IN VITRO ANALYSIS OF MECHANISMS OF MAMMALIAN MUTAGENESIS

暴露在某些人造和自然环境制剂中会造成 对人类健康构成重大威胁。越来越清楚的是， 我们需要扩大我们的知识，了解有毒物质和 致癌环境物质危害人类健康，以 使我们能够制定合理的政策来处理这个健康问题。 接触有害环境物质的主要风险之一是 癌症的发展。虽然致癌过程很复杂， 很明显，原癌基因的突变激活和失活 抑癌基因的研究起着重要的作用。了解 致癌剂引起突变的机制是长期的 这项研究提案的范围目标。许多致癌物质相互作用 会对DNA结构造成破坏。如果这一损害不是 通过细胞DNA修复过程移除，损伤会干扰 正常的DNA复制并导致突变。我们一直在使用In 体外DNA复制试验及猴病毒40(SV40)基质粒 载体(PZ189)研究哺乳动物的复制机制 细胞对DNA损伤做出反应，以及复制过程中突变是如何产生的。 我们已经证明了紫外线损伤的模板可以在体外复制，并且 这会导致固定的突变类似于 都是由体内的紫外线辐射诱导的。这些结果建立了实用程序 体外DNA复制系统作为研究工具的研究 分子水平上的诱变。我们现在提议重组 使用来自于 哺乳动物细胞提取物，以确定哪些成分是必需的 观察复制和突变固定情况。此外，我们建议 确定如何影响这些复制因子的活动 通过处理细胞时引起的细胞范围的变化 致癌物质。我们将特别关注 立即抑制在G1停滞期间发生的DNA复制，以及 细胞复制和复制能力的明显增加 随后发生的损坏模板的突变 致癌物治疗。这些研究的结果应该为我们提供 更好地理解DNA模板中的损伤过程 被转化为在关键基因中观察到的序列改变 癌细胞。最终，这些研究的结果可能会导致 制定可提供保护的干预战略 对抗致癌环境物质的诱变作用。

Exposure to certain man-made and natural environmental agents poses a significant threat to human health. It is becoming increasingly clear that we need to expand our knowledge about the mechanisms by which toxic and carcinogenic environmental agents compromise human health, in order to enable us to establish rational policies to deal with this health issue. One of the major risks of exposure to harmful environmental agents is the development of cancer. Although the process of carcinogenesis is complex, it is clear that mutagenic activation of proto-oncogenes and inactivation of tumor suppressor genes plays an important role. Understanding the mechanisms by which carcinogenic agents cause mutagenesis is the long range goal of this research proposal. Many carcinogenic agents interact with DNA, causing damage to the DNA structure. If this damage is not removed by cellular DNA repair processes, the damage interferes with normal DNA replication and leads to mutations. We have been using an in vitro DNA replication assay and a simian virus 40 (SV40)-based plasmid vector (pZ189) to investigate how the replication machinery of mammalian cells responds to DNA damage and how mutations arise during replication. We have shown that UV-damaged templates can be replicated in vitro, and that this results in fixation of mutations that are similar to those that are induced by UV radiation in vivo. These results establish the utility of the in vitro DNA replication system as a tool for investigating mutagenesis on the molecular level. We now propose to reconstitute replication of damaged templates using protein fractions derived from the mammalian cell extract, to determine which components are required for the observed replication and mutation fixation. Further we propose to determine how the activities of these replication factors are influenced by the cell-wide changes that are induced when cells are treated with carcinogenic agents. In particular, we will focus on the mechanisms of the immediate inhibition of DNA replication that occurs during Gl arrest, and the apparent increase in the capacity of cells for replication and mutagenesis of damaged templates that occurs at later times following carcinogen treatment. The results of these studies should provide us with a better understanding of the process by which damage in the DNA template is converted to the sequence alterations observed in critical genes in cancer cells. Ultimately, the results of these studies may lead to the development of intervention strategies that could provide protection against the mutagenic effects of carcinogenic environmental agents.