Mechanism of Closed Hairpin End Generation in Linear DNA

细菌的染色体和质粒通常是圆形的。伯氏疏螺旋体是莱姆病的病原，具有线形染色体和线形和环状两种质粒。这些线性复制子有闭合的发夹末端(一条DNA链翻转并继续，但成为互补链，形成没有自由端的线性双链)。在大肠杆菌N15和催产克雷伯氏菌的KO2中，溶原性的原噬菌体DNA没有整合到环状细菌染色体中，而是以发夹末端闭合的线状质粒形式存在。这种类型的DNA末端的出现虽然不常见，但最近在细菌界的不同分支中被发现。这项研究的目的是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。将对噬菌体编码系统和疏螺旋体细菌编码系统进行研究。一种整合酶/重组酶样蛋白称为端粒酶原，它是一种通过切割-重新连接机制以序列特异性的方式作用于产生闭合发夹末端的酶。将详细研究该蛋白质与靶部位之间的特定相互作用，以及发夹末端的产生途径。将进行饱和突变以确定功能所需的关键氨基酸。该项目还可以导致设计新的线性克隆工具，特别是用于克隆致命基因的工具。线形发夹末端复制子的研究将为染色体的维持提供新的视角。由于所有致病疏螺旋体都具有发夹末端的线性染色体，因此本研究也将有助于对疏螺旋体病生物学的理解。

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.