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Uracil in duplex DNA is a substrate for the human nucleotide incision repair pathway

Prorok, Paulina and Alili, Doria and Saint-Pierre, Christine and Gasparutto, Didier and Zharkov, Dimitry O. and Ischenko, Alexander A. and Tudek, Barbara and Saparbaev, Murat (2013) Uracil in duplex DNA is a substrate for the human nucleotide incision repair pathway. Proceedings of the National Academy of Sciences, 110 (39). E3695-E3703.

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Official URL: http://www.pnas.org/content/110/39/E3695.long


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 tumours. In the majority of species, uracil residues are removed from DNA by specific uracil-DNA glycosylases in the base excision repair (BER) pathway. Alternatively, in certain archaeal organisms uracil residues are eliminated by apurinic/apyrimidinic (AP) endonucleases in the nucleotide incision repair (NIR) pathway. Here, we characterized the substrate specificity of the major human AP endonuclease 1, APE1, towards 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 an 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 exonuclease III family of AP endonucleases is remarkably conserved from Archaea to human. We propose that NIR may act as a backup pathway to BER to remove uracils arising from cytosine deamination.

Item Type:Article
Subjects:Q Science > Q Science (General)
Divisions:Department of Molecular Biology
ID Code:647
Deposited By: Prof. Barbara Tudek
Deposited On:14 Apr 2014 11:32
Last Modified:12 Nov 2014 09:28

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