Highly mutagenic exocyclic DNA adducts are substrates for the human nucleotide incision repair pathway

Background: Oxygen free radicals induce lipid peroxidation (LPO) that damages and breaks polyunsaturated fatty acids in cell membranes. LPO-derived aldehydes and hydroxyalkenals react with DNA leading to formation of etheno(ε)-bases including 1,N6-ethenoadenine (εA) and 3,N4-ethenocytosine (εC). The εA and εC residues are highly mutagenic in mammalian cells and eliminated in the base excision repair (BER) pathway and/or by AlkB family proteins in the direct damage reversal process. BER initiated by DNA glycosylases is thought to be the major pathway for the removal of non-bulky endogenous base damage. Alternatively, in the nucleotide incision repair (NIR) pathway, the apurinic/apyrimidinic (AP) endonucleases can directly incise DNA duplex 5’ to a damaged base in a DNA glycosylase-independent manner. Methodology/Principal Findings: Here, we characterized the substrate specificity of human major AP endonuclease 1, APE1, towards εA, εC, thymine glycol (Tg) and 7,8-dihydro-8-oxoguanine (8oxoG) residues when present in duplex DNA. APE1 cleaves oligonucleotide duplexes containing εA, εC and Tg, but not those containing 8oxoG. The activity depends strongly on sequence context. The apparent kinetic parameters of the reactions suggest that APE1 has high affinity to DNA containing ε-bases but cleaves DNA duplex at an extremely slow rate. Consistent with this observation, the oligonucleotide duplexes containing an ε-base strongly inhibit AP site nicking activity of APE1 with IC50 values in the range of 5-10 nM. MALDI-TOF MS analysis of the reaction products demonstrated that APE1-catalyzed cleavage of εA•T and εC•G duplexes generates as expected DNA fragments containing 5’-terminal ε-base residue. Conclusions/Significance: The fact that ε-bases and Tg in duplex DNA are recognized and cleaved by APE1 in vitro, suggest that NIR may act as a backup pathway to BER one to remove a large variety of genotoxic base lesions in human cells

Réparation de l'ADN et mutagenèse dans les mitochondries des vertébrés : preuve de l'asymétrie de l'héritage des brins d'ADN

International audienceA variety of endogenous and exogenous factors induce chemical and structural alterations in cellular DNA in addition to the errors occurring throughout DNA synthesis. These types of DNA damage are cytotoxic, miscoding or both and are believed to be at the origin of cancer and other age-related diseases. A human cell, aside from nuclear DNA, contains thousands of copies of mitochondrial DNA (mtDNA), a double-stranded, circular molecule of 16,569 bp. It has been proposed that mtDNA is a critical target of reactive oxygen species: by-products of oxidative phosphorylation that are generated in the organelle during aerobic respiration. Indeed, oxidative damage to mtDNA is more extensive and persistent as compared to that to nuclear DNA. Although transversions are the hallmark of mutations induced by reactive oxygen species, paradoxically, the majority of mtDNA mutations that occur during ageing and cancer are transitions. Furthermore, these mutations show a striking strand orientation bias: T→C/G→A transitions preferentially occur on the light strand, whereas C→T/A→G on the heavy strand of mtDNA. Here, we propose that the majority of mtDNA progenies, created after multiple rounds of DNA replication, are derived from the heavy strand only, owing to asymmetric replication of the DNA strand anchored to the inner membrane via the D-loop structure.Divers facteurs endogènes et exogènes induisent des altérations chimiques et structurelles dans l'ADN cellulaire, en plus des erreurs qui se produisent tout au long de la synthèse de l'ADN. Ces types de dommages à l'ADN sont cytotoxiques, dus à un mauvais codage ou aux deux, et on pense qu'ils sont à l'origine du cancer et d'autres maladies liées à l'âge. Une cellule humaine, outre l'ADN nucléaire, contient des milliers de copies de l'ADN mitochondrial (ADNmt), une molécule circulaire double brin de 16 569 pb. Il a été proposé que l'ADNmt soit une cible critique des espèces réactives de l'oxygène : des sous-produits de la phosphorylation oxydative qui sont générés dans l'organelle pendant la respiration aérobie. En effet, les dommages oxydatifs de l'ADNmt sont plus étendus et plus persistants que ceux de l'ADN nucléaire. Bien que les transversions soient la marque des mutations induites par les espèces réactives de l'oxygène, paradoxalement, la majorité des mutations de l'ADNmt qui se produisent au cours du vieillissement et du cancer sont des transitions. De plus, ces mutations présentent un biais d'orientation des brins frappant : A→G/G→A transitions se produisent de préférence sur le brin léger, tandis que T→C/A→G sur le brin lourd de l'ADNmt. Ici, nous proposons que la majorité des descendants de l'ADNmt, créés après plusieurs cycles de réplication de l'ADN, soient dérivés du brin lourd uniquement, en raison de la réplication asymétrique du brin d'ADN ancré à la membrane interne via la structure en boucle D.Traduit avec www.DeepL.com/Translator (version gratuite