RECOMBINATION INTERMEDIATES: MECHANISMS IN RESOLUTION

重组中间体：解决机制

• 批准号: 3284124
• 负责人: ARTHUR LANDY
• 金额: $ 19.71万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-01-01 至 1989-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3284124
• 关键词: Escherichia coli; bacteriophage lambda; complementary DNA; genetic crossing over; genetic manipulation; genetic recombination; microorganism genetics; nucleic acid sequence; tissue /cell culture; transposon /insertion element

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.

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 中间体在一种特定情况下的分解(和形成) 重组途径。我们使用的系统是依赖于Int的 噬菌体Lambda的途径就是一个例子 保守的换位。提议的实验取决于我们的 最近的研究发现，人工合成的CHI形成了ATT位点DNA，这是正式的 类似于Holliday类型的重组中间体，通过 将纯化的Int蛋白转化为特定的预测产物。 这项提案的具体目标是：1)说明决议的特点 由纯化的Int蛋白合成的Holliday中间体，具有特殊的 注意解决问题的特殊性和方向性。 2)确定Holliday结构的分辨率如何受 辅助蛋白IHF和Xis。3)确定如何解决 Holliday结构受Int蛋白的影响 链交换区域。4)研究四者之间的关系 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)将 Holliday中间体具有特定结构域的Int蛋白。6)找到 有利于隔离“自然”假日的条件或建筑 中间体。