major pathways for repair of oxidative DNA base damage.

Optimisation of dendrimer-mediated gene transfer by anionic oligomers.

BACKGROUND: The application of synthetic vectors for gene transfer has potential advantages over virus-based systems. Their use, however, is limited since they generally lack the efficiency of gene transfer achieved with recombinant viral vectors such as adenovirus. Polyamidoamine (PAMAM) and phosphorus-containing dendrimers (P-dendrimers) are specific polymers with a defined spherical structure. They bind to DNA through electrostatic interactions thus forming complexes that efficiently transfect cells in vitro. METHODS AND RESULTS: The influence of anionic oligomers (oligonucleotides, dextran sulfate) on dendrimer-mediated polyfection of cultured cells has been studied. Anionic oligomers have been found to increase significantly the capacity of the PAMAM and P-dendrimers for DNA delivery into cells when they were mixed with plasmid DNA before addition of dendrimers. The efficiency of the DNA/dendrimer penetration depends on the size, structure and charge of anionic oligomers. CONCLUSIONS: Our results represent an important step towards the optimisation of gene transfer mediated by two types of dendrimers. The use of anionic oligomers improves the efficiency of gene expression within cells. As a consequence, a very efficient cell polyfection can be achieved with a lower plasmid quantity for the PAMAM dendrimer greatly increasing the gene expression level for P-dendrimers.