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MECHANISMS OF METAL MUTAGENESIS: CR, NI, & BE

金属诱变机制：CR、NI、

基本信息

批准号：
3458492
负责人：
ELIZABETH T SNOW
金额：
$ 11.4万
依托单位：
NEW YORK UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1987
资助国家：
美国
起止时间：
1987-07-06 至 1992-05-31
项目状态：
已结题

项目摘要

A number of industrially important metals and carcinogens. Nickel and chromium stand out because they are human and animal carcinogens and also positive in short-term toxicity tests. Beryllium, an animal carcinogen and mutagen, is also positive in short-term tests. The molecular mechanisms by which these three metals act are not known. These metal-DNA interactions will be examined in a series of in vitro and in vivo experiments designed to measure the mechanisms and importance of metals in mutagenesis, DNA damage, polymerase function, and DNA-protein crosslinking. Metal mutagenesis, will be assayed in vitro with the M13mp2 forward-mutation assay using eukaryotic DNA polymerase-alpha. Mutations produced in vitro by the interactions of Be, Ni, or Cr will be quantitated, sequenced, and compared. The metal-induced mutations will be compared with the types of metal-induced DNA damage; base- and/or sequence-specifically will be assessed. Metal-induced nonspecific DNA damage also will be identified as DNA polymerase pause sites. Because DNA polymerase require divalent metal cations for activity and can be adversely affected by the wrong cations, the kinetics of DNA polymerase activity will be determined in the presence of the metal ions using kinetic primer-extension techniques. Since both Ni and Cr produce DNA- protein crosslinking in vitro, the effects of crosslinking on polymerase activity and mutagenesis will be studied using nonsequence-specific DNA binding proteins. Each metal's mutation spectrum will also be analyzed in vivo using an integrated shuttle-vector. Comparison of metal mutagenesis in vivo with the damage and mutation spectrum in vitro will provide evidence of whether the mechanisms are the same or not.

许多工业上重要的金属和致癌物质。镍和铬之所以脱颖而出，是因为它们是人类的，动物致癌物，短期毒性试验也呈阳性。铍是一种动物致癌物和诱变剂，在短期测试。这些作用的分子机制三种金属的作用尚不清楚。这些金属-DNA 相互作用将在一系列研究中得到检验旨在测量的体外和体内实验金属在诱变、DNA 中的机制和重要性损伤、聚合酶功能和 DNA-蛋白质交联。金属诱变，将使用 M13mp2 进行体外测定使用真核 DNA 聚合酶-α 进行正向突变测定。 Be、Ni 或 Cr 相互作用在体外产生的突变将被定量、测序和比较。金属诱导突变将与金属诱导的 DNA 类型进行比较损害;将评估碱基和/或序列特异性。金属引起的非特异性 DNA 损伤也将被确定为 DNA 聚合酶暂停位点。因为DNA聚合酶需要二价金属阳离子的活性并可能受到不利影响通过错误的阳离子，DNA聚合酶活性的动力学将在金属离子存在的情况下使用动力学测定引物延伸技术。由于 Ni 和 Cr 都会产生 DNA- 蛋白质体外交联，交联对蛋白质的影响将使用以下方法研究聚合酶活性和诱变非序列特异性 DNA 结合蛋白。每种金属的突变谱也将使用体内分析综合穿梭载体。金属诱变的比较体内损伤和体外突变谱将提供机制是否相同的证据。