RECOMBINATION INTERMEDIATES-- PROPERTIES & PROCESSING

我们建议继续和扩大我们对Holliday结(HJS)的形成、性质和分辨的研究。我们正在使用的系统，即噬菌体lambda的Int依赖的位点特异性途径，已经在遗传和生化水平上进行了广泛的研究。它属于来自古细菌、真细菌、线粒体和酵母的蛋白质大家族，在没有高能辅助因子的情况下，催化DNA序列之间的重排，彼此之间的同源性最低。来自该家族的重组酶具有产生和分解HJS的独特能力。Holliday连接是“DNA新陈代谢”的重要中间环节，对其特性的了解应有助于形成基础知识体系，作为一般遗传学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关于位点特定重组过程中HJ形成和异构化机制的发现。其中一些最新的见解使合成底物的设计成为可能，因为它们特别监控了短暂的异构化步骤的动力学。这一提议涉及六个具体问题：1)什么蛋白质-DNA相互作用控制Holliday连接的分辨？2)什么蛋白质-蛋白质相互作用影响分辨反应？3)什么动力学特征在Holliday连接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”(与化学无关的)Ints是否模仿(替代)那些本来会产生HJ的Ints？C)哪些特定的蛋白质-蛋白质相互作用在将HJ从一种同分异构体“向前”移动到另一种异构体的过程中发挥了作用？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?