Role of Mismatch Repair in Maintaining Genome Stability

描述（由申请人提供）：错配修复（MMR）途径可减少DNA复制期间发生的错误掺入错误。MMR蛋白还阻止不同DNA序列之间的重组，在单链退火（SSA）期间处理含有非同源末端的重组中间体，以及向细胞周期检查点和细胞凋亡途径发出DNA损伤信号。这些蛋白质识别重组和复制中间体中的错配和其他结构并将其汇集到下游因子的机制还不清楚;我们正在S.酿酒酵母通过以下目的：1. MMR和SGS 1解旋酶突变的影响将在一个新的分析中进行检查，该分析使用物理和遗传方法分析同源重组。这些研究应该提供新的见解重组是如何调节的。2.在酵母中，MSH 2-MSH 3错配识别复合物和RAD 1-AD 10核酸内切酶在SSA期间去除非同源3'单链尾中起关键作用。由于SSA是酵母和哺乳动物细胞中重要的重组途径，我们正在开发一种体外核酸内切酶测定法来研究这些和其他DNA修复因子之间的相互作用。3.使用染色质免疫沉淀（CHIP），遗传和物理检测的组合，我们正在测试的模型，旨在了解如何MSH 2 MMR蛋白调节重组交配型转换过程中，一个明确的重组事件。4.我们已经鉴定了来源于S288 C酵母菌株的MLH 1 MMR基因中的突变，该突变仅在引入SK 1菌株时破坏MMR。当S288 C PMS 1 MMR基因与S288 C mlh 1等位基因共同引入时，在SK 1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S中共同进化。啤酒。它还表明，MMR缺陷表型如何在人类中出现，作为自然发生的多态性分离的结果。

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.