Highly Mutagenic Exocyclic DNA Adducts Are Substrates for the Human Nucleotide Incision Repair Pathway

作者：Prorok Paulina; Saint Pierre Christine; Gasparutto Didier; Fedorova Olga S; Ishchenko Alexander A; Leh Herve; Buckle Malcolm; Tudek Barbara^*; Saparbaev Murat

来源：PLos One, 2012, 7(12): e51776.

DOI：10.1371/journal.pone.0051776

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 the formation of etheno(e)-bases including 1, N-6-ethenoadenine (epsilon A) and 3,N-4-ethenocytosine (epsilon C). The epsilon A and epsilon C 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 59 to a damaged base in a DNA glycosylase-independent manner. %26lt;br%26gt;Methodology/Principal Findings: Here we have characterized the substrate specificity of human major AP endonuclease 1, APE1, towards epsilon A, epsilon C, thymine glycol (Tg) and7,8-dihydro-8-oxoguanine (8oxoG) residues when present in duplex DNA. APE1 cleaves oligonucleotide duplexes containing epsilon A, epsilon C and Tg, but not those containing 8oxoG. Activity depends strongly on sequence context. The apparent kinetic parameters of the reactions suggest that APE1 has a high affinity for DNA containing e-bases but cleaves DNA duplexes at an extremely slow rate. Consistent with this observation, oligonucleotide duplexes containing an e-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 epsilon A.T and epsilon C.G duplexes generates, as expected, DNA fragments containing 5%26apos;-terminal epsilon-base residue. %26lt;br%26gt;Conclusions/Significance: The fact that epsilon-bases and Tg in duplex DNA are recognized and cleaved by APE1 in vitro, suggests that NIR may act as a backup pathway to BER to remove a large variety of genotoxic base lesions in human cells. Citation: Prorok P, Saint-Pierre C, Gasparutto D, Fedorova OS, Ishchenko AA, et al. (2012) Highly Mutagenic Exocyclic DNA Adducts Are Substrates for the Human Nucleotide Incision Repair Pathway. PLoS ONE 7(12): e51776. doi:10.1371/journal.pone.0051776

• 出版日期2012-12-14
• 单位中国地震局