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位点特异性重组反应。位点特异性重组是用于描述仅需要有限同源区域的DNA片段的遗传重排的术语。这项工作是围绕一个接合转座子称为CTnDOT编码的重组酶，称为IntDOT，进行位点特异性重组反应。CTnDOT存在于临床上重要的拟杆菌属菌株的染色体中。它携带编码对抗生素四环素和红霉素的抗性的基因。CTnDOT还编码切除所需的多种辅助蛋白，称为Orf2c、Orf2d和Exc。在切除过程中，IntDOT、辅助蛋白和宿主因子促进元件的切除以形成元件的环状拷贝。通过缀合将CTnDOT的单链转移至受体菌株。在DNA环化和复制后，CTnDOT的拷贝通过IntDOT和宿主因子促进的反应整合到受体染色体中。本文主要研究了IntDOT的催化机理和切除反应的机理。我们将对IntDOT进行结构分析。野生型和突变体蛋白质将在DNA底物存在下结晶。这项工作将有助于了解IntDOT和其他酪氨酸重组酶的催化位点的差异。第二个目的是分析切除反应的机制。我们将纯化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反应过程中形成的霍利迪结（HJ）中间体的形成和分辨率。我们将分析辅助蛋白与HJs的相互作用以及辅助蛋白对HJs分解为底物或产物的影响。最终目的是分析突变的IntDOT蛋白。我们已经分离出了大量的突变体，这些突变体产生在体内重组中有缺陷的IntDOT蛋白。我们将鉴定突变体在重组途径中的缺陷。这些研究将补充从以前的生化研究中获得的结果，并为解释物理研究提供信息。

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.