MECHANISM OF SITE SPECIFIC DNA RECOMBINATION

描述：位点特异性DNA重组作为发育开关 在原核生物和真核生物中。 原则上，该反应可以， 通过切除、倒位或易位重新排列遗传信息， 通过开启或关闭启动子来控制基因表达模式。 的 重组的基本化学过程涉及到细胞内的磷酰基转移， 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PI的化学和机械的研究 由Flp位点特异性介导的链断裂和重新连接的特征 重组酶。啤酒。 Flp是整合酶超家族的成员 重组酶蛋白，包括λ整合酶，Flp，cre P1的重组酶和接合酵母的R重组酶。 这些 重组酶在40个氨基酸区域内具有有限的序列相似性 在它们的羧基末端，有四个不变的残基：两个精氨酸，一个精氨酸， 组氨酸和酪氨酸。 酪氨酸位于Flp的活性位点， 通过3 '-磷酸酪氨酰与DNA共价连接的残基 联动 Arg-His-Arg（RHR）三联体似乎形成了催化的 口袋也是。 已经确定Flp单体仅具有 部分活性部位。 一个完整的活性部位是由部分 蛋白质的至少两个单体的活性位点，其中一个单体贡献蛋白质的活性位点。 RHR三联体，另一个是Tyr。 所提出的实验集中在与 Flp介导的反应机制。 第一个目标是确定 是Flp的低聚状态，其与每个酶的催化相关， 反应的两个步骤，链断裂和重聚。 这里的问题是 Flp的重组单元是否是所提出的不对称三聚体 由Qian和考克斯提出，或者如所提出的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三联体突变体与酪氨酸突变体互补，即活性位点可以 由两种突变蛋白的异二聚体组装。 这个想法是 问是否解决一个霍利迪路口，这需要两个积极的 位点，可以通过三联体突变体与酪氨酸 变种人 如果活性单元是不对称三聚体，则拆分应 不可能 或者，如果活性单元是四聚体，则一些 决议应该发生。 进一步的测试包括构建复合物 其中三个活性单体和一个Y343 F单体位于特定位置， 位置并查看分辨率事件的位置。 第二个目的是了解活性位点排斥的机制 在Flp介导的重组过程中。 Flp重组分两步进行 这意味着四种可能的单链交换中只有两种 Flp四聚体中的活性位点每次都起作用。 第一对 的网站是用来形成霍利迪交界处中间，第二个 要检验的具体假设是， 由两个Flp单体结合到一个包含两个Flp单体的载体上诱导的>140度的弯曲。 在该活性位点排除中涉及完整的位点。 测试涉及 构建一组含有凸起的Flp基底， 臂以弯曲的形式堆叠，并通过以下方式确定解理的不对称性： Flp.将用野生型Flp和突变体Flp检测底物。 在DNA弯曲中有缺陷的变体。 本发明公开了一种含凸起的结构， 将通过NMR和FRET两者的物理分析来确认底物。 第三个目的是了解DNA突触在这两个过程中的作用。 Flp和Holliday连接催化的起始链断裂反应 决议事件。 关键问题是基材能否进入 突触复合物处于预裂解状态，或者，可替代地，是否裂解 在被Flp二聚体占据的完整位点内是失败的，具有生产性 切割，能够作为重组体解析，仅可能在 一对突触完整位点。 测试包括建筑合成物 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.