Mechanisms of the Two-Step Transposition Pathway of IS2

IS2 两步转座途径的机制

• 批准号: 6767028
• 负责人: LESLIE A LEWIS
• 金额: $ 16.84万
• 依托单位: YORK COLLEGE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6767028
• 关键词: DNA binding protein; DNA footprinting; DNA replication; Escherichia coli; bacterial DNA; bacterial genetics; bacterial proteins; cell free system; chemical structure function; gel mobility shift assay; genetic recombination; intermolecular interaction; molecular assembly /self assembly; nucleic acid sequence; transposon /insertion element

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).

该提案的长期目标是了解 IS2 等转座元件的遗传转座机制。转座元件——独立的移动遗传单位，已知在先天性和传染性人类疾病中发挥特定作用，并且它们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了重要作用。 IS2 属于细菌插入序列的 IS3 家族。它具有该家族特有的新型两步转座途径，由该元件的单一功能重组酶——其转座酶催化。路径是 突出显示的是该元素的圆形切除版本——小圆的形成。该途径的第一步（小环形成）是通过不对称裂解和连接反应实现的，其中右端（排他供体）毗邻被动左端（排他目标）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标，从而将元件插入到新位点。最近的体内遗传分析得出这样的假设：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。这种新颖的双转座体模型将通过三个具体目标进行测试：（i）从以下来源获得可溶性且高活性的转座酶制剂 高活性突变体（ii）开发体外凝胶阻滞，以及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，以及（iii）确定是否不同与小环形成复合物相比，蛋白质/DNA复合物在小环插入复合物中形成。凝胶阻滞技术将与 DNase I、羟自由基、甲基干扰或铜菲咯啉和高锰酸盐 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。