Recombination Intermediates - Properties and Processing

描述（由申请人提供）：被称为霍利迪连接（HJ）的四向DNA连接在DNA重组、复制和修复中占有重要地位;它是同源重组和位点特异性重组的中心中间体，其中HJ的遗传、生物化学和结构研究特别富有成效。我们一直在研究的形成，性质和决议，这种重组中间体？噬菌体重组途径，一个位点特异性重组酶大家族的范式，在原核生物、真核生物和古细菌中发挥广泛的功能。这个家族的酶催化DNA序列之间的重排（在DNA序列中称为att位点）。系统），彼此具有非常小的同源性，并且具有在不输入能量的情况下产生和分辨HJ的能力。在这个建议中的实验成长出一个庞大的身体的生化，遗传和结构数据，提供深入了解如何产生和解决的HJ？噬菌体编码的Int蛋白，其也是作为异二价DNA结合蛋白的病毒编码的家族成员的大子集的模型。这四个具体目标涉及上一个项目期间取得的成果和/或实地长期存在的问题。它们是：1）测试与Int和臂型寡核苷酸复合的HJ的晶体结构的功能含义2）确定形成稳定的突触复合物所需的化学和/或构象步骤; 3）确定HJ形成中的限速步骤; 4）确定attL结合和attR结合的整合酶亚基的数量分布，所述整合酶亚基导致成功的突触事件和随后稳定的Holliday连接的形成。目标1-3取决于上一个项目期间开发和使用的方法。目标4涉及其中一些技术的逻辑扩展。目标2-4中提出的几个问题将极难使用整体生物化学回答，因为许多相关中间体是短暂的和/或难以验证为途径上的事件。我们建议使用我们在单分子方法中的投资的逻辑扩展来解决这些问题，以研究位点特异性重组（迄今为止在该领域中是独一无二的）。被称为霍利迪连接的四路DNA连接在DNA重组、复制和修复中占有重要地位。我们一直在研究这种重组中间体的形成和解决方案？噬菌体编码的重组途径是一个大家族的位点特异性重组酶的范例，其在自然界中普遍存在并管理广泛的功能，其中许多功能在健康相关问题的各个方面都很突出。

DESCRIPTION (provided by applicant): The four-way DNA junction known as the Holliday junction (HJ) figures prominently in DNA recombination, replication and repair; it is the central intermediate in both homologous and site-specific recombination, where genetic, biochemical and structural studies of the HJ have been particularly fruitful. We have been studying formation, properties, and resolution of this recombination intermediate in the ? phage recombination pathway, a paradigm for a large family of site-specific recombinases that administers a wide range of functions in prokaryotes, eukaryotes, and archaea. Recombinases of this family catalyze rearrangements between DNA sequences (called att sites in the ? system) with very little homology to each other and have the ability to generate and resolve HJs without the input of energy. Experiments in this proposal grow out of a large body of biochemical, genetic, and structural data that afford insights into how HJs are generated and resolved by the ? phage-encoded Int protein, which is also a model for the large subset of virally-encoded family members that are heterobivalent DNA binding proteins. The four specific aims address questions growing out of results obtained during the previous project period and/or of long standing in the field. They are: 1) to test the functional implications of the crystal structure of HJ complexed with Int and arm-type oligonucleotides (HJ-Int-Arm complex); 2) to determine which chemical and/or conformational steps are required to form a stable synaptic complex; 3) to determine the rate limiting step in formation of the HJ; 4) to determine the numerical distribution of attL-bound and attR-bound Integrase subunits that lead to successful synaptic events and the subsequent formation of stable Holliday junction. Aims 1-3 depend upon approaches developed and utilized during the previous project period. Aim 4 involves a logical extension of some of those techniques. Several of the questions posed in aims 2-4 would be extremely difficult to answer using ensemble biochemistry since many of the relevant intermediates are transient and/or difficult to verify as being on-pathway events. We propose to address these questions using logical extensions of our investment in single molecule approaches to studying site-specific recombination (something which is thus far unique in the field). The four-way DNA junction known as the Holliday junction figures prominently in DNA recombination, replication and repair. We have been studying formation and resolution of this recombination intermediate in the ? phage-encoded recombination pathway, a paradigm for a large family of site-specific recombinases that is ubiquitous in nature and administers a wide range of functions, many of which figure prominently in various aspects of health-related issues.