Analyses of Site-Specific Recombination

描述(申请人提供)：定点重组系统在控制不同的过程中非常重要，如噬菌体插入和切除、质粒保持、DNA扩增、染色体分离和接合转座子的移动。这项研究的一个长期目标是了解特定部位重组反应的机制。本提案的重点是由共轭转座子CTnDOT编码的整合酶(DOT Int)。Dot Int是重组酶酪氨酸家族的成员之一，但其重组反应的机制不同于该家族大多数其他成员所使用的机制。我们将使用多种方法研究DOT Int酶的反应机理。DNA裂解和连接试验将被用来研究反应机制，确定裂解和连接的位置，并表征突变蛋白的缺陷。Holliday连接中间体将使用合成DNA组装，或使用新发现的多肽抑制剂通过体外反应积累。将研究在没有和存在影响方向性的辅助因素的情况下解决Holliday交叉口的问题。整合和切割反应中的链切割顺序将通过分析每个反应产生的Holliday连接来确定。遗传和体外技术将被用来产生突变的DOT Int蛋白。重组缺陷突变体将从体内筛选中分离出来，其缺陷将通过生化技术来确定。我们将使用同源建模方法来识别和突变与DNA接触的残基。将对Dot Int蛋白进行纯化和结晶，以解决其结构问题，并更好地了解Dot Int与其他酪氨酸重组酶催化区域的差异。最后，我们将描述切除反应的特征。Orf2c、Orf2d和exc蛋白将被纯化，并确定它们在切除反应中的作用。 接合转座子，如CTnDOT，携带各种编码抗生素耐药性的基因，在肠道细菌中广泛存在。由于CTnDOT及其近亲通过接合和重组将自身转移到受体细胞，它们是抗生素耐药性转移的重要来源。这些研究将增加我们对重组机制的理解，并有助于开发抑制这些元素在自然界中传播的策略。

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.