Misincorporation of dAMP opposite 2-hydroxyadenine, an oxidative form of adenine

Abstract

Nucleotide incorporation opposite an oxidative form of adenine, 2-hydroxyadenine (2-OH-Ade) was investigated. When a primed template with 2-OH-Ade was treated with an exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase I (KFexo-), recombinant rat DNA polymerase beta (pol beta) or calf thymus DNA polymerase alpha (pol alpha), incorporation of dTMP and dAMP was observed. In addition, KFexo- inserted dGMP as well. A steady-state kinetic study indicated that the insertion of dAMP and dTMP opposite the DNA lesion occurred with similar frequency with KFexo- and pol beta. Insertion of dTMP opposite 2-OH-Ade was favored to that of dAMP by pol alpha. Chain extension from the A.2-OH-Ade pair is less favored than that from the T.2-OH-Ade pair by all three DNA polymerase. Analysis of full-length products of in vitro DNA synthesis showed that dTMP and dAMP were incorporated by DNA polymerases and that exonuclease-proficient and -deficient Klenow fragments also inserted dGMP opposite 2-OH-Ade. These results suggest that formation of 2-OH-Ade from A in DNA will induce A-->T and A-->C transversions in cells.

UvrA and UvrB enhance mutations induced by oxidized deoxyribonucleotides

“…These purified deoxyribonucleotides each eluted as a single peak in both reverse-phase and anion-exchange high performance liquid chromatography (HPLC), and their purities were estimated to be more than 99% (data not shown). Oligodeoxyribonucleotides with 8-OH-Gua and 2-hydroxyadenine (2-OH-Ade) were synthesized by the phosphoramidite method, and were purified using both reverse-phase and anion-exchange HPLC, as described previously [29]. Other oligodeoxyribonucleotides were purchased from Sigma Genosys Japan (Ishikari, Japan) in purified forms.…”

Mutagenicities of 8-Hydroxyguanine and 2-Hydroxyadenine Produced by Reactive Oxygen Species

“…30,31) DNA polymerases inserted various nucleotides opposite 2-OH-Ade in the presence of a single dNTP, depending upon the sequence contexts and the DNA polymerases used. The two mammalian DNA polymerases (a and b) incorporated dCTP most frequently among the 'incorrect' nucleotides, while dGTP was inserted more efficiently than dCTP and dATP by the E. coli Klenow fragment.…”

“…4 length products obtained by in vitro DNA synthesis in the presence of the four nucleotides. 30,31) The mutations induced by 2-OH-Ade in E. coli and mammalian cells were analyzed using a ds shuttle vector (Table 3). 32,33) The oxidized adenine was incorporated into 5Ј-G(2-OH-Ade)C-3Ј and 5Ј-T(2-OH-Ade)A-3Ј sequences, based on the results of the in vitro experiments.…”

Neighbouring bases in template influence base-pairing of isoguanine

“…It demonstrates, however, that sequence context exerts an influence on basepairing in general, which in turn can alter the mutagenic potential of iG. In fact, studies performed by Kasai and co-workers [7,48] gave experimental evidence that incorporation of four natural dNTPs opposite iG is also sequence dependent. In addition to incorporation of dTTP occurring in each case, preferred incorporation of dCTP and dGTP opposite iG was observed along several contexts tested, with the exception of the 3h-AiGT-5h sequence, in which iG preferentially directs dATP incorporation by α and β mammalian DNA polymerases and by the exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase I.…”

“…However, iG (1,2-dihydro-2-oxoadenine or 2-hydroxyadenine) is formed in DNA, among other oxidized bases, in the reaction of reactive oxygen species with adenine [5]. The miscoding, mutagenicity and repair of this oxidized adenine derivative have been extensively studied [6][7][8][9][10][11][12].…”