Mechanism of Closed Hairpin End Generation in Linear DNA

细菌的染色体和质粒通常是圆形的。莱姆病病原体伯氏疏螺旋体具有线状染色体，并携带线状质粒和圆形质粒。这些线性复制子具有闭合的发夹末端（一条DNA链旋转并继续，但成为互补链，形成没有自由末端的线性双链）。在大肠杆菌的温带类lambda噬菌体N15和氧化克雷伯菌的KO2中，溶原后的前噬菌体DNA并没有整合到细菌的环状染色体中，而是以具有封闭发夹末端的线性质粒的形式存在。这种类型的DNA末端虽然不常见，但最近在细菌王国的不同分支中被发现。本研究的目的是利用遗传和分子生物学方法研究这些闭合发夹末端产生的机制。噬菌体编码和伯氏疏螺旋体编码系统都将被研究。一种被称为端粒前酶的整合酶/重组酶样蛋白已被确定为通过切割-重新连接机制以序列特异性方式起作用以产生闭合的发夹末端的酶。该蛋白与靶位点的特异性相互作用以及发夹末端产生的途径将被详细研究。将进行饱和诱变以确定功能所需的关键氨基酸。这个项目也可以导致新的线性克隆载体的设计，特别是克隆致命基因。线状发夹端复制子的研究将为染色体维持提供新的见解。由于所有致病性疏螺旋体都有带发夹末端的线状染色体，本研究也将有助于了解疏螺旋体疾病的生物学。

Bacterial chromosomes and plasmids are usually circular. Borrelia burgdorferi, the Lyme disease agent, has linear chromosomes and harbor linear as well as circular plasmids. These linear replicons have closed hairpin ends (one DNA strand turns around and continues but becomes the complementary strand to form a linear duplex without free ends). In the temperate lambda-like phages N15 of E. coli and KO2 of Klebsiella oxytoca, upon lysogeny the prophage DNA's are not integrated into the circular bacterial chromosome, but exist as linear plasmids with closed hairpin ends. Occurrences of this type of DNA ends, though not common, have recently been recognized in different branches of the bacterial kingdom. The goal of this research is to study the mechanism with which these closed hairpin ends are generated using both genetic and molecular biological approaches. Both the phage encoded as well as the Borrelia bacterial encoded systems will be investigated. An integrase/recombinase-like protein called protelomerase has been identified as the enzyme that acts in a sequence-specific manner via a cleavage-rejoining mechanism to generate the closed hairpin ends. The specific interaction between the protein and the target site, as well as the pathway of hairpin end generation will be investigated in detail. A saturation mutagenesis will be conducted to identify critical amino acids needed for function. This project can also lead to the design of new linear cloning vehicles especially for cloning lethal genes. The study of linear hairpin-ended replicons will provide new insight into chromosome maintenance. Since all pathogenic Borrelias have linear chromosomes with hairpin ends, this research will also contribute to the understanding of the biology of Borrelia diseases.