Structural Studies of Site-Specific Recombination

描述(申请人提供)：噬菌体Lambda整合酶(Int)是一大类位点特异性DNA重组酶的原型，它在分离质粒、病毒和细胞染色体、调节基因表达和程序性基因重排中发挥作用。这些酶不需要添加高能辅因子就可以催化DNA的裂解和宗教化。它们是已知的唯一既能产生分支Holliday连接中间体又能将它们分解成重组DNA双链的酶。最近相关的P1Cre重组酶和酵母FLP蛋白与Holliday连接的晶体结构为重组反应提供了许多新的见解。与这些简单的重组酶不同，lambda Int在包含多个DNA位点和将DNA弯曲成紧凑形状的辅助因子的高阶结构中发挥作用。这些额外的相互作用对于Int催化的体内重组是必不可少的，它们以变构方式调节DNA切割和链转移的效率和保真度。我们已经确定了一个共价Int-DNA复合体的晶体结构，与早期的未结合Int的结构相比，它揭示了酶活性部位结构中的DNA介导的开关。我们现在提议对更高阶的Int-DNA复合体进行结晶学研究，这将解决通过Int的两个自主DNA结合域的相互作用来调节重组的变构的物理基础。生长了几个较大的Int-Holliday结络合物的衍射晶，并鉴定了同构的重原子衍生物。拟议的晶体结构测定，以及对这些复合体中蛋白质亚单位界面的定点突变研究，将解决DNA切割和连接活性如何受Int-DNA复合体的物理组织调控的问题。对Holliday连接和其他类型的DNA重组关节的酶处理的物理描述是从机制上理解维持染色体结构或创造遗传多样性的各种生物过程的关键。Lambda Int重组系统是一个研究转酯化反应和两条DNA链交换的编排化学的模型，这些交换产生Holliday连接中间体，然后将其分解为重组DNA产物。

DESCRIPTION (provided by applicant): Phage lambda integrase (Int) is the archetype of a large family of site-specific DNA recombinases that function in the segregation of plasmids, viral and cellular chromosomes, the regulation of gene expression, and programmed gene rearrangements. These enzymes catalyze DNA cleavage and religation without the addition of high energy cofactors. They are the only enzymes known to both create branched Holliday junction intermediates and to resolve them into recombinant DNA duplexes. Recent crystal structures of the related P 1 Cre recombinase and the yeast Flp protein complexed to Holliday junctions have provided many insights into the recombination reaction. In contrast to these simple recombinases, lambda Int functions in higher order structures comprising multiple DNA sites and accessory factors that bend the DNA into a compact shape. These additional interactions, which are essential for Int-catalyzed recombination in vivo, allosterically regulate the efficiency and fidelity of DNA cleavage and strand transfer. We have determined a crystal structure of a covalent Int-DNA complex that, in comparison to an earlier structure of unbound Int, reveals a DNA-mediated switch in the structure of the enzyme active site. We now propose crystallographic studies of the higher order Int-DNA complexes that will address the physical basis for the allosteric regulation of recombination through interactions of Int's two autonomous DNA binding domains. Diffracting crystals of several of these larger Int-Holliday junction complexes have been grown and isomorphous heavy atom derivatives have been identified. The proposed crystal structure determinations, together with site-directed mutational studies of the protein subunit interfaces in these complexes, will address how DNA cleavage and ligation activities are regulated by the physical organization of the Int-DNA complexes. A physical description of the enzymatic processing of Holliday junctions and other types of DNA recombination joints is a key to mechanistic understanding of a variety of biological processes that maintain chromosome structure or create genetic diversity. The lambda Int recombination system is a model for studying the chemistry of the trans-esterification reactions and the orchestration of pairwise DNA strand exchanges that create the Holliday junction intermediate and then resolve it into recombinant DNA products.