RECOMBINATION INTERMEDIATES--MECHANISMS IN RESOLUTION

这个项目源于我们对机构的长期兴趣，通过 这些遗传物质被重新分类和重新排列。我们建议研究 Holliday复合的形成、性质和分解 中间体。我们正在使用的系统，依赖于Int的站点特定 噬菌体lambda的途径已经被广泛地研究在两个 遗传和生化水平。因为霍利迪的中间数字 在如此多的重组途径中，突出的是，在 拟议的研究应该具有普遍意义--除了它们的 特定位点重组的重要性，以及(不断增长的)Int家族 重组酶。 提出的实验依赖于我们最近的发现，这一途径 通过顺序高度有序的一对相互作用的链进行 交流。他们还利用了我们的演示，证明了适当的 “合成”重组中间体提供了非常有用的工具 研究霍利迪交汇点。 这项提案涉及六个具体问题：1)什么是 DNA的作用：DNA同源性在启动链转移中的作用？2)什么是 分支机构迁移在解决方案中的作用和属性？3)什么是 结合模式和结合的Int蛋白之间的相互作用 Holliday连接？4)臂型远端部位在 Holliday结点的分辨率？5)什么是结构特征 与重组有关的Holliday连接？6)Int介导的 链转移具有与非法有关的机械特征 重组？

This project is derived from our long term interest in the mechanisms by which genetic material is reassorted and rearranged. We propose to study the formation, properties and resolution of Holliday recombination intermediates. The system we are using, the Int-dependent site-specific pathway of bacteriophage lambda, has been extensively studied at both the genetic and biochemical levels. Because the Holliday intermediate figures prominently in so many recombination pathways, the results obtained in the proposed studies should be of general significance -- in addition to their importance for site-specific recombination, and the (growing) Int family of recombinases. The proposed experiments depend upon our recent finding that this pathway proceeds via a sequential highly ordered pair of reciprocal strand exchanges. They also draw on our demonstration that the appropriate "synthetic" recombination intermediates afford a very useful tool for studying Holliday junctions. Six specific questions are addressed in this proposal: 1) What is the role of DNA:DNA homology in initiating strand transfer? 2) What are the role and properties of branch migration in resolution? 3) What are the binding patterns and interactions among the Int proteins bound at the Holliday junction? 4) What is the role of arm-type distal sites in the resolution of Holliday junctions? 5) what are the structural features of the Holliday junction in relation to recombination? 6) Does Int-mediated strand transfer have mechanistic features related to illegitimate recombination?