Analyses of Site-Specific Recombination

描述(由申请人提供)： 酪氨酸重组酶在多种细胞过程中进行定点重组反应。定点重组是一个用来描述只需要有限同源区域的DNA片段的遗传重排的术语。这项工作是围绕一种名为CTnDOT的接合转座子展开的，该转座子编码一种名为IntDOT的重组酶，该重组酶可执行特定部位的重组反应。CTnDOT存在于临床上重要的类杆菌菌株的染色体中。它携带对抗生素四环素和红霉素的抗药性编码基因。CTnDOT还编码各种称为Orf2c、Orf2d和exc的附属蛋白，这些蛋白是切除所必需的。在切除过程中，IntDOT、辅助蛋白和宿主因子(S)促进该元件的切除，形成该元件的环形拷贝。通过接合将CTnDOT的单链转移到受体菌株。在DNA循环和复制后，在IntDOT和宿主因子的推动下，CTnDOT的副本被整合到受体染色体中。本工作重点研究了IntDOT的催化机理和切割反应的机理。我们将对IntDOT进行结构分析。野生型和突变型蛋白将在DNA底物存在的情况下结晶。这项工作将有助于理解IntDOT和其他酪氨酸重组酶催化位点的差异。第二个目的是分析切除反应的机理。我们将提纯辅助蛋白，并研究它们与DNA和IntDOT的相互作用。我们还将分析在IntDOT反应中形成的Holliday Junction(HJ)中间体的形成和分解。我们将分析辅助蛋白与HJS的相互作用，以及辅助蛋白对HJS拆分底物或产物的影响。最终目标是分析突变的IntDOT蛋白。我们已经分离出一大组突变株，这些突变株产生的IntDOT蛋白在体内重组时存在缺陷。我们将对重组途径中缺陷的突变体进行特征分析。这些研究将补充以前生化研究的结果，并为解释物理研究提供信息。 公共卫生相关性： 增加抗生素耐药性的机会病原体和重要的微生物组分，如拟杆菌属。正在成为一个严重的临床问题。这项拟议的工作将为研究接合转座子的整合和切除机制提供新的见解，接合转座子是耐药基因在类杆菌和其他重要病原体(如革兰氏阳性球菌)中传播的主要贡献者。

DESCRIPTION (provided by applicant): Tyrosine recombinases perform site-specific recombination reactions in a variety of cellular processes. Site-specific recombination is a term used to describe genetic rearrangements of DNA segments that require only limited regions of homology. This work is centered around a conjugative transposon called CTnDOT that encodes a recombinase, called IntDOT, that performs a site-specific recombination reaction. CTnDOT resides in the chromosomes of clinically important Bacteroides strains. It carries genes encoding resistances to the antibiotics tetracycline and erythromycin. CTnDOT also encodes a variety of accessory proteins called Orf2c, Orf2d and Exc that are required for excision. During excision, IntDOT, the accessory proteins and a host factor(s) promote the excision of the element to form a circular copy of the element. A single strand of CTnDOT is transferred to a recipient strain by conjugation. After the DNA is circularized and replicated a copy of CTnDOT is integrated into the recipient chromosome by a reaction promoted by IntDOT and a host factor. This works focuses on the mechanism of catalysis by IntDOT and the mechanism of the excision reaction. We will perform a structural analysis of IntDOT. The wild type and mutant proteins will be crystallized in the presence of DNA substrates. This work will help understand the differences in the catalytic sites of IntDOT and other tyrosine recombinases. A second aim is to analyze the mechanism of the excision reaction. We will purify the accessory proteins and study their interactions with DNA and with IntDOT. We will also analyze the formation and resolution of Holliday Junction (HJ) intermediates formed during the IntDOT reaction. We will analyze the interactions of accessory proteins with HJs and the effects of accessory proteins on the resolution of HJs to substrates or products. The final aim is to analyze mutant IntDOT proteins. We have isolated a large set of mutants that produce IntDOT proteins that are defective in recombination in vivo. We will characterize the mutants for defects in the recombination pathway. These studies will complement results obtained from previous biochemical studies and provide information for interpreting physical studies. PUBLIC HEALTH RELEVANCE: Increasing antibiotic resistance in opportunistic pathogens and prominent microbiota components such as Bacteroides spp. is becoming a serious clinical problem. The proposed work will provide new insights into the mechanisms of integration and excision of conjugative transposons, which are major contributors to the spread of resistance genes among Bacteroides and other important pathogens such as the gram positive cocci.