Biochemical behavior of N-oxidized cytosine and adenine bases in DNA polymerase-mediated primer extension reactions
| Autor: | Mitsuo Sekine, Yoshiaki Masaki, Akihiro Ohkubo, Tomomi Kudo, Kohji Seio, Hirosuke Tsunoda |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2011 |
| Předmět: |
DNA polymerase
Base pair Stereochemistry Deoxyribonucleotides DNA-Directed DNA Polymerase Nucleic Acid Denaturation Primer extension Substrate Specificity Cyclic N-Oxides Cytosine chemistry.chemical_compound Genetics Base Pairing DNA Primers Klenow fragment biology Adenine Temperature DNA Templates Genetic DNA Polymerase I Terminator (genetics) Oligodeoxyribonucleotides chemistry Biochemistry Synthetic Biology and Chemistry biology.protein DNA polymerase I |
| Zdroj: | Nucleic Acids Research |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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