Mechanism of Termination of DNA Replication

我们建议继续我们的调查，以复制终止蛋白的作用机制， 酵母S.酿酒酵母和远亲裂殖酵母粟酒裂殖酵母， 酵母遗传学，体内和体外生物化学的结合，并开始朝着更长期的工作 目的是建立与末端DMA结合的终止子蛋白的结晶试验。In S. 我们希望通过体外实验揭示Fob 1终止蛋白的作用机制， 生物化学分析以研究蛋白质是否是拮抗以下活性的逆解旋酶： 真核生物复制解旋酶。我们亦建议制订检查站的运作机制 蛋白Tofl和Csm3在复制末端。我们发现，S. 粟酒裂殖酵母有四个而不是三个复制抑制位点。四个位点中的两个需要有趣的蛋白质Reb1 p 它不仅以极性模式阻止了叉，而且终止了rDNA的转录， 作为一个转录因子启动转录。我们希望研究出 Reb1p。我们已经缩小了S的Ter 1的位置。pombe的约30bp，我们希望克隆该基因 其编码同源终止子蛋白并研究其作用模式。最后，我们希望揭开 Swi1和Swi3蛋白的作用模式，以确定它们是否作为“抗分泌酶”。

We propose to continue our investigations into the mechanism of action of replication terminator proteins of the yeasts S. cerevisiae and the distantly related fission yeast Schizosaccharomyces pombe by using a combination of yeast genetics, in vivo and in vitro biochemistry and to start work towards the longer term goals of setting up crystallization trials of the terminator proteins bound to the terminus DMA. In S. cerevisiae we wish to uncover the mechanism of action of the Fob1 terminator protein by performing in vitro biochemical analysis to investigate if the protein is a contrahelicase that antagonizes the activities of eukaryotic replicative helicases. We also propose to work out the mechanism of action of the checkpoint proteins Tofl and Csm3 at the replication termini. We have discovered that the spacer region of rDNA of S. pombe has four not three replication arrest sites. Two of the four sites require the interesting protein Reb1 p that not only arrests forks in a polar mode but also terminates the transcription of rDNA and probably also acts as a transcription factor that turns on transcription. We wish to work out the mechanism of action of Reb1p. We have narrowed down the location of Ter1 of S. pombe to ~30bp and we wish to clone the gene that encodes the cognate terminator protein and investigate its mode of action. Finally, we wish to uncover the mode of action of the Swi1 and Swi3 proteins to determine if these act as "antisweepases".