Recombination Intermediates - Properties and Processing

描述(申请人提供)：被称为Holliday连接(HJ)的四向DNA连接在DNA重组、复制和修复中起着重要的作用；它是同源重组和位点特异性重组的中心中间体，对HJ的遗传、生化和结构研究特别有成果。我们一直在研究这种复合中间体的形成、性质和拆分。噬菌体重组途径，一大类位点特异性重组酶的范例，在原核生物、真核生物和古生物中管理着广泛的功能。该家族的重组酶催化DNA序列之间的重排(称为？系统)，彼此几乎没有同源性，并且有能力在不输入能量的情况下产生和分解HJS。这个建议中的实验来自大量的生化、遗传和结构数据，这些数据为深入了解HJS是如何产生和解决的？噬菌体编码的Int蛋白，它也是病毒编码的家族成员的一个大子集的模型，这些成员是异二价DNA结合蛋白。这四个具体目标涉及在前一个项目期内取得的成果和(或)长期在外地取得的成果所产生的问题。它们是：1)测试HJ与Int和臂型寡核苷酸(HJ-Int-Arm复合体)晶体结构的功能意义；2)确定形成稳定的突触复合体需要哪些化学步骤和/或构象步骤；3)确定HJ形成过程中的限速步骤；4)确定导致成功突触事件和随后形成稳定的Holliday连接的attL结合和Attr结合的整合酶亚基的数量分布。目标1-3取决于在前一个项目期间开发和利用的方法。目标4涉及其中一些技术的合乎逻辑的扩展。在AIMS 2-4中提出的几个问题将极难用系综生物化学来回答，因为许多相关的中间体是瞬时的和/或难以验证为途径上的事件。我们建议通过对单分子方法的投资的合理扩展来解决这些问题，以研究特定部位的重组(这在该领域迄今是独一无二的)。被称为Holliday连接的四向DNA连接在DNA重组、复制和修复中发挥着重要作用。我们一直在研究这种复合中间体的形成和分解。噬菌体编码的重组途径，是一大类位点特异性重组酶的范例，在自然界中普遍存在，并管理着广泛的功能，其中许多在与健康相关的问题的各个方面都发挥着重要作用。

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.