RECOMBINATION INTERMEDIATES: MECHANISMS IN RESOLUTION

本项目源于我们对以下机制的长期兴趣： 哪些遗传物质被重新组合和重新排列。 我们建议研究 在一个特定的过程中， 重组途径。 我们使用的系统，Int-dependent 噬菌体λ的途径，是一个被称为 保守转座 这些实验取决于我们的 最近的发现是，合成chi形式的att位点DNA，形式上是 类似于Holliday型重组中间体，通过 纯化的Int蛋白与特定的预测产物。 本提案的具体目标是：1）确定决议的性质 通过纯化的Int蛋白质，与特殊的“合成”霍利迪中间体 注意解决的具体性和方向性问题。 2)为了确定霍利迪结构的分辨率如何受到 辅助蛋白IHF和Xis。 3)为了确定如何解决 Holliday结构受Int蛋白在远离细胞的位点的影响。 链交换区。 4)为了研究这四个方面之间的关系， Holliday交叉区的Int蛋白结合位点及其作用 DNA：该区域的DNA同源性。 5)要将 具有Int蛋白特异结构域的Holliday中间体。 6)找到 有利于隔离“自然”霍利迪的条件或结构 中间体的

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 resolution (and the formation) of intermediates in one particular pathway of recombination. The system we are using, the Int-dependent pathway of bacteriophage lambda, is an example of what is called conservative transposition. The proposed experiments depend upon our recent finding that synthetic chi forms of att site DNA, which are formally analogous to Holliday-type recombination intermediates, are resolved by purified Int protein to specific predicted products. The specific aims of this proposal are: 1) To characterize the resolution of "synthetic" Holliday intermediates by purified Int protein, with special attention to the questions of specificity and directionality in resolution. 2) To determine how the resolution of Holliday structures is influenced by the accessory proteins IHF and Xis. 3) To determine how the resolution of Holliday structures is influenced by Int protein at sites distant from the region of strand exchange. 4) To study the relationships among the four Int protein binding sites in the Holliday crossover region and the role of DNA:DNA homology in this region. 5) To associate resolution of the Holliday intermediate with a specific domain of Int protein. 6) To find conditions or constructions that favor the isolation of "natural" Holliday intermediates.