title: Highly mutagenic exocyclic DNA adducts are substrates for the human nucleotide incision repair pathway.
abstract: 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(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.
  
  
  RESULTS
  Here 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 IC(50) 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
  The 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.
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Paulina
authors.initials: P
authors.lastname: Prorok
authors.forename: Christine
authors.initials: C
authors.lastname: Saint-Pierre
authors.forename: Didier
authors.initials: D
authors.lastname: Gasparutto
authors.forename: Olga S
authors.initials: OS
authors.lastname: Fedorova
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Ishchenko
authors.forename: Hervé
authors.initials: H
authors.lastname: Leh
authors.forename: Malcolm
authors.initials: M
authors.lastname: Buckle
authors.forename: Barbara
authors.initials: B
authors.lastname: Tudek
authors.forename: Murat
authors.initials: M
authors.lastname: Saparbaev
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000378:metabolism; D055162:Biocatalysis; D002457:Cell Extracts; D002474:Cell-Free System; D003596:Cytosine / Q000031:analogs & derivatives / Q000378:metabolism; D018736:DNA Adducts / Q000737:chemistry / Q000378:metabolism*; D045647:DNA Glycosylases / Q000378:metabolism; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D009153:Mutagens / Q000378:metabolism*; D009841:Oligonucleotides / Q000378:metabolism; D010084:Oxidation-Reduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013941:Thymine / Q000031:analogs & derivatives / Q000378:metabolism; D013997:Time Factors
chemical_list: C018289:3,N(4)-ethenocytosine; D002457:Cell Extracts; D018736:DNA Adducts; D009153:Mutagens; D009841:Oligonucleotides; C004981:1,N(6)-ethenoadenine; C029389:thymine glycol; D003596:Cytosine; D045647:DNA Glycosylases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D000225:Adenine; D013941:Thymine