STRUCTURAL BASIS OF CRE RECOMBINASE DNA SPECIFICITY

说明(由申请人提供)：歧视的结构基础 使用Cre重组酶的DNA底物将被研究。Cre重组酶 促进两个34碱基鸟的loxP DNA之间的特异性交叉反应 体外和体内测序。CRE-LOX系统在以下方面显示出巨大的实用价值 在活细胞和生物体中产生特定的染色体重排。 它的最大限制是将lox序列引入 通过相对低效的方法获得目标基因组。尽管与鲑鱼有关 序列(LRS)存在于哺乳动物基因组中，它们不能有效地发挥作用 足以满足大多数应用程序的需求。如果歧视的结构性机制 来自其他DNA的IoxP序列可以被识别，它们可以通过以下方式重定向 蛋白质工程使Cre能够识别现有的基因组LRSs和非LOX 序列。LOX序列的大小，多聚体活性复合体， 并且多步骤反应允许几种水平的底物区分。 定义loxP高效重组的序列要求 在核苷酸水平上，一组系统性的 将对单取代和双取代的loxP序列进行定量评估。 对于低活性底物，参与的反应步骤(S) 区分将通过分析DNA结合、活性络合物来确定 组装和催化。要了解不活动的结构基础 结合但不发生复合的衬底，X射线晶体 受损的Cre变异的lox复合体的结构将被分析为 与同系物loxP络合物的结构差异。八的角色 LoxP的核苷酸区域，分离两个Cre结合部位 将从反应中间体中确定有效的复合 包含这一区域的结构。这些结构也将作为 与突变复合体比较的参考文献。因为Cre-lox 反应是特定部位重组的范例，这些结果如下 工作应适用于理解其他人对专一性的控制 酪氨酸重组酶。

DESCRIPTION (provided by applicant): The structural basis for discrimination of DNA substrates by Cre recombinase will be investigated. Cre recombinase promotes a specific crossing-over reaction between two 34 basepair loxP DNA sequences in vitro and in vivo. The Cre-lox system has shown great utility for generating specific chromosomal rearrangements in living cells and organisms. Its greatest limitation is the requirement for introducing lox sequences into the target genomes by relatively inefficient methods. Although lox-related sequences (LRSs) occur in mammalian genomes, they do not function efficiently enough for most applications. If the structural mechanisms for discriminating IoxP sequences from other DNA can be identified, they could be redirected by protein engineering to allow Cre to recognize existing genomic LRSs and non-lox sequences. The large size of the lox sequence, the multimeric active complex, and multi-step reaction allow for several levels of substrate discrimination. To define the sequence requirements for efficient recombination of the loxP site at the nucleotide level, the recombination activity of a systematic set of singly and doubly substituted loxP sequences will be quantitatively assessed. For low activity substrates, the reaction step(s) that is(are) involved in discrimination will be determined by analysis of DNA binding, active complex assembly and catalysis. To understand the structural basis for inactivity of substrates which are bound but do not undergo recombination, X-ray crystal structures of the impaired Cre-variant lox complexes wilt be analyzed for structural differences from the cognate loxP complexes. The role of an eight nucleotide region of LoxP that separates the two Cre binding sites in promoting efficient recombination will be ascertained from reaction intermediate structures incorporating this region. These structures wilt also act as references for comparisons with the mutant complexes. Since the Cre-lox reaction is paradigmatic for site-specific recombination, these results of this work should be applicable to understanding control of specificity by other tyrosine recombinases.