Major oxidative products of cytosine are substrates for the nucleotide incision repair pathway.
Most common point mutations occurring spontaneously or induced by ionizing radiation are C-->T transitions implicating cytosine as the target. Oxidative cytosine derivatives are the most abundant and mutagenic DNA damage induced by oxidative stress. Base excision repair (BER) pathway initiated by DNA glycosylases is thought to be the major pathway for the removal of these lesions. However, in alternative nucleotide incision repair (NIR) pathway the apurinic/apyrimidinic (AP) endonucleases incise DNA duplex 5' to an oxidatively damaged base in a DNA glycosylase-independent manner. Here, we characterized the substrate specificity of human major AP endonuclease, Ape1, towards 5-hydroxy-2'-deoxycytidine (5ohC) and alpha-anomeric 2'-deoxycytidine (alphadC) residues. The apparent kinetic parameters of the reactions suggest that Ape1 and the DNA glycosylases/AP lyases, hNth1 and hNeil1 repair 5ohC with a low efficiency. Nevertheless, due to the extremely high cellular concentration of Ape1, NIR was the major activity towards 5ohC in cell-free extracts. To address the physiological role of NIR function, we have characterized naturally occurring Ape1 variants including amino acids substitutions (E126A, E126D and D148E) and N-terminal truncated forms (NDelta31, NDelta35 and NDelta61). As expected, all Ape1 mutants had proficient AP endonuclease activity, however, truncated forms showed reduced NIR and 3'-->5' exonuclease activities indicating that these two functions are genetically linked and governed by the same amino acid residues. Furthermore, both Ape1-catalyzed NIR and 3'-->5' exonuclease activities generate a single-strand gap at the 5' side of a damaged base but not at an AP site in duplex DNA. We hypothesized that biochemical coupling of the nucleotide incision and exonuclease degradation may serve to remove clustered DNA damage. Our data suggest that NIR is a backup system for the BER pathway to remove oxidative damage to cytosines in vivo.
Daviet
Stéphane
S
Groupe Réparation de l'ADN, UMR 8126 CNRS, Univ Paris-Sud, Institut Gustave Roussy, 94805 Villejuif Cedex, France.
Couvé-Privat
Sophie
S
Gros
Laurent
L
Shinozuka
Kazuo
K
Ide
Hiroshi
H
Saparbaev
Murat
M
Ishchenko
Alexander A
AA
D045647:DNA Glycosylases / Q000378:metabolism; D004260:DNA Repair; D045643:DNA Repair Enzymes; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics / Q000378:metabolism*; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000378:metabolism; D043223:Deoxyribonuclease IV (Phage T4-Induced) / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D015398:Signal Transduction; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D029701:Saccharomyces cerevisiae Proteins; D003841:Deoxycytidine; C074071:5-hydroxy-2'-deoxycytidine; D004706:Endodeoxyribonucleases; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; C083274:Apn1 protein, S cerevisiae; D043224:Deoxyribonuclease (Pyrimidine Dimer); C468347:NTHL1 protein, human; D045647:DNA Glycosylases; C473634:NEIL1 protein, human; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D045643:DNA Repair Enzymes