PERSISTENT RNA/DNA HYBRID AT A T4 REPLICATION ORIGIN

目的是进一步确定DNA复制的分子机制 入会仪式 复制起始是一个重要的事件， 在所有的生物系统中都是如此。复制叉形成和 维护对于准确复制遗传信息至关重要， 下一代体内结构分析支持一个模型， 一个持久的RNA-DNA杂交体（R环）的形成是一个 复制起始重要中间体。 的格局 T4起源的起始可以作为比较 其他起源的结构和启动机制 原核和真核基因组。 原始质粒拓扑结构将是 用作RNA-DNA杂交体形成的标记。来源质粒是 在T4噬菌体感染后的早期提取， 在有或没有RNase H的二维氯喹琼脂糖凝胶上 治疗 由RNase H引起的任何拓扑变化都将是 这是由于一个持久的混合体的去除。 其他相关方法 涉及使用拓扑异构酶来检测RNA-DNA杂合体。 的 杂合中间体可用于暴露非模板链和非模板链。 RNA本身作为引物。

The aim is to further define the molecular mechanisms of DNA replication initiation. Replication initiation is an essential event and is closely regulated in all biological systems. Replication fork formation and maintenance is critical to accurately duplicate genetic information for the next generation. In vivo structural analysis support a model in which the formation of a persistent RNA-DNA hybrid (R-loop) is an important intermediate in replication initiation. The pattern of initiation at T4 origins may serve as a basis for comparison of the structures and mechanisms of initiation at the origins of other prokaryotic and eukaryotic genomes. Origin plasmid topology will be used as a marker for RNA-DNA hybrid formation. Origin plasmid is extracted at early times after T4 phage infection and electrophoresed on two-dimensional chloroquine agarose gels with or without RNase H treatment. Any topological changes caused by the RNase H will be attributed to the removal of a persistent hybrid. Other related methods involve the use of topoisomerases to detect the RNA-DNA hybrid. The hybrid intermediate may serve to expose the nontemplate strand and the RNA itself as a primer.