Helical and Superhelical Structure of DNA

在这个续订申请中提出的实验围绕三个主要目标。第一个是设计一种酶，用来制造正向超螺旋DNA。它的基础是，在转录过程中，正的超螺旋债务建立在复制叉之前，而负的超螺旋张力积累在复制叉的后面。当DNA和聚合酶在转录过程中都受到限制时，这种情况是戏剧性的。这种酶的模型是一种融合蛋白，它能产生这两种条件，也就是说，一种含有连接到RNA聚合酶的序列特定DNA结合域的蛋白质。第二个目标是分析体内的染色质结构。在酵母和高等真核细胞中，DNA核酸酶将在可诱导调控序列的控制下进行克隆。为了避免在体内制造核酸酶的问题，策略是构建在低温下不活跃的冷敏感突变体，但当介质转移到更高温度时，能够在体内切割DNA。第三个目标是制造超级卷曲的RNA。这项研究的结果应该会加强我们对基因结构的理解，以及它们在生命系统中如何在分子水平上发挥作用。

The experiments proposed in this renewal application revolve around three primary goals. The first is to design an enzyme for making positively supercoiled DNA. It is based on the thesis that during transcription a positive superhelical debt is built ahead of the replication fork while a negative superhelical tension accumulates behind the replication fork. The situation is dramatic when both the DNA and polymerase are constrained during the act of transcription. The model for such an enzyme is a fusion protein that generates both conditions, that is, a protein containing a sequence-specific DNA binding domain joined to RNA polymerase. The second goal is to analyze chromatin structure in vivo. DNA nucleases will be cloned under the control of inducible regulatory sequences in yeast and in higher eukaryotic cells. To obviate problems in making nucleases in vivo, the strategy is to construct cold sensitive mutants that are inactive at low temperature, but able to cleave DNA in vivo when the medium is shifted to a higher temperature. The third goal is to make supercoiled RNA. The results of the research should enhance our understanding of the structure of genes and how they function at the molecular level in living systems.