METAL MUTAGENESIS IN VITRO--POLYMERASE KINETICS

体外金属诱变--聚合酶动力学

• 批准号: 3254738
• 负责人: ELIZABETH T SNOW
• 金额: $ 12.57万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3254738
• 关键词: DNA directed DNA polymerase; DNA replication; Escherichia coli; SDS polyacrylamide gel electrophoresis; bacteriophage M13; carcinogens; chemical binding; chemical kinetics; chromium; deoxyguanosine; enzyme activity; magnesium; manganese; methylguanidine; molecular cloning; molecular site; mutagens; nickel; nucleic acid sequence; nucleoside triphosphate; oligonucleotides; site directed mutagenesis; spleen exonuclease; thin layer chromatography

The goals of this research are two-fold: to determine the mechanisms of mutagenesis induced by the carcinogenic metals nickel and chromium and to use Ni(II) and Cr(III) ions as tools to investigate the enzymatic mechanisms responsible for DNA polymerase function and fidelity. Although the mechanisms of carcinogenesis induced by these metals are complex and still largely unknown, both metals can produce DNA base damage (via the intracellular production of active oxygen species) and both Ni(II) and Cr(III) can interact with DNA polymerases in vitro in a biphasic manner to enhance and inhibit polymerase function and fidelity. Do these metal ions also enhance the mutagenic potential of other forms of DNA damage and can they induce mutagenesis by the direct inhibition of polymerase function? The specific aims of this research are: to establish whether either 1) Cr(III) or 2) Ni(II) can alter the kinetics and fidelity of DNA replication past a model mutagenic lesion, O6-methyl guanine; 3) to investigate the effects of these metal ions on the kinetics of DNA polymerase 3'-5' exonuclease function; 4) to determine whether Cr(III) affects the use of deoxynucleotide triphosphate substrates by forming a beta, gamma-Cr(III)-dNTP complex and/or interacting with the Mg+2 binding site on the polymerase; and 5) to examine the biological consequences of DNA replication across an oxidized DNA lesion (8-oxo dG) in the presence of Cr(III) or Ni(II) using a site modified bacteriophage DNA template. The primary approach will be to study the effect of these metal ions on the kinetics of DNA polymerase function in vitro and to use the results to evaluate the polymerase mechanism. O6-methyl guanine was chosen as a model DNA lesion because it can basepair with both dT and dC with different efficiencies and because it acts as a replication block for some, but not all, polymerases. Two eukaryotic and two prokaryotic DNA polymerases will be used: calf thymus DNA polymerase alpha, cloned human polymerase beta, T7 polymerase and exonuclease free T7 (Sequenase). 8-Oxo dG was chosen to look at biological consequences because chromium and nickel both increase the amount of 8-oxo dG in cellular DNA and because 8-oxo dG is weakly mutagenic in the absence of added metals. Do Ni(II) or Cr(III) increase the mutagenic potential of 8-oxo dG by increasing the mutagenic bypass of this lesion? The results of these investigations will increase our fundamental knowledge of the mechanisms of carcinogenesis of an important class of environmental agents and will provide new information on the mechanisms by which DNA polymerases achieve their high degree of replication fidelity.

这项研究的目标有两个：确定 由致癌金属镍和铬引起的突变 使用 Ni(II) 和 Cr(III) 离子作为工具来研究酶促反应 负责 DNA 聚合酶功能和保真度的机制。 尽管这些金属致癌的机制尚不明确 复杂且仍知之甚少，这两种金属都可以产生 DNA 碱基 损伤（通过细胞内活性氧的产生）和 Ni(II) 和 Cr(III) 均可在体外与 DNA 聚合酶相互作用 双相方式增强和抑制聚合酶功能和保真度。 这些金属离子是否也会增强其他形式的致突变潜力 DNA 损伤，它们能否通过直接抑制诱导突变 聚合酶功能？ 本研究的具体目标是： 确定 1) Cr(III) 或 2) Ni(II) 是否可以改变动力学 和 DNA 复制经过模型诱变病变 O6-甲基 的保真度 鸟嘌呤； 3）研究这些金属离子对 DNA聚合酶3'-5'核酸外切酶功能的动力学； 4）确定 Cr(III)是否影响脱氧核苷酸三磷酸的使用 通过形成 β、γ-Cr(III)-dNTP 复合物和/或 与聚合酶上的 Mg+2 结合位点相互作用； 5) 到 检查 DNA 复制跨过氧化的生物学后果 在 Cr(III) 或 Ni(II) 存在下使用位点进行 DNA 损伤 (8-oxo dG) 修饰的噬菌体 DNA 模板。 主要方法是 研究这些金属离子对 DNA 聚合酶动力学的影响 体外功能并使用结果来评估聚合酶 机制。 选择 O6-甲基鸟嘌呤作为 DNA 损伤模型是因为 它可以以不同的效率与 dT 和 dC 进行碱基配对，并且 因为它充当某些人（但不是全部）的复制块， 聚合酶。 两种真核 DNA 聚合酶和两种原核 DNA 聚合酶将 使用：小牛胸腺 DNA 聚合酶 α、克隆人聚合酶 β、 T7 聚合酶和无核酸外切酶的 T7（测序酶）。 选择8-Oxo dG 看看生物学后果，因为铬和镍都 增加细胞 DNA 中 8-oxo dG 的量，因为 8-oxo dG 在没有添加金属的情况下具有弱致突变性。 Ni(II) 或 Cr(III) 通过增加诱变能力来增加 8-oxo dG 的诱变潜力 绕过这个病变？ 这些调查的结果将会增加 我们对癌症发生机制的基本了解 重要的一类环境剂，将提供新的信息 DNA聚合酶实现高度聚合的机制 复制保真度。