STRUCTURAL BASIS OF CRE RECOMBINASE DNA SPECIFICITY

描述（由申请人提供）： 将研究Cre重组酶的DNA底物。Cre重组酶 促进两个34碱基对loxP DNA之间的特异性交换反应 体外和体内的序列。Cre-lox系统已显示出巨大的效用， 在活细胞和生物体中产生特定的染色体重排。 其最大的限制是需要将lox序列引入到 通过相对低效的方法获得目标基因组。虽然与lox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， 对于大多数应用来说足够了。如果歧视的结构机制 来自其他DNA的IoxP序列可以被识别，它们可以被重定向。 蛋白质工程，使Cre能够识别现有的基因组LRS和非lox 序列的大尺寸的lox序列，多聚体活性复合物， 和多步反应允许几个水平的底物区分。 为了定义loxP有效重组的序列要求， 在核苷酸水平上，一组系统的重组活性， 单取代和双取代的loxP序列将被定量评估。 对于低活性底物，涉及的反应步骤 将通过分析DNA结合、活性复合物 组装和催化。为了理解不活动的结构基础， 结合但不经历复合的基质，X射线晶体 将分析受损的Cre变体LOX复合物的结构， 与同源loxP复合物的结构差异。8的作用 LoxP的核苷酸区域，其在促进中分开两个Cre结合位点。 将从反应中间体中确定有效的重组 这一地区的结构。这些结构也将作为 用于与突变体复合物进行比较的参考。自从Cre-lox 反应是位点特异性重组的范例，这一结果 工作应该适用于理解其他人对特异性的控制 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.