Analysis of Cdc20-Mad2 in spindle checkpoint signalling

Mad 1、Mad 2和Cdc 20在有丝分裂的纺锤体检查点中具有重要作用。在哺乳动物细胞中，Mad 1负责Mad 2定位于动粒。Mad 1-Mad 2复合物已被证明具有“安全带”结合构象。该络合物在4 M尿素或2 M NaCl中也能保持稳定。不管这种极其稳定的相互作用，在动粒处Mad 1和Cdc 20之间存在Mad 2开关。在Mad 2的存在下，Cdc 20不能激活后期促进复合物（APC）。这种抑制导致细胞在中期仍然停滞。我们将 利用四个cdc 20突变体的集合，分析Cdc 20-Mad 2在有丝分裂中抑制细胞周期的机制。cdc 20突变体中的三个在Mad 2结合中是缺陷的，并且另一个对Mad 2具有增加的亲和力。这些突变体也将被用来确定其他基因参与Mad 1和Cdc 20之间的Mad 2开关。该提议的另一个目的是鉴定参与纺锤体检查点的张力感测方面的基因。我们将使用温度敏感mcd 1 -1突变体来筛选参与这一过程的基因。mcd 1是内聚复合体的成员，该复合体对于姐妹染色单体联合至关重要。

It has been well-documented that Mad1, Mad2, and Cdc20 have important roles in the spindle checkpoint in mitosis. In mammalian cells, Mad1 is responsible for Mad2 localization at the kinetochore. The Mad1-Mad2 complex has been shown to have a 'seat belt' binding conformation. This complex can also remain stable in 4m urea or 2M NaCI. Regardless of this extremely stable interaction, there is a Mad2 switch between Mad1 and Cdc20 at the kinetochore. In the presence of Mad2, Cdc20 is not able to activate the anaphase-promoting complex (APC). This inhibition causes cells to remain arrested in metaphase. We will use a collection of four cdc20 mutants to analyze the mechanism of Cdc20-Mad2 inhibition of the cell cycle in mitosis. Three of the cdc20 mutants are deficient in Mad2 binding, and the other has an increased affinity for Mad2. These mutants will also be used to identify other genes involved in the Mad2 switch between Mad1 and Cdc20. Another aim of this proposal is to identify genes involved in the tension-sensing aspect of the spindle checkpoint. We will use temperature sensitive mcd1-1 mutants to screen for genes involved in this process. Mcd1 is a member of the cohesion complex that is essential for sister chromatid association.