Recombination Intermediates - Properties and Processing

描述（由申请人提供）：被称为Holliday结（HJ）的四向DNA结在DNA重组、复制和修复中起着重要作用；它是同源重组和位点特异性重组的中心中间体，在这方面，对HJ的遗传、生化和结构研究特别富有成果。我们一直在研究这种复合中间体的形成、性质和分辨率。噬菌体重组途径，是一大类位点特异性重组酶的范例，在原核生物、真核生物和古细菌中具有广泛的功能。这个家族的重组酶催化DNA序列之间的重排（在DNA中称为att位点）。系统)，彼此几乎没有同源性，并且能够在没有能量输入的情况下产生和分解hj。本提案中的实验源于大量的生化、遗传和结构数据，这些数据可以深入了解hj是如何产生和解决的。噬菌体编码的Int蛋白，这也是病毒编码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。这四项具体目标涉及上一个项目期间取得的成果和（或）在实地长期存在的问题。它们是：1)测试HJ与Int和臂型寡核苷酸络合（HJ-Int- arm complex）的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和/或构象步骤；3)确定HJ形成的限速步长；4)确定attl - binding和attr - binding Integrase亚基的数量分布，这些亚基导致成功的突触事件和随后形成稳定的Holliday连接。目标1-3取决于在前一个项目期间开发和使用的方法。目标4涉及对其中一些技术的逻辑扩展。目标2-4中提出的几个问题很难用集合生物化学来回答，因为许多相关的中间体是短暂的和/或难以验证为通路上的事件。我们建议使用我们在单分子方法上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（这在该领域迄今为止是独一无二的）。被称为霍利迪结的四向DNA连接在DNA重组、复制和修复中起着重要作用。我们一直在研究这种重组中间体在？噬菌体编码重组途径，是自然界中普遍存在的一大家族位点特异性重组酶的范例，具有广泛的功能，其中许多功能在健康相关问题的各个方面都占有突出地位。

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.