Persistence of Alkylated DNA Carcinogenesis

烷基化 DNA 致癌作用的持续存在

• 批准号: 7749554
• 负责人: Sreenivas Kanugula
• 金额: $ 32.38万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-12-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7749554
• 关键词: Abbreviations; Active Sites; Address; Alkylating Agents; Alkylation; Amino Acid Sequence; Area; Binding; Carcinogens; Cells; Chimeric Proteins; Cysteine; DNA; DNA Damage; DNA Repair; DNA repair protein; Defense Mechanisms; Environmental Carcinogens; Escherichia coli; Exposure to; Fission Yeast; Genes; Genome; Grant; Human; Individual; Investigation; Kinetics; Knowledge; Laboratories; Lesion; Life; Mammalian Cell; Mediating; Modeling; Mutagenesis; Mutation; Nitrosamines; Nucleotide Excision Repair; O(6)-Methylguanine-DNA Methyltransferase; Pathway interactions; Peptide Sequence Determination; Process; Property; Protein Binding; Proteins; Reaction; Reagent; Research Personnel; Risk; Role; Site-Directed Mutagenesis; Specificity; Substrate Specificity; Techniques; Therapeutic; Toxic effect; Tryptophan; Variant; Work; adduct; alkyl group; alkyltransferase; base; cancer risk; carcinogenesis; cell injury; cytotoxic; enhancing factor; follow-up; genotoxicity; improved; in vitro Assay; in vivo; inhibitor/antagonist; microorganism; mutant; nitrosamides; novel; programs; repaired; research study; resistance mechanism; response; vector

O^-Alkylguanine-DNA alkyltransferase (AGT) is an important DMArepair protein that is a major resistance mechanism protecting from the carcinogenic, cytotoxic and mutagenic effects of alkylating agents including nitrosamines and nitrosamides, which are important human carcinogens. It acts to repair O^-alkylguanine adducts by transferring the alkyl group to a cysteine acceptor residue in the protein. The proposed experiments are focussed on the properties of AGT and related proteins and aimed at providing a more complete understanding of the manner by which damage to DNA is influenced by these proteins. The planned investigations build on advances based on studies carried out in the previous period of support. These include: the description of a model for the mechanism of the AGT reaction based on structural studies and the properties of AGT mutants generated by site-directed mutagenesis; studies of the substrate specificity, kinetics and directionality of repair by AGT; investigation of the interactions of AGT with itself in the presence of DNA and with other proteins; the characterization of several proteins related to AGT that also protect against alkylation damage; and studies showing the mechanism by which AGT paradoxically enhances the genotoxicity of dihalplalkanes. Most of the experimental techniques and reagents needed including mutant AGT proteins, vectors for expressing them and the AGT like proteins and cells expressing them are already available in the Pis laboratory and the proposed experiments are a logical extension of the ongoing work. There are 4 interrelated specific aims: (1) To study the mechanism and importance of the paradoxical AGT-mediated increase in DNA damage caused by dihalohaloalkanes and other bifunctional agents. (2) To continue studies on a fusion protein that we have found in a microorganism that lives under extreme conditions what has both AGT and EndoV activity. (3) To investigate the function of ALS (alkyltransferase like sequence), a novel protein that binds to O6- alkylguanine and protects from alkylation damage but does not by itself repair these adducts. (4) To investigate the mechanism of AGT repair of DNA and variations in repair by polymorphic variants of hAGT. These studies will focus on understanding how AGT finds and recognizes lesions and how the inactivated alkylated form of AGT is disposed of. These experiments will aid in the understanding of the individual risks associated with exposure to alkylating agents and an important class of environmental carcinogens.

O^-烷基鸟嘌呤-DNA 烷基转移酶 (AGT) 是一种重要的 DMA 修复蛋白，是一种重要的 DMA 修复蛋白。 抵抗机制可防止烷基化的致癌、细胞毒性和致突变作用 包括亚硝胺和亚硝酰胺在内的物质，它们是重要的人类致癌物。它的作用是 通过将烷基转移至半胱氨酸受体残基来修复O^-烷基鸟嘌呤加合物 蛋白质。拟议的实验集中于 AGT 和相关蛋白质的特性以及 旨在更全面地了解 DNA 损伤的方式 受这些蛋白质的影响。 计划中的调查建立在前一时期进行的研究的基础上 的支持。其中包括： 基于 AGT 反应机制的模型描述 通过定点诱变产生的 AGT 突变体的结构研究和特性； 研究 AGT 修复的底物特异性、动力学和方向性；的调查 DNA 存在下 AGT 与其自身以及与其他蛋白质的相互作用；的表征 几种与 AGT 相关的蛋白质也能防止烷基化损伤；和研究表明 AGT 矛盾地增强了二卤代烷的遗传毒性的机制。大部分的 所需的实验技术和试剂，包括突变AGT蛋白、表达载体 它们以及类似 AGT 的蛋白质和表达它们的细胞已经在 Pis 实验室中可用 拟议的实验是正在进行的工作的逻辑延伸。 有 4 个相互关联的具体目标：（1）研究 反常的 AGT 介导的二卤代烷烃和其他物质引起的 DNA 损伤增加 双功能剂。 (2) 继续研究我们在a中发现的融合蛋白 生活在极端条件下的微生物，具有 AGT 和 EndoV 活性。 (3) 至 研究 ALS（烷基转移酶样序列）的功能，这是一种与 O6- 结合的新型蛋白质 烷基鸟嘌呤并防止烷基化损伤，但其本身不能修复这些加合物。 (4) 至 研究 DNA 的 AGT 修复机制以及多态性变异的修复变化 hAGT。这些研究将侧重于了解 AGT 如何发现和识别病变以及如何 失活的烷基化形式的 AGT 被丢弃。 这些实验将有助于了解与接触环境相关的个人风险 烷化剂和一类重要的环境致癌物。