STUDIES ON MECHANISMS OF IS2 TRANSPOSITION

该项目的长期目标是了解像IS2这样的插入序列中的转位是如何发生的。IS2属于广泛分布的细菌元件家族，其中一种新的两步转位途径最近被描述。到目前为止，有两种经典范式描述了原核和真核元素的转位机制。在IS3家族的两个成员IS2和IS911中发现了这种新途径，从而使人们普遍认识到第三种转位机制。本实验室已经确定了IS2中两步转位途径的一些机制细节。在第一步中，(i)仅在元件的右端由一个新的不对称单链缺口启动转位。（ii）左端附近的单链连接反应在分支图8 （F-8）内形成单链环。复制或修复将F-8转化为双链迷你环，其中相邻的两端由单个碱基对分开。在第二步（iv）中，在两端的连接处（小圆连接处）有两个一致的单链刻痕，使元件线性化，以便插入目标中。最近的证据表明，迷你圆结（mj）是一个新的杂交启动子的位置，它被假设驱动转位的第二步。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)微环要么是途径中的强制性中间体，要么是线性切除产物的副产品，其mj启动子对转位至关重要，要么是一个伪产物。(b)步骤1的单链切割和步骤2的两条协调的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这个项目。(i)研究微圆结及其启动子在转位途径中的作用。（ii）探索第一步解理反应不对称的基础，（iii）破译决定第一步会发生单链刻痕和第二步会发生两个协同单链刻痕的属性。

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.