Mechanism of Closed Hairpin End Generation in Linear DNA

细菌的染色体和质粒通常是环状的。莱姆病病原体伯氏疏螺旋体（Borrelia burgdorferi）具有线性染色体，并且具有线性以及环状质粒。这些线性复制子具有闭合的发夹末端（一条DNA链转向并继续，但成为互补链以形成没有自由末端的线性双链体）。在E. coli和产酸克雷伯氏菌KO 2中，在溶原性时，原噬菌体DNA不整合到环状细菌染色体中，而是作为具有闭合发夹末端的线性质粒存在。这种类型的DNA末端的出现，虽然不常见，但最近在细菌王国的不同分支中被认识到。本研究的目的是利用遗传学和分子生物学方法研究这些封闭发夹末端的产生机制。将研究噬菌体编码以及疏螺旋体细菌编码系统。被称为原核端粒酶的整合酶/重组酶样蛋白已被鉴定为通过切割-重新连接机制以序列特异性方式起作用以产生闭合发夹末端的酶。蛋白质和靶位点之间的特异性相互作用，以及发夹末端产生的途径将被详细研究。将进行饱和诱变以鉴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，特别是用于克隆致死基因。线性发夹末端复制子的研究将为染色体的维持提供新的见解。由于所有致病性疏螺旋体都具有发夹末端的线性染色体，因此这项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.