METAL MUTAGENESIS IN VITRO--POLYMERASE KINETICS

体外金属诱变--聚合酶动力学

• 批准号: 2155343
• 负责人: ELIZABETH T SNOW
• 金额: $ 12.79万
• 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/2155343
• 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.

这项研究的目的有两个：确定心力衰竭的机制 致癌金属镍和铬的致突变作用 以Ni(II)、Cr(III)离子为工具研究酶促反应 负责DNA聚合酶功能和保真度的机制。 虽然这些金属的致癌机制是 这两种金属很复杂，但在很大程度上仍不为人所知，它们都能产生DNA碱基 损害(通过细胞内产生的活性氧物种)和 镍(II)和铬(III)在体外均能与DNA聚合酶相互作用 增强和抑制聚合酶功能和保真度的双相方式。 这些金属离子是否也增强了其他形式的诱变潜力？ DNA损伤以及它们能否通过直接抑制诱导突变 聚合酶的功能？这项研究的具体目的是： 确定1)Cr(III)或2)Ni(II)是否能改变动力学 和DNA复制通过模型突变损伤O6-甲基后的保真度 3)研究这些金属离子对酶活性的影响。 DNA聚合酶3‘-5’外切酶功能的动力学；4)测定 铬(III)是否影响脱氧核苷酸三磷酸的使用 通过形成β，γ-铬(III)-dNTP络合物和/或 与聚合酶上的Mg+2结合部位相互作用；以及5) 检查DNA复制跨氧化的生物后果 铬(III)或镍(II)存在下的DNA损伤(8-oxo DG) 修饰的噬菌体DNA模板。主要的方法将是 这些金属离子对DNA聚合酶动力学影响的研究 体外功能，并用结果来评估聚合酶 机制。O6-甲基鸟嘌呤被选为DNA损伤的模型是因为 它可以与DT和DC进行基本通信，具有不同的效率和 因为它充当了一些但不是全部的复制块， 聚合酶。两个真核DNA聚合酶和两个原核DNA聚合酶 用途：小牛胸腺DNA聚合酶α，克隆人聚合酶β， T7聚合酶和不含核酸外切酶的T7(序列酶)。选择8-氧代DG 观察生物后果，因为铬和镍都 增加细胞DNA中8-oxo DG的量，因为8-oxo DG是 在没有添加金属的情况下，致突变性较弱。镍(II)或铬(III) 通过增加诱变剂提高8-氧代DG的诱变潜力 绕过这块损伤？这些调查的结果将会增加 我们对AN致癌机制的基础知识 重要的环境代理人类别，并将提供新的信息 DNA聚合酶获得高活性的机制 复制保真度。