STUDIES ON MECHANISMS OF IS2 TRANSPOSITION

该项目的长期目标是从机械上了解转座如何在插入序列（如IS 2）中发生。IS 2属于广泛分布的细菌元件家族，最近描述了其中一种新的两步转座途径。到目前为止，两个经典的范式描述了原核和真核元件中的转座机制。在IS 3家族的两个成员IS 2和IS 911中鉴定出这种新途径，导致了对第三种转座机制的普遍认识。本实验室已经确定了IS 2中两步转座途径的一些机制细节。 在第一步中，（i）转座仅由元件右端的新型不对称单链切口启动。(ii)靠近左端的单链连接反应在分支图-8（F-8）内产生单链环。复制或修复将F-8转化为双链小环，其中双链末端由单个碱基对分开。在第二步骤（iv）中，在两端的接合处（小环接合处）的两个协同单链切口使元件线性化以插入靶中。最近的证据表明，小环连接（mj）是一个新的杂合启动子，它已被假设，驱动转座的第二步的网站。两个假设构成了本研究的基础，以进一步探索这一新途径的机制细节。(a)小环或者是途径中的必需中间体，或者是线性切除产物的副产物，其MJ启动子对于转座是必需的，或者是人工产物。(b)步骤1中的单链切割和步骤2中的两个协同单链切割需要不同的蛋白质-DNA相互作用。三个具体目标将推动该项目。(i)研究小环连接及其启动子在转座途径中的作用。(ii)探索第一步切割反应的不对称性的基础，和（iii）解释决定第一步中将出现单链切口和第二步中将出现两个协同单链切口的属性。

The long-term goal of this project is to understand mechanistically how transposition occurs in insertion sequences like IS2. IS2 belongs to the wide spread family of bacterial elements, in which a novel 2-step pathway of transposition has recently been described. Until now two classical paradigm described transpositional mechanisms in prokaryotic and eukaryotic elements. The identification of this new pathway in two members of the IS3 family, IS2 and IS911, has led to the general recognition of a 3rd mechanism of transposition. This lab has determined some of the mechanistic details of the 2-step transpositional pathway in IS2. In the first step, (i) transposition is initiated by a novel asymmetric single strand nick at the right end of the element only. (ii) a single stranded joining reaction near the left end creates a single strand circle within a branched figure-8 (F-8). Replication or repair convert F-8 into a double stranded mini-circle in which the abutted ends are separated by a single base pair. In the second step, (iv), two concerted single strand nicks at the junction of the two ends (the mini-circle junction) linearize the element for insertion into the target. Recent evidence has indicated that the minicircle junction (mj) is the site of a new hybrid promoter which, it has been hypothesized, drives the 2nd step of transposition. Two hypotheses form the basis of this study to explore further the mechanistic details of this novel pathway. (a) the minicircle is either a mandatory intermediate in the pathway or it is by product of a linear excision product, and its mj promoter is essential for transposition, or it is an artifact. (b) different protein-DNA interactions are required for the single strand cleavage in step 1 and the two concerted single strand cleavages in step 2. Three specific objectives will drive this project. (i) to examine the role of the minicircle junction and its promoter in the transposition pathway. (ii) to explore the basis for the asymmetry of the 1st step cleavage reaction, and (iii) to decipher the attributes which determine that, a single strand nick will occur in the 1st step and two concerted single strand nicks will occur in the second step.