Role of Mismatch Repair in Maintaining Genome Stability

描述(由申请人提供)：错配修复(MMR)途径减少了DNA复制过程中发生的错配错误。MMR蛋白还可以防止不同DNA序列之间的重组，在单链退火(SSA)过程中处理含有非同源末端的重组中间产物，并将DNA损伤信号转导到细胞周期检查点和细胞凋亡途径。这些蛋白质识别重组和复制中间产物中的错配和其他结构并将它们输送到下游因子的机制尚不清楚；我们正在通过以下目标在酿酒酵母中解决这些问题：1.将在一种新的分析同源重组的新方法中检验MMR和SGS1解旋酶突变的影响，该方法使用物理和遗传方法分析同源重组。这些研究应该会为重组如何受到监管提供新的见解。2.在酵母中，MSH2-MSH3错配识别复合体和Rad1-AD10内切酶在SSA过程中起着去除非同源3‘单链尾巴的关键作用。由于SSA在酵母和哺乳动物细胞中都是一条重要的重组途径，我们正在开发一种体外核酸内切酶试验来研究这些和其他DNA修复因子之间的相互作用。3.利用染色质免疫沉淀(CHIP)、遗传和物理分析相结合的方法，我们正在测试旨在了解MSH2 MMR蛋白在交配类型转换过程中如何调控重组的模型，这是一种定义明确的重组事件。4.我们已经鉴定了来源于S288C酵母菌株的MLH1 MMR基因突变，该突变仅当被导入SK1菌株时才会中断MMR。当S288C PMS1 MMR基因与S288C MLH1等位基因共导入时，SK1中观察到的MMR缺陷被消除。这一结果鼓励我们测试MMR基因是否在酿酒酵母中共同进化。这也表明MMR缺陷表型如何在人类中作为自然发生的多态分离的结果而出现。

DESCRIPTION (provided by applicant): The mismatch repair (MMR) pathway reduces mis-incorporation errors that occur during DNA replication. MMR proteins also prevent recombination between divergent DNA sequences, process recombination intermediates containing non-homologous ends during single strand annealing (SSA), and transduce DNA damage signals to cell cycle checkpoint and apoptosis pathways. The mechanisms by which these proteins recognize mismatches and other structures within recombination and replication intermediates and funnel them to downstream factors are not well understood; we are addressing these issues in S. cerevisiae through the following aims: 1. The effect of MMR and SGS1 helicase mutations will be examined in a new assay that analyzes homeologous recombination using both physical and genetic approaches. These studies should provide new insights into how recombination is regulated. 2. In yeast, the MSH2-MSH3 mismatch recognition complex and the RAD1-AD10 endonuclease play critical roles in removing nonhomologous 3' single strand tails during SSA. Because SSA is an important recombination pathway in both yeast and mammalian cells, we are developing an in vitro endonuclease assay to study interactions between these and other DNA repair factors. 3. Using a combination of chromatin-immunoprecipitation (CHIP), genetic, and physical assays, we are testing models aimed at understanding how the MSH2 MMR protein regulates recombination during mating type switching, a well defined recombination event. 4. We have identified mutations in the MLH1 MMR gene derived from the S288C yeast strain that disrupt MMR only when introduced into SK1 strains. The MMR defects observed in SK1 were eliminated when the S288C PMS1 MMR gene was co-introduced with the S288C mlh1 alleles. This result is encouraging us to test whether the MMR genes are co-evolving in S. cerevisiae. It also suggests how a MMR defective phenotype could arise in humans as the result of the segregation of naturally occurring polymorphisms.