Mechanism of Termination of DNA Replication

我们建议继续研究复制终止子蛋白的作用机制。 酿酒酵母和远缘裂殖酵母Pombe 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方向工作 建立与DMA末端结合的终止子蛋白的结晶试验的目标。在S。 我们希望通过体外实验来揭示Fob1终止子蛋白的作用机制 生化分析，以调查蛋白质是否是一种对抗螺旋酶的活性 真核复制型解旋酶。我们还建议制定检查站的行动机制。 复制末端的Tof1和Csm3蛋白。我们发现沙门氏菌rDNA的间隔区。 庞贝有四个而不是三个复制捕获点。四个位点中的两个需要有趣的蛋白质Reb1 p 这不仅在极性模式下阻止了分叉，而且还终止了rDNA的转录，可能还 起到转录因子的作用，启动转录。我们希望制定出……的作用机制。 报告1p.我们已经将S.pombe的Ter1基因的位置缩小到~30bp，并希望克隆该基因 编码同源终止子蛋白，并研究其作用方式。最后，我们希望揭开 Swi1和Swi3蛋白的作用模式，以确定它们是否起到“反扫描酶”的作用。

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".