Exonuclease 1 in DNA Meiosis, Mismatch Repair and Canc

DNA 减数分裂、错配修复和癌症中的核酸外切酶 1

• 批准号: 6694091
• 负责人: WINFRIED EDELMANN
• 金额: $ 37.03万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2006-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6694091
• 关键词: DNA repair; Saccharomyces cerevisiae; cancer risk; cell line; enzyme activity; enzyme mechanism; exonuclease; gene mutation; genetic recombination; immunoprecipitation; intermolecular interaction; laboratory mouse; meiosis; neoplastic transformation; tumor suppressor genes; yeast two hybrid system

The DNA mismatch repair system (MMR) is essential for maintaining the integrity of the mammalian genome. Mutations in MMR genes can lead to increased cancer susceptibility and meiotic failure. At present most of our understanding of the MMR is limited to the early steps of MMR. The initial events include the recognition of mispaired nucleotides by the mammalian homologs of the E. coli mutS gene. Subsequent to the mismatch recognition event, the MutS proteins bind to MutL proteins. This interaction activates events that lead to the excision of the misincorporated nucleotide(s) and the filling of the resulting single strand gap by DNA resynthesis. These later MMR events are less well understood and many of the enzymes facilitating these reactions are unknown. An essential step for late MMR is the removal of the newly synthesized DNA strand by exonucleases. Data in S. cerevisiae indicate that 5'-3' as well as 3'-5' exonucleases are involved in this process. Recently, yeast Exonuclease l (EXO1), a 5'-3' exonuclease, has been identified due to its interaction with MSH2 in two hybrid screens. EXO1 deficient yeast strains display a mutator phenotype that is similar to MSH2 deficient strains, providing strong evidence for a role in MMR. In addition to its role in MMR, EXO1 is also required for mitotic and meiotic recombination in yeast. Based on the studies in yeast, we hypothesize that EXO1 is an essential component of mammalian MMR and targeted disruption of the Exonuclease 1 gene in mice will result in MMR deficiency leading to cancer and infertility. The specific aims of this proposal are: 1. To investigate the role of EXO1 in mammalian Meiosis: We have generated a mouse line carrying an inactivating mutation. Homozygous mutant Exo1 mice are infertile and we will analyze meiosis in the Exo1 mutant mice. We propose to identify the meiotic MMR partners of EXO1 and analyze their interactions. 2. To examine the role of mammalian EX01 in mitotic MMR and recombination: We will provide a detailed study of the role of EX01 in MMR and recombination in EXO1 deficient embryonic stem cell lines. 3. To study the cancer susceptibility of EXO1 deficient mice: We will analyze the cancer susceptibility phenotype of Exo1 mutant mice to determine the involvement of EX01 in the initiation and progression of cancer. 4. To determine the relationship between EXO1 and other MMR genes: In order to position EXO1 in the MMR pathway, we will examine the repair activity of the MSH2-MSH6 and MSH2-MSH3 complexes on a mutant Exo1 background to determine whether it is necessary and sufficient for the activity of either or both of these complexes.

DNA 错配修复系统 (MMR) 对于维持哺乳动物基因组的完整性至关重要。 MMR 基因突变可导致癌症易感性增加和减数分裂失败。 目前我们对MMR的了解大多仅限于MMR的早期步骤。 最初的事件包括大肠杆菌 mutS 基因的哺乳动物同源物识别错配的核苷酸。 错配识别事件之后，MutS 蛋白与 MutL 蛋白结合。 这种相互作用激活了一些事件，导致错误掺入的核苷酸被切除，并通过 DNA 重新合成来填充由此产生的单链间隙。 这些后来的 MMR 事件尚不清楚，并且许多促进这些反应的酶也是未知的。 晚期 MMR 的一个重要步骤是通过核酸外切酶去除新合成的 DNA 链。酿酒酵母中的数据表明5'-3'以及3'-5'核酸外切酶参与该过程。 最近，酵母核酸外切酶 I (EXO1) 是一种 5'-3' 核酸外切酶，因其与 MSH2 的相互作用而在两次杂交筛选中被鉴定出来。 EXO1 缺陷酵母菌株表现出与 MSH2 缺陷菌株相似的突变表型，为 MMR 中的作用提供了强有力的证据。 除了在 MMR 中发挥作用外，EXO1 也是酵母有丝分裂和减数分裂重组所必需的。 基于对酵母的研究，我们假设 EXO1 是哺乳动物 MMR 的重要组成部分，并且有针对性地破坏小鼠的核酸外切酶 1 基因将导致 MMR 缺陷，从而导致癌症和不孕不育。 该提案的具体目标是： 1. 研究 EXO1 在哺乳动物减数分裂中的作用：我们已经产生了携带失活突变的小鼠品系。纯合突变 Exo1 小鼠是不育的，我们将分析 Exo1 突变小鼠的减数分裂。 我们建议鉴定 EXO1 的减数分裂 MMR 伴侣并分析它们的相互作用。 2. 检查哺乳动物 EX01 在有丝分裂 MMR 和重组中的作用：我们将在 EXO1 缺陷胚胎干细胞系中详细研究 EX01 在 MMR 和重组中的作用。 3. 研究EXO1缺陷小鼠的癌症易感性：我们将分析Exo1突变小鼠的癌症易感性表型，以确定EX01在癌症发生和发展中的参与。 4.确定EXO1与其他MMR基因之间的关系：为了将EXO1定位在MMR途径中，我们将在突变的Exo1背景上检查MSH2-MSH6和MSH2-MSH3复合物的修复活性，以确定是否有必要并且足以满足这些复合物中的一个或两个的活性。