RECOMBINATION INTERMEDIATES-- PROPERTIES & PROCESSING

我们建议继续和扩大我们的研究的形成，性质和决议的霍利迪路口（HJ）。 我们正在使用的系统，int依赖的位点特异性途径的噬菌体λ，已被广泛研究在遗传和生物化学水平。 它属于来自古细菌、真细菌、线粒体和酵母的蛋白质大家族，其在不存在高能量辅因子的情况下催化彼此具有最小同源性的DNA序列之间的重排。来自该家族的内切酶具有产生和解决HJ的独特能力。 霍利迪连接是“DNA代谢”的重要中间体，对其性质的理解应有助于基础知识的主体，作为遗传学一般领域与健康相关的进展的平台。拟议的实验取决于我们最近的研究结果，在特定位点的重组过程中的HJ形成和异构化的机制。 这些最近的一些见解，使设计的合成基板，数字突出在这个建议，因为他们专门监测的动力学短暂的异构化步骤。在这个提议中解决了六个具体的问题：1）什么蛋白质-DNA相互作用支配霍利迪连接的分辨率？2)什么样的蛋白质-蛋白质相互作用会影响拆分反应？3)什么样的动力学特征在霍利迪结的形成和分辨中是重要的，它们依赖于什么？a）第一链交换的连接是HJ异构化的先决条件（触发）吗？B）“促进”（化学上无关）的Int是否模拟（替代）了那些会产生HJ的Int？c）哪些特定的蛋白质-蛋白质相互作用在将HJ从一种异构形式“向前”移动到另一种异构形式中起作用？d）方向性的调节是否通过异构化步骤进行？

We propose to continue and extend our studies on the formation, properties and resolution of Holliday junctions (HJs). 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. It belongs to a large family of proteins from archaebacteria, eubacteria, mitochondria and yeast that catalyze rearrangements, in the absence of high energy cofactors, between DNA sequences with minimal homology to each other. Recombinases from this family have the unique capacity to both generate and resolve HJs. The Holliday junction is an important intermediate in "DNA metabolism" and an understanding of its properties should contribute to the body of basic knowledge that serves as a platform for health-related advances in the general area of genetics. The proposed experiments depend upon our recent findings regarding the mechanisms of HJ formation and isomerization during site-specific recombination. Some of these recent insights have enabled the design of synthetic substrates that figure prominently in this proposal because they specifically monitor the dynamics of the ephemeral isomerization step. 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 dynamic features are important in the formation and resolution of Holliday junctions and what do they depend upon? a) Is ligation of the first strand-exchange a prerequisite (trigger) for HJ isomerization? b) Do the "facilitating" (chemically uninvolved) Ints mimic (substitute for) those Ints that would have generated the HJ? c) Which specific protein-protein interactions play a role in moving the HJ "forward" from one isomeric form to the other? d) Is the regulation of directionality exerted through the isomerization step?