Exonuclease 1 in DNA Meiosis, Mismatch Repair and Canc

DNA错配修复系统（MMR）是维持哺乳动物基因组完整性的关键。 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。 初始事件包括E. colimutS基因。 在错配识别事件之后，MutS蛋白结合MutL蛋白。 这种相互作用激活导致错误掺入的核苷酸的切除和通过DNA再合成填充所得单链缺口的事件。 这些后来的MMR事件不太清楚，许多促进这些反应的酶是未知的。 晚期MMR的重要步骤是通过核酸外切酶去除新合成的DNA链。数据在S。酿酒酵母的研究表明，5 '-3'以及3 '-5'外切核酸酶参与了这一过程。 最近，酵母外切核酸酶1（EXO 1），一种5 '-3'外切核酸酶，由于其与MSH 2的相互作用而在两个杂交筛选中被鉴定。 EXO 1缺陷型酵母菌株显示出与MSH 2缺陷型菌株相似的增变子表型，为在MMR中的作用提供了强有力的证据。 除了在MMR中的作用外，EXO 1也是酵母中有丝分裂和减数分裂重组所必需的。 基于在酵母中的研究，我们假设EXO 1是哺乳动物MMR的重要组成部分，并且在小鼠中靶向破坏外切核酸酶1基因将导致MMR缺陷，从而导致癌症和不育。 该提案的具体目标是：1.为了研究EXO 1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系。纯合子突变型Exo 1小鼠是不育的，我们将分析Exo 1突变型小鼠的减数分裂。 我们建议确定减数分裂MMR合作伙伴EXO 1和分析他们的相互作用。 2.为了研究哺乳动物EX 01在有丝分裂MMR和重组中的作用：我们将提供EX 01在EXO 1缺陷胚胎干细胞系中MMR和重组中的作用的详细研究。 3.为了研究EXO 1缺陷小鼠的癌症易感性：我们将分析Exo 1突变小鼠的癌症易感性表型，以确定EX 01在癌症发生和发展中的参与。 4.为了确定EXO 1和其他MMR基因之间的关系：为了在MMR通路中定位EXO 1，我们将检查MSH 2-MSH 6和MSH 2-MSH 3复合物在突变型EXO 1背景下的修复活性，以确定这两种复合物中的一种或两种的活性是否必要和充分。

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.