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 (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 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 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.

Mechanism of the Formation of Electronically Excited Species by Oxidative Metabolic Processes: Role of Reactive Oxygen Species

“…Whereas the endoperoxide decomposition leads to the termination of lipid peroxidation and protein oxidation, the decomposition of ROOH leads to the propagation of oxidizing chain reactions. It has been reported that the oxidation of nucleic acids by 1 O 2 results in the alternation of the bases [87,88,89]. A high reactivity of 1 O 2 towards guanine has been shown, resulting in the formation of 8-oxo-7,8-dihydro-2-deoxyguanosine [87,90,91].…”

Double-headed nucleotides as xeno nucleic acids: information storage and polymerase recognition

“…The study was conducted on a primer-template system consisting of a previously studied 21,22 18 nt primer (P4) that was fluorescently labelled with fluorescein (FAM) at the 5′-position and a 26 nt template: the unmodified T1 or the U A -modified T2, in which a 5′-TA dinucleotide have been exchanged with the U A double-headed nucleotide. The primertemplate duplex (0.1 µM) was incubated in the presence of the particular DNA polymerase and U A TP (1000 µM), and the products of the reaction were separated by denaturing 20% polyacrylamide gel electrophoresis (PAGE) and visualized by fluoroimaging (Fig.…”

La pharmacogénétique/pharmacogénomique en psychiatrie, perspectives théoriques et perspectives pour l'avenir

“…Quelques principes de base de la génomique L'encodage des informations contenues dans l'acide désoxyribonucléique (ADN) est effectué par l'intermédiaire de quatre bases azotées, les nucléotides adénine (A), cytosine (C), guanine (G) et thymine (T) qui sont placés linéairement sur la séquence d'ADN, et sont complémentaires à une deuxième séquence, l'ensemble formant la double hélice bien connue de l'ADN [9]. La double hélice est formée selon le modèle de complémentarité de Watson et Crick qui implique une liaison non covalente du nucléotide A de la séquence -« sens » avec son homologue T de la séquence -« anti sens » et celle de C à G et vice versa [10].…”