MISMATCH REPAIR AND MAINTAINING GENOME STABILITY

描述：本提案涉及错配修复机制 以及这一过程在基因组维护中的作用。焦点 是在MSH2基因产物(Msh2)上的，它是E.coliMutS的类似物。Dr。 Alani和他的同事证明，像MutS一样，Msh2直接 参与与不匹配的碱基结合。他们还展示了 Msh2与MLH1和Pms1的相互作用 MutL是大肠杆菌错配修复系统的另一个组成部分。一个 建议对msh2基因进行突变，目的是识别 对错配修复有不同影响的等位基因。这个 初步检测基于MSH2突变体(10-30X)的突变子表型 用于CanR和1-700用于LacZ二核苷酸重复逆转试验)。 寻找条件突变和显性突变以及 过度表达时显性(GAL10：：MSH2)。抑制者分析 这些突变体也被描述，目的是进一步定义 参与特定活动的Msh2结构域和其他具有 其中Msh2可能会相互作用。Msh2的生化特性研究 建议结合携带错配的寡核苷酸来定义 可以与突变蛋白质进行比较的参数。以此类推 对MutS和Msh1(酵母线粒体同源物)的ATPase活性 将对Msh2进行调查。突变的Msh2蛋白将被比较 到野生型，用于结合不匹配的寡核苷酸，结合到Pms1 和MLH1，ATPase活动，目标是定义负责的域 为这些活动做准备。Msh2与已知重组的关系 蛋白质将通过亲和层析进行监测， 免疫沉淀和免疫共定位。免疫定位 这种方法将扩展到减数分裂细胞。 Alani还谈到了Msh2在限制 同源DNA之间链交换中间体的形成 终止或逆转错配附近的链交换 本垒打。一个相关的建议是，失配检测是一个中心 抑制交叉事件的机制的组成部分 同源序列，因此禁止易位和缺失 这将通过异位站点之间的交叉发生。进一步测试以 确定错配检测在限制同源基因中的作用 对复合进行了描述。阿拉尼将决定 错配修复缺陷对同源基因转化效率的影响 以及此类事件与使用 吉姆·哈伯实验室检测到同源重组的化验 事件，并通过简单的 复制测试。大肠杆菌纠错能力的关键组成部分 源自复制错误的不匹配碱基是它检测 由于半甲基化位点的存在而形成的老链。穆特兄弟 蛋白质负责将错配检测偶联到链上 歧视。酵母没有甲基化的DNA，也没有同源的 已检测到Muth。阿拉尼建议确定这些功能 参与链识别，与错配校正相结合 在观察后建立的合成致命性的基因筛查 来自E.Coli。RecA-Dam-细胞是不活的，但MutS-recA-Dam-细胞 是可行的。一种解释是错配修复，在 没有大坝系统提供的钢绞线歧视，造成了缺口 在成为DSB并依赖于重组的两条链上 他们的修缮。从携带mh2条件性病毒的rad52突变体开始 等位基因，在MSH2不允许的条件下繁殖的菌落将 对无法在允许的条件下生长的人进行筛查 MSH2。阿拉尼希望，在这一屏幕上合成的致命突变中 以确定与链识别有关的基因突变。

DESCRIPTION: This proposal addresses the mechanism of mismatch repair in yeast and the role of this process in genome maintenence. The focus is on the MSH2 gene product (Msh2) an analogue of E. coli mutS. Dr. Alani and co-workers demonstrated that like mutS, Msh2 is directly involved in binding to mismatched bases. They also demonstrated interaction between Msh2 and Mlh1 and Pms1, the yeast homologues of MutL, another component of the E. coli mismatch repair system. A mutagenesis of the MSH2 gene is proposed with the goal of identifying alleles that differentially affect aspects of mismatch repair. The initial assay is based on the mutator phenotype of msh2 mutants (10-30X for CanR and 1-700 for a lacZ dinucleotide repeat reversion assay). Conditional and dominant mutants are sought as well as mutations that are dominant when over expressed (GAL10::MSH2). A suppressor analysis of these mutants is also described with the goal of further defining domains of Msh2 involved in specific activities and other proteins with which Msh2 might interact. A biochemical characterization of Msh2 binding to oligonucleotides carrying mismatches is proposed to define parameters that could be compared to the mutant proteins. By analogy to MutS and Msh1 (the yeast mitochondrial homologue) an ATPase activity for Msh2 will be investigated. Mutant Msh2 proteins will be compared to wildtype for binding to mismatched oligonucleotides, binding to Pms1 and Mlh1, ATPase activity with the goal of defining domains responsible for these activities. Association between Msh2 and known recombination proteins will be monitored both by affinity chromatography, immunoprecipitation and co-immunolocalization. The immunolocalization approach will be extended to meiotic cells. Alani also addresses the proposal that Msh2 has a role in restricting the formation of strand exchange intermediates between homologous DNAs by terminating or reversing strand exchange in the vicinity of mismatched bases. A related suggestion is that mismatch detection is a central component of the mechanisms that inhibit crossover events between homeologous sequences, hence prohibiting translocations and deletions that would occur by crossover between ectopic sites. Further tests to determine the role of mismatch detection in restricting homeologous recombination are described. Alani will determine the consequences of mismatch repair defects on the efficiency of homeologous gene conversion and the proportion of such events associated with crossovers using an assay from Jim Haber's laboratory that detects homeologous recombination events and distinguished those associated with crossovers by a simple replicating test. A key component of the ability of E. coli to correct mismatched bases derived from replication errors is its ability to detect the old strand by the presence of hemimethylated sites. The mutH protein is responsible for coupling mismatch detection to strand discrimination. Yeast do not have methylated DNA and no homologue of mutH has been detected. Alani proposes to determine the functions involved in strand discrimination as coupled to mismatch correction by a genetic screen for synthetic lethality patterned after an observation from E. coli. recA- dam- cells are inviable but mutS- recA- dam- cells are viable. One interpretation is that the mismatch repair, in the absence of strand discrimination provided by the dam system, makes nicks on both strandswhich become DSBs and are dependent on recombination for their repair. Starting with a rad52 mutant carrying a msh2 conditional allele, colonies propagated at the nonpermissive condition for msh2 will be screened for the inability to grow at the permissive condition for msh2. Among the synthetic lethal mutations from this screen, Alani hopes to identify mutations in genes involved in strand discrimination.