STUDIES ON MECHANISMS OF IS2 TRANSPOSITION

这个项目的长期目标是从机械上了解在插入序列中如何发生转座，如IS2。IS2属于分布广泛的细菌分子家族，最近描述了一种新的两步转座途径。到目前为止，有两种经典的范式描述了原核生物和真核生物中的转座机制。在IS3家族的两个成员IS2和IS911中发现了这一新途径，这导致了人们对转座的第三种机制的普遍认识。本实验已经确定了IS2中两步转位途径的一些机制细节。在第一步中，(I)仅在元件的右端通过新的不对称单链缺口来启动转座。(2)靠近左端的单链连接反应在分枝的图-8(F-8)内产生单链环。复制或修复将F-8转化为一个双链迷你圈，其中相邻的两端由单个碱基对隔开。在第二步，(Iv)，在两端交界处的两个协调一致的单链缺口(微圆交界处)线性化元件以插入靶中。最近的证据表明，微环连接(MJ)是一个新的杂交启动子的位置，它被假设为驱动第二步转座。两个假设构成了这项研究的基础，以进一步探索这一新途径的机制细节。(A)微环要么是通路中的强制性中间产物，要么是线性切除产物的副产物，其MJ启动子对于转座是必不可少的，或者它是一种人工制品。(B)步骤1中的单链切割和步骤2中的两个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一项目。(I)研究微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不对称的基础，以及(Iii)破译决定第一步将发生单链缺口，第二步将发生两个协同单链缺口的属性。

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.