RECOMBINATION INTERMEDIATES -- PROPERTIES & PROCESSING

该建议是对DNA重组中间体（四向DNA连接）的形成、性质和分辨率的持续研究的延伸，称为Holliday连接（HJ）。本文使用的模型系统，即噬菌体lambda的int依赖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。它属于一个来自古细菌、真细菌和酵母的蛋白质大家族，催化DNA序列之间的重排，彼此之间的同源性最小。来自该家族的重组酶具有独特的能力，可以在不输入能量的情况下产生和分解hj。Holliday连接是许多生物途径中重排DNA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，作为遗传学领域与健康相关的进展的平台。提出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hj如何被Int蛋白产生和分解的见解，这也是病毒编码的家族成员中大子集的异质二价DNA结合蛋白的范例。推动这一提议的特别见解涉及在DNA切割、连接和异构化的严格有序序列中，Int原体之间的通信机制，以及包含Int、HJ和“臂型”DNA位点的三元复合物的结构。实验可分为四个一般性问题：a)在HJ中间体形成过程中，是什么决定了链交换的精确顺序？b)在HJ解决过程中，什么机制确保两个伙伴Int对中只有一个在同一时间“登台”（活动）？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？

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?