MECHANISMS OF SITE-SPECIFIC RECOMBINATION

描述（由申请人提供）：Flp（“翻转”）位点特异性重组酶由2微米环状酵母质粒编码。重组中的两个关键化学前步骤是：（1）Flp识别靶DNA位点和（2）在Flp相邻物之间建立功能性二聚体界面。通过定向进化，有可能放松或改变Flp的DNA特异性，并抑制削弱亚基间相互作用的突变。我们将确定晶体结构的“改变特异性”和“抑制”Flp变体与其同源的DNA底物。酵母中2微米环的持久性是由分配系统和扩增系统介导的。Flp重组是后者的核心。我们将应用拓扑方法来确定潜在的新活动的Flp，decatenation（和unknotting），这可能会进一步帮助平等的质粒分区。Flp重组受到重组配偶体（FRT位点）之间完美间隔区同源性的需要的限制。通常，在平面重组突触内，这些位点呈现反平行的几何形状。一项证据表明，间隔区异质性并不消除重组;相反，它促进甚至多轮重组以恢复间隔区同源性和亲本构型。此外，FRT网站建议具有平行的几何形状。我们将设计拓扑测试来解决这个明显的矛盾。Flp的所有生物化学分析均采用裸DNA底物进行。然而，2微米的质粒是核居民，包装在染色质中。我们将研究在核小体组装和染色质重塑的背景下进行重组的功能。我们将完成几项正在进行的机制研究，并启动新的研究，以阐明Flp在酵母体内的作用和调节。这些研究将促进我们对位点特异性重组过程中大分子识别和变构的理解。它们将为改进位点特异性重组作为基因工程工具的应用提供新的见解。最后，他们将为在高阶染色质和天然2微米环生理学的背景下分析重组奠定基础。

DESCRIPTION (provided by applicant): The Flp ('flip') site-specific recombinase is coded for by the 2 micron circle yeast plasmid. Two key pre- chemical steps in recombination are: (1) recognition of the target DNA site by Flp and (2) establishing a functional dimer interface between Flp neighbors. By directed evolution, it is possible to relax or alter the DNA specificity of Flp and to suppress mutations that weaken intersubunit interactions. We will determine the crystal structures of 'altered specificity' and 'suppressor' Flp variants in association with their cognate DNA substrates. The persistence of the 2 micron circle in yeast is mediated by a partitioning system and an amplification system. Flp recombination is at the heart of the latter. We will apply topological methods to identify potential novel activities of Flp, decatenation (and unknotting), which may further assist in equal plasmid partitioning. Flp recombination is constrained by the need for perfect spacer homology between recombination partners (FRT sites). Normally, the sites assume antiparallel geometry within the planar recombination synapse. One piece of evidence suggests that spacer heterology does not abolish recombination; rather it promotes even rounds of recombination to restore spacer homology and parental configuration. Furthermore, the FRT sites are suggested to have a parallel geometry. We will devise topological tests to resolve this apparent contradiction. All biochemical analyses of Flp have been carried out with naked DNA substrates. The 2 micron plasmid, though, is a nuclear resident, packaged into chromatin. We will study features of recombination carried out in the context of nucleosome assembly and chromatin remodeling. We will complete several mechanistic studies in progress and initiate new studies to shed light on the action and regulation of Flp in vivo in yeast. These investigations will advance our understanding of macromolecular recognition and allostery during site-specific recombination. They will provide new insights for improved application of site-specific recombination as a tool for genetic engineering. Finally, they will set the stage for analyzing recombination in the context of high-order chromatin and native 2 micron circle physiology.