IN VITRO ANALYSIS OF MECHANISMS OF MAMMALIAN MUTAGENESIS

暴露于某些人造和自然环境因素会造成 严重威胁人类健康。越来越清楚的是， 我们需要扩大我们对有毒和 致癌环境因子危害人类健康， 使我们能够制定合理的政策来处理这一健康问题。 接触有害环境因子的主要风险之一是 癌症的发展。虽然致癌过程很复杂， 很明显，原癌基因突变激活和失活 肿瘤抑制基因的表达起着重要的作用。了解 致癌剂引起诱变的机制是长期的 这是本研究计划的目标。许多致癌物质相互作用 与DNA接触，导致DNA结构受损。如果这种伤害不是 通过细胞DNA修复过程去除，损伤干扰了 正常的DNA复制并导致突变。我们一直在用一个 体外DNA复制试验和基于猿猴病毒40（SV40）的质粒 载体（pZ189），以研究哺乳动物细胞的复制机制 细胞对DNA损伤的反应以及复制过程中突变是如何产生的。 我们已经证明，紫外线损伤的模板可以在体外复制， 这会导致类似于 是由体内紫外线辐射诱导的。这些结果建立了效用 体外DNA复制系统作为研究 分子水平上的突变。我们现在提议重组 使用衍生自DNA的蛋白质级分复制受损的模板 哺乳动物细胞提取物，以确定哪些成分是必需的， 观察到复制和突变固定。此外，我们建议， 确定这些复制因子的活动如何受到影响 当细胞被处理时， 致癌物质特别是，我们将重点讨论 在G1停滞期间发生的DNA复制的立即抑制，和 细胞复制能力的明显增加， 在随后的时间发生的受损模板的诱变 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 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 制定干预战略， 对抗致癌环境因子的诱变效应。

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.