RECOMBINATION INTERMEDIATES -- PROPERTIES & PROCESSING

这项提议是对DNA重组中间体(四向DNA结)的形成、性质和分辨的正在进行的研究的延伸，被称为Holliday结(HJ)。这里使用的模型系统，噬菌体lambda的Int依赖的位点特异性重组途径，已经在遗传和生化水平上得到了广泛的表征。它属于来自考古细菌、真细菌和酵母的蛋白质大家族，这些蛋白质催化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在不需要能量输入的情况下产生和分解HJS的独特能力。Holliday连接是许多重排DNA的生物途径中的重要中介，对其性质的了解应该有助于形成基础知识体系，作为遗传学领域与健康相关的进展的平台。拟议的实验源于最近的生化、遗传和结构数据，这些数据提供了对HJS是如何由Int蛋白产生和分解的见解，Int蛋白也是病毒编码的家族成员的大子集的范例，这些成员是异源二价DNA结合蛋白。推动这一建议的特殊见解涉及DNA切割、连接和异构化的严格有序序列中Int原型之间的通信机制，以及包含Int、Hj和“臂型”DNA位点的三元复合体的结构。这些实验可以归结为四个一般问题：a)是什么决定了HJ中间体形成过程中链交换的准确顺序？B)在HJ解析期间，如何确保一次只有两个合作伙伴Int对中的一个处于“舞台”(活动)状态？C)臂型DNA位点如何提高HJ分辨率的保真度？D)三元HJInt-臂型DNA复合体在高分辨率下是什么样子的？

This proposal is an extension of ongoing studies on the formation, properties and resolution of the DNA recombination intermediate (four-way DNA junction) known as a Holliday junction (HJ). The model system used here, the Int-dependent site-specific recombination pathway of bacteriophage lambda, has been extensively characterized at both the genetic and biochemical levels. It belongs to a large family of proteins from archaebacteria, eubacteria, and yeast that catalyze rearrangements between DNA sequences with minimal homology to each other. Recombinases from this family have the unique capacity to both generate and resolve HJs without the input of energy. Holliday junctions are an important intermediate in many biological pathways that rearrange DNA and an understanding of their properties should contribute to the body of basic knowledge that serves as a platform for health-related advances in the area of genetics. The proposed experiments grow out of recent biochemical, genetic, and structural data that afford insights into how HJs are generated and resolved by the Int protein, which is also a paradigm for the large subset of virally-encoded family members that are hetero-bivalent DNA binding proteins. The particular insights which drive this proposal concern the mechanisms of communication between Int protomers during the strictly ordered sequence of DNA cleavages, ligations and isomerizations and the architecture of a ternary complex containing Int, HJ, and "arm-type" DNA sites. The experiments can be grouped into four general questions: a) What determines the precise order of strand exchanges during formation of the HJ intermediate? b) During HJ resolution, what is the mechanism that ensures only one of the two partner Int pairs will be "on stage" (active) at one time? c) How do arm-type DNA sites increase the fidelity of HJ resolution? d) What does the ternary HJInt- arm-type DNA complex look like at high resolution?