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: 6419099
• 负责人: WINFRIED EDELMANN
• 金额: $ 36.52万
• 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/6419099
• 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的了解大多局限于MMR的早期阶段。最初的事件包括大肠杆菌mutS基因的哺乳动物同源物对错配核苷酸的识别。在错配识别事件之后，MutS蛋白结合到MutL蛋白上。这种相互作用激活了一些事件，导致错误结合的核苷酸被切除，并通过DNA再合成填充由此产生的单链间隙。这些后期的MMR事件尚不清楚，许多促进这些反应的酶是未知的。晚期MMR的一个重要步骤是外切酶去除新合成的DNA链。在S. cerevisiae中的数据表明，5‘-3’和3‘-5’外切酶参与了这一过程。最近，酵母外切酶1 （EXO1）是一种5‘-3’外切酶，由于其与MSH2的相互作用，在两个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，为其在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在酵母有丝分裂和减数分裂重组中也是必需的。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，在小鼠中靶向破坏外切酶1基因会导致MMR缺乏，从而导致癌症和不育。本建议的具体目的是：1。为了研究EXO1在哺乳动物减数分裂中的作用：我们产生了一个携带失活突变的小鼠系。纯合子突变的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伙伴并分析它们的相互作用。2. 为了研究哺乳动物EX01在有丝分裂MMR和重组中的作用：我们将详细研究EX01在EXO1缺陷胚胎干细胞系的MMR和重组中的作用。3. 为了研究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EX01参与癌症的发生和发展。4. 为了确定EXO1和其他MMR基因之间的关系：为了定位EXO1在MMR通路中的位置，我们将在突变的EXO1背景下检查MSH2-MSH6和MSH2-MSH3复合物的修复活性，以确定它是否是这些复合物活性的必要和充分条件。