Biochemical behavior of N -oxidized cytosine and adenine bases in DNA polymerase-mediated primer extension reactions

Author: Tsunoda Hirosuke   Kudo Tomomi   Masaki Yoshiaki   Ohkubo Akihiro   Seio Kohji   Sekine Mitsuo  

Publisher: Oxford University Press

ISSN: 1362-4962

Source: Nucleic Acids Research, Vol.39, Iss.7, 2011-04, pp. : 2995-3004

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Abstract

To clarify the biochemical behavior of 2-deoxyribonucleoside 5-triphosphates and oligodeoxyribonucleotides (ODNs) containing cytosine N-oxide (Co) and adenine N-oxide (Ao), we examined their base recognition ability in DNA duplex formation using melting temperature (Tm) experiments and their substrate specificity in DNA polymerase-mediated replication. As the result, it was found that the Tm values of modified DNADNA 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 Co and Ao 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 Ao 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.

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