Mechanisms of the Two-Step Transposition Pathway of IS2

这项建议的长期目标是了解转座因子如IS 2的遗传转座机制。转座因子是一种独立的移动的遗传单位，在人类先天性疾病和感染性疾病中发挥着特殊的作用，在脊椎动物免疫系统的进化、基因组进化以及生物进化中发挥着重要作用。IS 2属于细菌插入序列的IS 3家族。它有一个新的2步转座途径的特点，这是由单一功能的重组酶的元件-它的转座酶催化的家庭。述途径被 突出的是形成了一个圆形的切除版本的元素-小环。该途径的第一步（小环形成）是通过不对称切割和连接反应实现的，其中右端（唯一供体）与被动左端（唯一靶标）连接以形成小环连接。在第二步（小环插入）中，两端都成为供体并转移到分子间靶点，导致在新位点插入元件。最近的体内遗传分析已经导致了这样的假设，即所提出的两个步骤中的每一个都发生在本质上不同的蛋白质/DNA复合物-突触复合物或转座体中。将通过三个具体目标来测试这种新的双转座体模型：（i）从转座体中获得转座酶的可溶性和高活性制剂， 超活性突变体，（ii）开发体外凝胶阻滞，和DNA足迹分析，以检查转座酶和小环形成复合物中元件的自由端之间发生的蛋白质/DNA相互作用的类型，和（iii）确定与小环形成复合物相比，在小环插入复合物中是否形成不同的蛋白质/DNA复合物。凝胶阻滞技术将与DNA酶I、羟基自由基、甲基干扰或铜邻菲咯啉和高锰酸盐DNA足迹相结合，以探测复合物中的特异性和非特异性蛋白质/DNA接触。

The long-term objective of this proposal is to understand the mechanisms of genetic transposition in transposable elements like IS2. Transposable elements - independent mobile genetic units, are known to play specific roles in both congenital and infectious human diseases, and they have played an important role in the evolution of vertebrate immune systems, genome evolution and thus biological evolution. IS2 belongs to the IS3 family of bacterial insertion sequences. It has a novel 2-step transposition pathway characteristic of this family that is catalyzed by the single functional recombinase of the element -its transposase. The pathway is highlighted by the formation of a circularized excised version of the element - the minicircle. The first step of the pathway (minicircle formation) is achieved by an asymmetric cleavage and joining reaction in which the right end (the exclusive donor) is abutted to the passive left end (the exclusive target) to form the mini-circle junction. In the second step (minicircle insertion) both ends become donors and are transferred to an intermolecular target, resulting in the insertion of the element at a new site. Recent in vivo genetic analysis has led to the hypothesis that each of the two proposed steps occurs in intrinsically different protein/DNA complexes - synaptic complexes or transpososomes. This novel two-transpososome model will be tested through three specific aims: (i) to obtain soluble and highly active preparations of the transposase from hyperactive mutants (ii) to develop in vitro gel retardation, and DNA footprinting analyses to examine the types of protein/DNA interactions which occur between the transposase and the free ends of the element in the minicircle formation complex and (iii) to determine whether different protein/DNA complexes are formed in a minicircle insertion complex as compared to a minicircle formation complex. Gel retardation techniques will be coupled with DNase I, hydroxyl radical, methyl interference or copper phenanthroline and permanganate DNA footprinting, to probe the specific and non-specific protein/DNA contacts in the complex(es).