FORMATION OF 2-HYDROXYDEOXYADENOSINE TRIPHOSPHATE, AN OXIDATIVELY DAMAGED NUCLEOTIDE, AND ITS INCORPORATION BY DNA-POLYMERASES - STEADY-STATE KINETICS OF THE INCORPORATION
FORMATION OF 2-HYDROXYDEOXYADENOSINE TRIPHOSPHATE, AN OXIDATIVELY DAMAGED NUCLEOTIDE, AND ITS INCORPORATION BY DNA-POLYMERASES - STEADY-STATE KINETICS OF THE INCORPORATION
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DOI：
10.1074/jbc.270.33.19446
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发表时间：
1995-08-18
影响因子：
4.8
通讯作者：
KASAI, H
中科院分区：
文献类型：
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作者：
KAMIYA, H;KASAI, H
我们发现，通过氧自由基(Fe2+-EDTA)处理da、dATP以及单链和双链DNA，腺嘌呤的C-2位发生了羟基化。当单体和多核苷酸分别处理时，这种氧化损伤的碱基2-羟基腺嘌呤的产量分别是8-羟基鸟嘌呤的3-6倍和40倍。为了确定受损的核苷酸2-羟基脱氧腺苷三磷酸(2-OH-dATP)是否被掺入到生长中的DNA中，并揭示与2-OH-dATP掺入相反的核苷酸的种类，我们利用小牛胸腺DNA聚合酶α和大肠杆菌DNA聚合酶I的Klenow片段在2-OH-dATP存在的情况下进行了体外DNA合成。DNA聚合酶α将T和C相反的核苷酸掺入DNA模板中。另一方面，在使用Klenow片段的实验中，只观察到与T相反的2-OH-dATP的掺入。稳态动力学研究表明，与T相反的DNA聚合酶α对2-OH-dATP的掺入仅为C的4.5倍。这些结果表明，2-OH-dATP是DNA聚合酶的底物，是DNA聚合酶的底物，并被复制的DNA聚合酶错误地掺入。
We found that hydroxylation occurs at the C-2 position of adenine by oxygen radical treatment (Fe2+-EDTA) of dA, dATP, and single- and double-stranded DNA. This oxidatively damaged base, 2-hydroxyadenine, was produced 3-6-fold and 40-fold less than 8-hydroxyguanine when monomers and polynucleotides, respectively, were treated. To determine whether the damaged nucleotide, 2-hydroxydeoxyadenosine triphosphate (2-OH-dATP), is incorporated into a growing DNA, and to reveal the kinds of nucleotides opposite which 2-OH-dATP is incorporated, calf thymus DNA polymerase alpha and the Klenow fragment of Escherichia coli DNA polymerase I were used in in vitro DNA synthesis in the presence of 2-OH-dATP. DNA polymerase alpha incorporated the nucleotide opposite T and C in the DNA template. On the other hand, in an experiment using the Klenow fragment, incorporation of 2-OH-dATP was observed only opposite T. Steady-state kinetic studies indicated that incorporation of 2-OH-dATP by DNA polymerase alpha opposite T was favored over that opposite C by a factor of only 4.5, These results indicate that 2-OH-dATP, an oxidatively damaged nucleotide, is a substrate for DNA polymerases and is incorporated incorrectly by the replicative DNA polymerase.