title: Biochemical behavior of N-oxidized cytosine and adenine bases in DNA polymerase-mediated primer extension reactions.
abstract: To clarify the biochemical behavior of 2'-deoxyribonucleoside 5'-triphosphates and oligodeoxyribonucleotides (ODNs) containing cytosine N-oxide (C(o)) and adenine N-oxide (A(o)), we examined their base recognition ability in DNA duplex formation using melting temperature (T(m)) experiments and their substrate specificity in DNA polymerase-mediated replication. As the result, it was found that the T(m) values of modified DNA-DNA duplexes incorporating 2'-deoxyribonucleoside N-oxide derivatives significantly decreased compared with those of the unmodified duplexes. However, single insertion reactions by DNA polymerases of Klenow fragment (KF) (exo(-)) and Vent (exo(-)) suggested that C(o) and A(o) selectively recognized G and T, respectively. Meanwhile, the kinetic study showed that the incorporation efficiencies of the modified bases were lower than those of natural bases. Ab initio calculations suggest that these modified bases can form the stable base pairs with the original complementary bases. These results indicate that the modified bases usually recognize the original bases as partners for base pairing, except for misrecognition of dATP by the action of KF (exo(-)) toward A(o) on the template, and the primers could be extended on the template DNA. When they misrecognized wrong bases, the chain could not be elongated so that the modified base served as the chain terminator.
authors.affiliation: Department of Life Science, Tokyo Institute of Technology and CREST, JST, 4259 Nagatsuta, Midoriku, Yokohama 226-8501, Japan.
authors.forename: Hirosuke
authors.initials: H
authors.lastname: Tsunoda
authors.forename: Tomomi
authors.initials: T
authors.lastname: Kudo
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Masaki
authors.forename: Akihiro
authors.initials: A
authors.lastname: Ohkubo
authors.forename: Kohji
authors.initials: K
authors.lastname: Seio
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Sekine
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D020029:Base Pairing; D003497:Cyclic N-Oxides / Q000737:chemistry* / Q000378:metabolism; D003596:Cytosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000737:chemistry*; D004256:DNA Polymerase I / Q000378:metabolism; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D009691:Nucleic Acid Denaturation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D013379:Substrate Specificity; D013696:Temperature; D013698:Templates, Genetic
chemical_list: D003497:Cyclic N-Oxides; D017931:DNA Primers; D003854:Deoxyribonucleotides; D009838:Oligodeoxyribonucleotides; C030755:adenine-N-oxide; D003596:Cytosine; D004247:DNA; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; D000225:Adenine