METAL MUTAGENESIS IN VITRO--POLYMERASE KINETICS

本研究的目标有两个方面：确定 致癌金属镍和铬引起的诱变， 使用Ni（II）和Cr（III）离子作为工具来研究酶促 负责DNA聚合酶功能和保真度的机制。 虽然这些金属诱发的致癌机制是 这两种金属都很复杂，而且在很大程度上还不为人所知， 损伤（通过细胞内产生活性氧）， Ni（II）和Cr（III）都可以在体外与DNA聚合酶相互作用， 以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增强了其他形式的诱变潜力 DNA损伤，它们是否可以通过直接抑制 聚合酶的功能？ 这项研究的具体目标是： 确定1）Cr（III）或2）Ni（II）是否可以改变动力学 和DNA复制的保真度过去的模型诱变损伤，O 6-甲基 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;和5） 研究DNA复制的生物学后果， 在Cr（III）或Ni（II）存在下使用位点的DNA损伤（8-氧代dG） 修饰的噬菌体DNA模板 主要方法将是 研究这些金属离子对DNA聚合酶动力学的影响 功能，并使用结果来评估聚合酶 机制 选择O 6-甲基鸟嘌呤作为模型DNA损伤，因为 它可以以不同效率与dT和dC进行碱基配对， 因为它充当某些，但不是全部， 聚合酶 两种真核生物和两种原核生物的DNA聚合酶将被 使用：小牛胸腺DNA聚合酶α，克隆的人聚合酶β， T7聚合酶和无核酸外切酶的T7（测序酶）。 8-选择Oxo dG 因为铬和镍 增加细胞DNA中8-氧代dG的量，因为8-氧代dG是 在不添加金属的情况下具有弱致突变性。 做Ni（II）还是Cr（III） 通过增加8-氧代dG的致突变性， 绕过这个病变 这些调查的结果将增加 我们对肿瘤发生机制的基本认识 重要类别的环境因素，并将提供新的信息 关于DNA聚合酶实现其高度聚合的机制， 复制保真度

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.