MECHANISM OF SITE SPECIFIC DNA RECOMBINATION

描述：位点特异性DNA重组是一种发育开关 在原核生物和真核生物中都有。原则上，这种反应可以， 通过切除、倒置或易位来重新排列遗传信息，以及 通过打开或关闭启动子来控制基因表达的模式。这个 复合的基本化学包括或内的磷酰基转移 在核酸链之间，并且必须与 一般重组、RNA剪接和DNA转座。这项建议 寻求继续支持国际和平研究所在化学和机械方面的研究 FLP位点特异性介导的链断裂和重组的特征 酿酒酵母的重组酶。FLP是整合酶超家族的一员 重组酶蛋白，包括lambda整合酶、FLP、cre。 酵母菌的P1重组酶和R重组酶。这些 重组酶在40个氨基酸区域内的序列相似性有限 在它们的羧基末端，有四个不变残基：两个精氨酸，一个 组氨酸和酪氨酸。酪氨酸位于FLP的活性部位，是 通过3‘-磷酸酪氨酰与DNA共价连接的残基 联动。Arg-His-Arg(RHR)三联体似乎构成了催化剂的一部分 口袋里也有。众所周知，FLP单体只有一个 部分活动站点。一个完整的活动站点是通过共享部分 至少两个蛋白质单体的活性部位，其中一个贡献 RHR三合会，另一个是提尔。 建议的实验集中在三个不同的问题上，与 FLP介导的反应机理。第一个目标是确定 是FLP的低聚状态，它与每个 反应分两步进行，即链断裂和重聚。这里的问题是 FLP的重组单位是否为所提出的不对称三聚体 由钱和考克斯提出的或由两个不对称二聚体组成的四聚体 李和贾亚拉姆。拟议的测试是基于以下证明： RHR三联体突变体补充酪氨酸突变体，即活性位点可以 由两个突变蛋白的异源二聚体组装而成。我们的想法是 问一问霍利迪结的分辨率，这需要两个活跃的 位点，可以由三合一突变体结合酪氨酸进行 变种人。如果活性单元是不对称三聚体，则分辨率应为 不可能。或者，如果活性单元是四聚体，那么一些 应该会进行解析。另一项测试涉及建造复合体 三个活性单体和一个Y343F单体位于特定位置 位置，并查看解析事件的位置。 第二个目的是了解活性位点排除的机制 在FLP介导的重组过程中。FLP重组分两步进行 单链交换，这意味着只有四种潜在的 FLP四聚体中的活性位点同时起作用。第一双鞋 用于形成Holliday连接中间体，而第二个 配对来解决它。需要检验的具体假设是，DNA是否 由于两个FLP单体与一个FLP结合而引起的140度弯曲 此活动站点排除涉及完整站点。这项测试涉及 构建一组包含凸起的FLP基板，这些凸起将预先处理 手臂以弯曲的形式堆叠，并通过以下方式确定乳沟的不对称性 FLP。这些底物将用野生型FLP和突变体进行测试 变异体在DNA弯曲方面有缺陷。包含凸起的结构 底物将通过物理分析确认，包括核磁共振和FRET。 第三个目标是了解dna突触在这两种疾病中的作用。 FLP和Holliday连接催化的起始链切割反应 解析事件。关键问题是底物能否进入 突触复合体处于预裂状态，或者，或者，无论是裂解 在一个完整的位置内占据一个FLP二聚体是流产的，具有生产性 裂解，能够作为重组进行分解，只有在 两个突触完整的站点。测试涉及建筑合成材料 底物来测试这两个问题。

DESCRIPTION: Site specific DNA recombination acts as a developmental switch in both prokaryotes and eukaryotes. The reaction can, in principle, rearrange genetic information via excision, inversion, or translocation, and control patterns of gene expression by turning promoters on or off. The basic chemistry of recombination involves phosphoryl transfer within or between nucleic acid chains, and must share mechanistic similarities with general recombination, RNA splicing and DNA transposition. This proposal seeks continued support for the PI's studies on the chemical and mechanistic features of strand breakage and reunion mediated by the Flp site-specific recombinase of S. cerevisiae. Flp is a member of the Integrase superfamily of recombinase proteins, which includes lambda integrase, Flp, the cre recombinase of P1 and the R recombinase of Zygosaccharomyces. These recombinases have limited sequence similarity within a 40 amino acid region in their carboxyl termini, with four invariant residues: two arginines, a histidine and a tyrosine. The tyrosine is in the active site of Flp and is the residue that becomes covalently linked to DNA by a 3'-phosphotyrosyl linkage. The Arg-His-Arg (RHR) triad appears to form part of the catalytic pocket as well. It is well established that a Flp monomer has only a partial active site. A complete active site is formed by sharing partial active sites of at least two monomers of the protein, one contributing the RHR triad, and the other the Tyr. The proposed experiments focus on three different issues related to the mechanism of the Flp mediated reactions. The first aim is to determine what is the oligomeric state of Flp that is catalytically relevant to each of the two steps of the reaction, strand breakage and reunion. The issue here is whether the recombinational unit of Flp is an asymmetric trimer as proposed by Qian and Cox or a tetramer composed of two asymmetric dimers as proposed by Lee and Jayaram. The proposed test is based on the demonstration that RHR triad mutants complement tyrosine mutants, i.e. that an active site can be assembled by a heterodimer of the two mutant proteins. The idea is to ask whether the resolution of a Holliday junction, which requires two active sites, can be carried out by a triad mutant in combination with a tyrosine mutant. If the active unit is an asymmetric trimer, then resolution should not be possible. Alternatively, if the active unit is a tetramer, then some resolution should occur. A further test involves constructing complexes with three active monomers and one Y343F monomer positioned at specific places and looking at the position of the resolution event. The second aim is to understand the mechanism of active site exclusion during Flp-mediated recombination. Flp recombination proceeds in two steps of single strand exchanges, implying that only two of the four potential active sites within a Flp tetramer are function at a time. The first pair of sites is used to form the Holliday junction intermediate, and the second pair to resolve it. The specific hypothesis to be tested is whether the DNA bend of >140 degrees that is induced by binding of two Flp monomers to a complete site is involved in this active site exclusion. The test involves building a set of Flp substrates containing bulges that would predispose the arms to stack in a bent form, and determine the asymmetry of cleavage by Flp. The substrates will be tested with wild type Flp and with mutant variants defective in DNA bending. The structure of the bulge-containing substrates will be confirmed by physical analysis, both NMR and FRET. The third aim is directed at understanding the role of DNA synapsis in both the initial strand cleavage reaction catalyzed by Flp and Holliday junction resolution event. The key question is whether substrate can enter the synaptic complex in a pre-cleaved state, or, alternatively, whether cleavage within a full site occupied by a Flp dimer is abortive, with a productive cleavage, capable of being resolved as a recombinant, only possible within a pair of synapsed full sites. The tests involve the building synthetic substrates to test both questions.