MISMATCH REPAIR AND MAINTAINING GENOME STABILITY

描述：该提案涉及错配修复的机制 以及这一过程在基因组维持中的作用。 重点 在MSH 2基因产物（Msh 2）上是E. coli mutS. 博士 Alani和同事证明，像mutS一样，Msh 2直接 与错配碱基结合有关。 他们还证明 Msh 2和Mlh 1和Pms 1之间的相互作用， MutL是E. 大肠杆菌错配修复系统。 一 MSH 2基因的诱变被提出，目的是鉴定 差异影响错配修复方面的等位基因。 的 初始测定基于msh 2突变体的增变子表型（10- 30 X 对于CanR和1-700对于lacZ二核苷酸重复回复测定）。 寻找条件突变体和显性突变体以及 当过表达时为显性（GAL 10：：MSH 2）。 抑制因子分析 还描述了这些突变体，目的是进一步定义 Msh 2的结构域参与特定的活动和其他蛋白质 Msh 2可能会相互作用。 Msh 2的生化特性 提出了与携带错配的寡核苷酸的结合以定义 可以与突变蛋白质进行比较的参数。 通过类比 MutS和Msh 1（酵母线粒体同源物）的ATP酶活性 将对MSH 2进行研究。 将比较突变Msh 2蛋白 与野生型结合错配的寡核苷酸，结合Pms 1 和Mlh 1，ATP酶活性，目的是定义负责 对于这些活动。 Msh 2与已知重组之间的关联 蛋白质将通过亲和层析， 免疫沉淀和免疫共定位。 免疫定位 方法将扩展到减数分裂细胞。 Alani还提到了Msh 2在限制人类免疫力方面的作用。 同源DNA之间链交换中间体的形成， 终止或逆转错配位点附近的链交换 基地 一个相关的建议是，失配检测是一个核心的 抑制交叉事件的机制的组成部分， 同源异型序列，因此禁止易位和缺失 异位部位之间的交叉。 进一步检查以 确定不匹配检测在限制家庭暴力中的作用 描述了重组。 阿拉尼将决定 错配修复缺陷对同源异型基因转化效率的影响 以及与交叉相关的此类事件的比例， 吉姆·哈伯实验室的一种检测同源重组的方法 事件，并通过一个简单的 复制测试 E. 大肠杆菌校正 来自复制错误的错配碱基是它检测 旧链的半甲基化位点的存在。 关于MUH 蛋白质负责将错配检测偶联到链 歧视 酵母没有甲基化的DNA，也没有 已检测到mutH。 Alani建议确定 参与链辨别，与错配校正偶联， 在观察到一种合成致命性的基因后， 来自大肠 杆菌 recA- dam-细胞是不可存活的，但mutS- recA- dam-细胞 是可行的。 一种解释是，错配修复，在 大坝系统提供的钢绞线歧视的缺乏， 在成为DSB并依赖于重组的两条链上 他们的修复。 从携带msh 2条件突变的rad 52突变体开始， 等位基因，在msh 2的非允许条件下繁殖的菌落将 筛选不能在允许的条件下生长， msh2。 Alani希望在这次筛选的合成致命突变中， 来识别与链辨别有关的基因突变。

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.