title: Uracil in duplex DNA is a substrate for the nucleotide incision repair pathway in human cells.
abstract: Spontaneous hydrolytic deamination of cytosine to uracil (U) in DNA is a constant source of genome instability in cells. This mutagenic process is greatly enhanced at high temperatures and in single-stranded DNA. If not repaired, these uracil residues give rise to C → T transitions, which are the most common spontaneous mutations occurring in living organisms and are frequently found in human tumors. In the majority of species, uracil residues are removed from DNA by specific uracil-DNA glycosylases in the base excision repair pathway. Alternatively, in certain archaeal organisms, uracil residues are eliminated by apurinic/apyrimidinic (AP) endonucleases in the nucleotide incision repair pathway. Here, we characterized the substrate specificity of the major human AP endonuclease 1, APE1, toward U in duplex DNA. APE1 cleaves oligonucleotide duplexes containing a single U⋅G base pair; this activity depends strongly on the sequence context and the base opposite to U. The apparent kinetic parameters of the reactions show that APE1 has high affinity for DNA containing U but cleaves the DNA duplex at an extremely low rate. MALDI-TOF MS analysis of the reaction products demonstrated that APE1-catalyzed cleavage of a U⋅G duplex generates the expected DNA fragments containing a 5'-terminal deoxyuridine monophosphate. The fact that U in duplex DNA is recognized and cleaved by APE1 in vitro suggests that this property of the exonuclease III family of AP endonucleases is remarkably conserved from Archaea to humans. We propose that nucleotide incision repair may act as a backup pathway to base excision repair to remove uracils arising from cytosine deamination.
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland.
authors.forename: Paulina
authors.initials: P
authors.lastname: Prorok
authors.forename: Doria
authors.initials: D
authors.lastname: Alili
authors.forename: Christine
authors.initials: C
authors.lastname: Saint-Pierre
authors.forename: Didier
authors.initials: D
authors.lastname: Gasparutto
authors.forename: Dmitry O
authors.initials: DO
authors.lastname: Zharkov
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Ishchenko
authors.forename: Barbara
authors.initials: B
authors.lastname: Tudek
authors.forename: Murat K
authors.initials: MK
authors.lastname: Saparbaev
mesh_terms: D001483:Base Sequence; D055162:Biocatalysis; D002460:Cell Line; D003596:Cytosine / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism; D003641:Deamination; D006801:Humans; D007700:Kinetics; D017020:Methanosarcina / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism*; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013447:Sulfites; D044502:Thymine DNA Glycosylase / Q000378:metabolism; D014498:Uracil / Q000378:metabolism*
chemical_list: D009711:Nucleotides; D013447:Sulfites; D014498:Uracil; D003596:Cytosine; D004247:DNA; D044502:Thymine DNA Glycosylase; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; C009279:sodium bisulfite
keywords: alternative excision repair; evolution; spontaneous DNA base deamination
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