Mechanism of Termination of DNA Replication

描述（由申请人提供）：我们建议通过酵母遗传学，体内和体外生物化学的结合，继续研究酵母S. cerevisiae和远亲分裂酵母Schizosaccharomyces pombe的复制终止蛋白的作用机制，并开始朝着建立与末端DNA结合的终止蛋白结晶试验的长期目标工作。在酿酒酵母中，我们希望通过体外生化分析来揭示Fob1终止蛋白的作用机制，以研究该蛋白是否是一种拮抗真核生物复制解旋酶活性的对解旋酶。我们还提出了在复制末端找出检查点蛋白Tofl和Csm3的作用机制。我们发现S. pombe的rDNA间隔区有4个而不是3个复制阻滞位点。四个位点中的两个位点需要一种有趣的蛋白质reb1p，它不仅在极性模式下阻止分叉，而且还终止rDNA的转录，可能还作为转录因子开启转录。我们希望找出reb1p的作用机制，我们已经将S. pombe的Ter1的位置缩小到~30bp，我们希望克隆编码同源终止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，以确定它们是否作为“抗扫酶”。

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".