RECOMBINATION INTERMEDIATES

我们建议研究Holliday的形成、性质和分解 利用Int依赖的位点特异性途径重组中间体 噬菌体Lambda。有几个具体目标是有动机的，或者 将由以下结果和来自 项目前期：霍利迪交界处的两个家庭自杀 底物的开发是为了：a)将Int切割事件从 紧密耦合的连接反应和，b)指向特定的Int 到特定站点的代理；两个合作伙伴Int无法执行解析 除非还存在第三个Int(跨核心)；跨核心 解析力的刺激是由FLEE提高卵裂率的结果 合作伙伴Ints；Holliday连接解析涉及位点上的切割 Int结合，即在顺式中；辅助蛋白和臂型Int 结合部位影响拆分的效率和方向性； 重组中涉及的更高阶约束由特定的 辅助蛋白介导的INT桥：分支迁移不是 ALL分辨过程中DNA同源性检测的主要机制 霍利迪交叉口。 这项提案涉及六个具体问题： 1)什么蛋白质-DNA相互作用决定了Holliday的分辨率 交界处？ 2)什么蛋白质-蛋白质相互作用影响拆分反应？ 3)刺激分辨率所需的最低蛋白质是多少？ 通过跨核心的Int？ 4)Holliday连接蛋白复合体中的DNA是如何配置的？ 5)什么动态特征在形成和分解过程中是重要的 Holliday交汇点以及它们依赖于什么？ 6)ATT现场武器和附件的远程效果如何 蛋白质影响拆分的效率和/或方向性？

We propose to study the formation, properties and resolution of Holliday recombination intermediates using the Int-dependent site-specific pathway of bacteriophage lambda. Several of the specific aims are motivated, or will be facilitated, by the following results and observations from the previous project period: two families of Holliday junction suicide substrates were developed to: a) disengage Int cleavage events from the closely coupled ligation reactions and, b) to direct specific Int protomers to specific sites; two partner Ints cannot carry out resolution unless a third Int (cross-core) is also present; the cross-core stimulation of resolution is the result of enhanced cleavage rates by flee partner Ints; Holliday junction resolution involves cleavage at the site of Int binding, i.e., in cis; the accessory proteins and arm-type Int binding sites influence the efficiency and directionality of resolution; the higher-order strictures involved in recombination consist of specific Int bridges mediated by accessory proteins: branch migration is not the principal mechanism of sensing DNA homology during resolution of the all site Holliday junction. Six specific questions are addressed in this proposal: 1) What protein-DNA interactions govern resolution of the Holliday junction? 2) What protein-protein interactions influence the resolution reaction? 3) What are the minimal protein requirements for stimulation of resolution by a cross-core Int? 4) How is the DNA configured in the Holliday junction-protein complex? 5) What dynamic features are important in the formation and resolution of Holliday junctions and what do they depend upon? 6) How do the long-range effects of the att site arms and accessory proteins influence the efficiency and/or directionality of resolution?