Mechanism of Termination of DNA Replication

描述(申请人提供)：我们建议利用酵母遗传学、体内和体外生物化学相结合的方法，继续研究酿酒酵母和远缘裂解酵母复制终止子蛋白的作用机制，并开始进行与DNA末端结合的终止子蛋白结晶试验的长期目标。在酿酒酵母中，我们希望通过体外生化分析来揭示Fob1终止子蛋白的作用机制，以研究该蛋白是否是一种对抗真核复制解旋酶活性的反螺旋酶。我们还建议研究检查点蛋白TOF1和Csm3在复制末端的作用机制。我们发现S.pombe的rDNA间隔区有四个而不是三个复制停滞位点。四个位点中的两个需要有趣的蛋白质Reb1p，它不仅阻止极性模式的分叉，还终止rDNA的转录，并可能还作为转录因子启动转录。我们希望研究Reb1p的作用机制。我们已经将S.pombe的Ter1的位置缩小到~30bp，并希望克隆编码同源终止子蛋白的基因，并研究其作用方式。最后，我们希望揭示Swi1和Swi3蛋白的作用模式，以确定它们是否起到“抗扫描酶”的作用。

DESCRIPTION (provided by applicant): 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 DNA. 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 Reb1 p. 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".