Mechanism of Eukaryotic Environmental Mutagenesis

描述（由申请人提供）：DMA修复和损伤容忍过程对于在暴露于许多不同药剂后保持人体健康绝对至关重要。本研究的长期目标是对真核生物环境诱变的分子机制进行详细的综合理解。在复杂和精心控制的分子过程中，当特化翻译合成（TLS） DNA聚合酶复制由环境因子引起的DNA损伤时，就会引入突变。这项研究特别强调了Rev1，因为它既是其他TLS DNA聚合酶的支架，又是聚合酶本身，是真核突变的根源。我们将通过研究其细胞周期控制的基础来跟进我们意想不到的发现，即S. cerevisiae Rev1在G2/M期的表达比G1和S期的大部分时间高50倍。我们将对有希望的候选调控基因如UMP1和CDC7进行测试，对新的调控基因进行遗传筛选，并进行实验来确定这种控制的重要性。我们将使用生化和遗传方法，继续研究Rev1相互作用的结构和功能基础，特别关注Rev1 c端结构域、BRCT结构域和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可能具有一类同源病变，它复制得特别好。我们将研究S. pombe DinB的功能、调控及其与Rev1的关系。我们将开发一种基于Rev1-Rev7相互作用破坏的高通量测定方法，这将允许筛选真核环境诱变抑制剂。提出的研究将通过阐明仍然知之甚少的真核生物翻译合成机制负责大多数突变，对基础科学做出高度重要的贡献。这些突变导致衰老、癌症和各种人类疾病。确定在这些致突变过程中发挥作用的其他基因将有助于解决为什么只有一些人在暴露于环境毒素时发病的问题。TLS的小分子抑制剂可能导致新的“抗诱变”药物，对人类健康具有多种应用。

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.