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诸如噬菌体插入和切除、质粒维持、DNA扩增、染色体分离和接合转座子的移动等多种过程中是重要的。这项研究的一个长期目标是了解位点特异性重组反应的机制。该建议的焦点是由接合转座子CTnDOT编码的整合酶（DOT Int）。DOT Int是重组酶酪氨酸家族的成员，但它执行的重组反应的机制不同于该家族的大多数其他成员所使用的机制。我们将使用各种方法研究DOT Int酶的反应机理。将开发DNA切割和连接测定来研究反应机制，以鉴定切割和连接的位点并表征突变蛋白的缺陷。霍利迪连接中间体将使用合成DNA组装或使用新发现的肽抑制剂通过体外反应积累。决议霍利迪路口的辅助因素，影响方向性的存在和不存在将进行研究。整合和切除反应中链裂解的顺序将通过分析每个反应产生的Holliday连接来确定。遗传和体外技术将用于产生突变DOT Int蛋白。将从体内筛选中分离纯化缺陷突变体，并通过生物化学技术确定其缺陷。我们将使用同源性建模方法来识别和突变接触DNA的残基。将对Dot Int蛋白进行纯化和结晶，以解析其结构，并更好地了解DOT Int和其他酪氨酸重组酶的催化区域之间的差异。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。 接合转座子如CTnDOT携带编码抗生素抗性的各种基因，并且在肠道细菌中广泛存在。由于CTnDOT及其亲属通过接合和重组将自身转移到受体细胞，因此它们是抗生素抗性转移的重要来源。这些研究将增加我们对重组机制的理解，并有助于制定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.