Mechanism of Eukaryotic Environmental Mutagenesis

描述(由申请人提供)：在接触到许多不同的制剂后，DMA修复和损害容忍过程对于保护人类健康绝对至关重要。这项研究的长期目标是对真核生物环境诱变的分子机制有一个详细而完整的理解。在既复杂又受到严格控制的分子过程中，当专门的跨病变合成(TLS)DNA聚合酶复制环境因素造成的DNA损伤时，就会引入突变。这项拟议的研究特别强调Rev1，它既是其他TLS DNA聚合酶的支架，也是聚合酶本身，是真核细胞突变的根源。我们将继续研究我们意想不到的发现，酿酒酵母Rev1在G2/M期的表达是G1期和S期大部分时间的50倍，方法是通过研究其细胞周期控制的基础，这主要是转录后调控；将测试有希望的候选调控基因，如UMP1和CDC7，将进行新的调控基因的遗传筛选，并进行实验以确定这种调控的重要性：我们将使用生化和遗传学方法继续研究Rev1相互作用的结构和功能基础，特别是Rev1C-末端结构域、其BRCT结构域和其泛素结合基序的相互作用。我们将通过跟踪我们最近的观察结果来研究Rev1聚合酶活性的功能重要性，这些观察表明Rev1可能具有一类它复制得特别好的同源病变。我们将研究S.pombe DinB的功能和调控及其与Rev1的关系。我们将开发一种基于Rev1-Rev7相互作用中断的高通量分析方法，这将使我们能够筛选真核环境突变的抑制剂。这项拟议的研究将通过阐明导致大多数突变的真核跨损伤合成机制，对基础科学做出非常重要的贡献。这些突变会导致衰老、癌症和各种人类疾病。识别在这些诱变过程中发挥作用的其他基因将有助于解决为什么只有某些人在接触环境毒素时才会患上疾病的问题。TLS的小分子抑制剂可能导致新型的抗突变药物，对人类健康具有多方面的应用。

DESCRIPTION (provided by applicant): DMA repair and damage tolerance processes are absolutely critical to preserving human health following exposure to many different agents. The long term goal of this research is to develop a detailed integrated understanding of the molecular mechanisms responsible for environmental mutagenesis in eukaryotes. In molecular processes that are both complex and elaborately controlled, mutations are introduced when specialized translesion synthesis (TLS) DNA polymerases copy over DNA damage caused by environmental agents. The proposed research places a special emphasis on Rev1, which by virtue of acting both as a scaffold for other TLS DNA polymerases and as a polymerase itself, lies at the root of eukaryotic mutagenesis. We will follow up on our unanticipated finding that S. cerevisiae Rev1 is expressed 50 fold higher during G2/M than in G1 and most of S phase by investigating the basis of its cell cycle control," which is predominantly posttranscriptional; promising candidate regulatory genes such as UMP1 and CDC7 will be tested, genetic screens for new regulatory genes will be carried out, and experiments will be conducted to determine the importance Of this control: Using both biochemical and genetic approaches, we will continue to investigate the structural and functional basis of Rev1 interactions, focusing particularly on the interactions of the Rev1 C-terminal domain, its BRCT domain, and its ubiquitin-binding motifs. We will investigate the functional importance of the Rev1 polymerase activity by following up on our recent observations suggesting that Rev1 may have a class of cognate lesions it replicates particularly well. We will investigate the function and regulation of S. pombe DinB and its relationship to Rev1. We will develop a high-throughput assay, based on disruption of the Rev1-Rev7 interaction, that will allow screening for inhibitors of eukaryotic environmental mutagenesis. The proposed research will make a highly significant contribution to basic science by elucidating the still poorly understood eukaryotic translesion synthesis mechanisms responsible for most mutations. These mutations contribute to aging, cancer, and various human diseases. Identification of additional genes that play roles in these mutagenic processes will help make it possible to address the question of why only some people develop disease when exposed to an environmental toxin. Small molecule inhibitors of TLS could lead to novel "anti-mutagenesis" drugs with multiple applications to human health.