Abstract
BackgroundOxygen 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(ε)-bases including 1,N 6-ethenoadenine (εA) and 3,N 4-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 FindingsHere we have 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. Activity depends strongly on sequence context. The apparent kinetic parameters of the reactions suggest that APE1 has a high affinity for DNA containing ε-bases but cleaves DNA duplexes at an extremely slow rate. Consistent with this observation, 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/SignificanceThe fact that ε-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.
Highlights
The etheno (e) ring system is formed by the attack of reactive bifunctional epoxides or aldehydes at the exocyclic nitrogen atoms of DNA bases, followed by dehydration and ring closure [1,2]
Containing Ethenobases Previously, we have investigated the cleavage of eANT and eCNG duplexes by E. coli Nfo, S. cerevisiae Apn1 and human APE1 nucleotide incision repair (NIR)-AP endonucleases using a limited number of sequence contexts and short incubation times from 5 to 30 min
These results suggest that human APE1 has a broader substrate specificity compared to that of the endonuclease IV family of AP endonucleases, and that this property could be specific to mammalian Xth-family of AP endonucleases
Summary
The etheno (e) ring system is formed by the attack of reactive bifunctional epoxides or aldehydes at the exocyclic nitrogen atoms of DNA bases, followed by dehydration and ring closure [1,2]. The ederivatives of DNA bases such as 1,N6-ethenoadenine (eA) and 3,N4-ethenocytosine (eC) (Figure 1) are generated in cellular DNA either by reaction with epoxides that result from the metabolism of various industrial pollutants such as vinyl chloride (VC) or via endogenous processes through the interaction of lipid peroxidation (LPO)-derived aldehydes and hydroxyalkenals [3]. The eA residues are highly mutagenic in mammalian cells, where they mainly lead to eANT to TNA transitions, but are weakly mutagenic in E. coli [8,9]. LPO-derived aldehydes and hydroxyalkenals react with DNA leading to the formation of etheno(e)-bases including 1,N6-ethenoadenine (eA) and 3,N4-ethenocytosine (eC). The eA and eC 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. 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
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