Analyses of Site-Specific Recombination

描述（由申请人提供）：位点特异性重组系统在控制噬菌体插入和切除、质粒维持、DNA扩增、染色体分离和共轭转座子的运动等多种过程中很重要。本研究的一个长期目标是了解位点特异性重组反应的机制。本文的研究重点是共轭转座子CTnDOT编码的整合酶（DOT Int）。DOT Int是重组酶酪氨酸家族的一员，但它进行重组反应的机制与该家族的大多数其他成员使用的机制不同。我们将使用多种方法研究DOT Int酶的反应机制。DNA切割和连接试验将用于研究反应机制，确定切割和连接的位点，并表征突变蛋白的缺陷。Holliday连接中间体将使用合成DNA组装或通过使用新发现的肽抑制剂进行体外反应积累。在没有和存在影响方向性的辅助因素的情况下，假日路口的解析将被研究。在整合和切除反应中，链断裂的顺序将通过分析每个反应形成的霍利迪结来确定。遗传和体外技术将用于产生突变DOT Int蛋白。重组缺陷突变体将从体内筛选中分离出来，并通过生化技术确定其缺陷。我们将使用同源建模方法来识别和突变接触DNA的残基。Dot Int蛋白将被纯化和结晶，以解决其结构，并更好地了解Dot Int催化区域与其他酪氨酸重组酶的区别。最后，我们将描述切除反应。将纯化Orf2c、Orf2d和Exc蛋白，并确定它们在切除反应中的作用。

DESCRIPTION (provided by applicant): Site-specific recombination systems are important in controlling such diverse processes such as phage insertion and excision, plasmid maintenance, DNA amplification, chromosome segregation and movement of conjugative transposons. A long-range goal of this research is to understand the mechanism of site-specific recombination reactions. The focus of this proposal is the integrase (DOT Int) encoded by conjugative transposon CTnDOT. DOT Int is a member of the tyrosine family of recombinases but the mechanism of the recombination reaction it performs differs from the one used by most other members of the family. We will study the reaction mechanism of the DOT Int enzyme using a variety of approaches. DNA cleavage and ligation assays will be developed to study the reaction mechanism, to identify the sites of cleavage and ligation and to characterize defects of mutant proteins. Holliday junction intermediates will be assembled using synthetic DNA or accumulated by in vitro reactions using newly discovered peptide inhibitors. Resolution of Holliday junctions in the absence and presence of accessory factors that affect directionality will be studied. The order of strand cleavages in the integration and excision reactions will be determined by analysis of the Holliday junctions made by each reaction. Genetic and in vitro techniques will be used to generate mutant DOT Int proteins. Recombination-defective mutants will be isolated from an in vivo screen and their defects will be determined by biochemical techniques. We will use a homology modeling approach to identify and mutate residues that contact DNA. The Dot Int protein will be purified and crystallized to solve its structure and better to understand the differences between the catalytic region of DOT Int and other tyrosine recombinases. Finally, we will characterize the excision reaction. The Orf2c, Orf2d, and Exc proteins, will be purified and their roles in the excision reaction will be defined. Conjugative transposons such as CTnDOT carry various genes that encode resistance to antibiotics and are widespread amongst intestinal bacteria. Because CTnDOT and its relatives transfer themselves to recipient cells by conjugation and recombination they are an important source for the transfer of antibiotic resistance. These studies will increase our understanding of the recombination mechanism and help develop strategies to inhibit the spread of these elements in Nature.