RECOMBINATION INTERMEDIATES-- PROPERTIES & PROCESSING

我们建议继续和扩展我们对假日结（HJs）的形成、性质和分解的研究。我们正在使用的系统，噬菌体λ的int依赖位点特异性途径，已经在遗传和生化水平上得到了广泛的研究。它属于一个来自古细菌、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小。来自这个家族的重组酶具有产生和解决hj的独特能力。Holliday结是“DNA代谢”的重要中间体，对其特性的理解应该有助于建立基础知识体系，为遗传学一般领域与健康相关的进展提供平台。提出的实验取决于我们最近关于HJ形成和异构化机制的发现在位点特异性重组。最近的一些见解使得合成底物的设计在本提案中占有突出地位，因为它们专门监测短暂异构化步骤的动力学。在这个提议中解决了六个具体问题：1)什么蛋白质- dna相互作用决定了Holliday连接的分辨率？2)什么蛋白质-蛋白质相互作用影响分解反应？3)在假日结点的形成和解决中，哪些动态特征是重要的？它们依赖于什么？a)第一链交换连接是HJ异构化的先决条件（触发条件）吗？b)“促进”（不涉及化学成分）的int是否模仿（替代）了那些可能产生HJ的int ？c)哪种特定的蛋白质-蛋白质相互作用在将HJ从一种异构体形式“向前”移动到另一种异构体形式中起作用？d)是否通过异构化步骤对方向性进行调节？

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?