RECOMBINATION INTERMEDIATES

描述：Bacteriohpage lambda整合酶是一种特定于位点的 在形成过程中负责病毒整合的重组酶 关于Lambda前驱噬菌体的。对INT系统的研究提供了非常丰富的信息 就理解lambda集成的基础而言，但在更大的范围内 他们也被证明对这一领域非常有洞察力 一般的同源重组在理解 Holliday结形成和分辨的分子机制 (HJ)。这种四臂交叉链结构被认为是 在复合过程中形成的中心中间体。大量的什么 已知的关于Holliday结点的处理来自 Int蛋白的研究。 现已证实，Int蛋白是一种I型拓扑异构酶 进行负责重组的卵裂和连接。 众所周知，从DNA结构到核苷酸的分子细节 INT蛋白与同源结合位点的相互作用。INT作用于 核心DNA靶点，与蛋白质有两个结合位点，排列如下 颠倒的重复。顶股和底股的割裂点是 交错排列，并限定内部间隔区的边界 在重组DNA伴侣中是同源的。重组收益 通过一对顺序的相互链交换产生 解决一个四臂霍利迪交叉口。 已经积累了对反式切割模型的实验支持 两个Int原基贡献不同的氨基酸残基来产生 一个活动站点。换句话说，一个原型者提供域 用于核苷酸定位，而配对原始体则贡献 活性酪氨酸是裂解反应中的亲核剂。 兰迪实验室的实验更容易提供证据 根据单个活动站点进行解释。人为设计自杀 与来自相关lambda的Int蛋白结合的底物 核心DNA识别略有不同的噬菌体使 实验表明，相同的Int Protomer边界会使 瞬时蛋白质-DNA连锁。 第一个目的是分析蛋白质-DNA相互作用，以解决 HJS。而模型野生型HJS有四个核心Int结合位点和携带 在完全反应之外，具有一个和两个位点的HJS被切割，但是 未解决。添加第三个位点允许拆分；第三个分子 Int的被称为跨核异构体。他将执行几项 了解主动拆分复合体是否有三个或三个 四个国际传真机。 第二个目标涉及识别蛋白质-蛋白质相互作用， 影响拆分反应。这些实验的一个目的是 是试图解决这一发现之间的明显差异 Jayaram实验室FLP重组裂解反式和Landy Int在顺式中裂解的实验室结果。 第三个目标是定义最低蛋白质需要量 通过跨核心集成来提高分辨率。使用双特异性HJ 对于同源的配对位点，lambda和HK022 Int蛋白都是 有效解析所需；如果使用lambda Y342F突变体Int， 所有的决议都在香港的网站上。这一结果将扩大到 利用lambda Int片段刺激HK022 Int同源序列 网站。调查人员指出，其中最令人兴奋的结果是 实验将是鉴定和表征一种多肽 变构激活Int裂解。作为替代方案，这些 实验可以鉴定出一种含有反式亲核试剂的多肽。 第四个目标是了解DNA是如何在HJ-蛋白质中配置的 很复杂。不动4路HJS的DNA臂构型 在没有蛋白质的情况下进行了广泛的研究。一套 实验将确定HJ中长臂和短臂的配置。 蛋白质复合体的凝胶迁移率分析。兰迪博士指出， 初步研究表明，现有的无蛋白质HJS方法 也可以按此处建议的方式应用。他将控制数量和 在不同物种中插入非结合序列的结合Int的位置 通过使用双特异性Holliday连接。他还会问 在Int裂解后是否更改手臂的处置。 第五个目标是了解哪些动态特性对 Holliday结点的形成和分解。为了做到这一点，他将 使用不同的HJ对分辨率进行仔细的动力学测量 底物，特别是了解反应是否为两相反应 在某些条件下，以及操纵这一点的能力如何 对这种反应有不同的看法。 第六个目标是探讨美国航空运输协会ARM地点和 辅助蛋白对拆分效率和方向性的影响。 前面的所有目标都处理仅包含核心Int的HJ 但是，在核心区两侧还有许多其他的结合序列 Int的其他分子加上IHF等其他蛋白质的分子， XIS和FIS。因此，在att位dna中存在较高的顺序结构。 它们对重组很重要。此前的研究表明， 这些建筑群的基本建筑主题是由 高亲和力臂和低亲和力臂之间的二价INT 核心站点。DNA结合促进了这些桥的形成 使正确的臂和核心位置对接近的蛋白质 接近和适当的登记。

DESCRIPTION: Bacteriohpage lambda integrase is a site-specific recombinase that is responsible for viral integration during formation of lambda prophage. Studies on the Int system have been very informative as far as understanding the basis of lambda integration, but in a larger sense, they have also proven to be extremely insightful for the field of general homologous recombination in terms of understanding the molecular mechanism of formation and resolution of the Holliday junction (HJ). This four-armed crossed strand structure is considered to be the central intermediate formed during recombination. A great deal of what is known about the processing of the Holliday junction comes from studies on Int protein. It has been established that Int protein is a type I topoisomerase that carries out the cleavages and ligations responsible for recombination. Molecular details of DNA structure down to the nucleotide are known for Int protein interaction with the cognate binding site. Int acts on a core DNA target that has two binding sites for the protein arranged as an inverted repeat. Cleavage points on top and bottom strands are staggered and define the boundaries of an internal spacer region that is homologous in the recombining DNA partners. Recombination proceeds via a sequential pair of reciprocal strand exchanges that generate then resolve a 4-armed Holliday junction. Experimental support has accumulated for a trans cleavage model in which two Int protomers contribute different amino acid residues to generate a single active site. In other words, one protomer provides the domain for nucleotide positioning while the partner protomer contributes an active tyrosine as the nucleophile in the cleavage reaction. Experiments from Landy's laboratory have provided evidence more easily interpreted in terms of a single active site. Engineered suicide substrates in conjunction with an Int protein from a related lambda phage which has slightly different core DNA recognition have enabled experimentation showing that the same Int protomer bound makes the transient protein-DNA linkage. The first aim analyzes protein-DNA interactions needed for resolution of HJs. While model wildtype HJs have four core Int binding sites and carry out the complete reaction, HJs with one and two sites are cleaved, but not resolved. Adding a third site permits resolution; the third molecule of Int is called the cross-core protomer. He will carry out several experiments to learn whether the active resolution complex has three or four Int protomers. The second aim deals with identifying protein-protein interactions that influence the resolution reaction. One objective of these experiments is to try to resolve the apparent discrepancy between the finding of the Jayaram laboratory that Flp recombinanse cleaves in trans and the Landy laboratory result that Int cleaves in cis. The third aim is to define the minimal protein requirements for stimulation of resolution by a cross-core Int. Using the bispecific HJ with homologous partner sites, both lambda and HK022 Int proteins are required for efficient resolution; if lambda Y342F mutant Int is used, all of the resolution is at HK sites. That result will be extended by using fragments of lambda Int to stimulate HK022 Int at its cognate sites. The investigator notes that the most exciting outcome of these experiments would be to identify and characterize a peptide that allosterically activates Int cleavage. As an alternative these experiments could identify a peptide containing a trans nucleophile. The fourth aim is to learn how the DNA is configured in the HJ-protein complex. The configuration of the DNA arms in immobile 4-way HJs has been studied extensively in the absence of proteins. One set of experiments will determine the disposition of long and short arms in HJ- protein complexes by assaying gel mobility. Dr. Landy noted that preliminary studies indicate that existing methods for protein-free HJs can also be applied as proposed here. He will control the number and positions of bound Int by inserting non-binding sequences in various arms and by using bispecific Holliday junctions. He will also ask whether the disposition of the arms is altered after Int cleavage. The fifth aim is to learn which dynamic features are important for the formation and resolution of Holliday junctions. To do this, he will carry out careful kinetic measurements of resolution using different HJ substrates, in particular to learn whether the reaction is biphasic under certain conditions and how the ability to manipulate this fits with different views of the reaction. The sixth aim probes long-range effects of the att arm sites and accessory proteins on the efficiency and directionality of resolution. All of the previous aims deal with HJs that contain only the core Int sites; however, flanking the core are many other sequences that bind other molecules of Int plus molecules of other proteins such as IHF, Xis, and Fis. Thus, there are higher order structures in att site DNA that are important for recombination. Previous studies have shown that the basic architectural motif in these complexes is a bridge formed by the bivalent Int between a high-affinity arm site and a low-affinity core site. Formation of these bridges is facilitated by the DNA binding proteins which bring the correct pair of arm and core sites into close proximity and proper register.