DNA REPLICATION IN THE LYME DISEASE AGENT

莱姆病，由螺旋体感染引起 burgdorferi，是最常见的节肢动物传播的疾病， 美国的 B的基因组。burgdorferi是不寻常的一个 细菌，因为它主要由线性DNA分子组成。 端粒，线性DNA分子的物理末端， 这些分子的稳定性。 不同的蛋白质和DNA 序列是复制和维持端粒所必需的。 的 线性DNA复制和端粒维持的机制 细菌在很大程度上仍是未知的。 的复制和稳定性 具有发夹端粒的原核线性DNA以前没有被发现。 研究了 该提案的重点是反式作用蛋白和顺式- DNA复制和端粒所需的作用DNA序列 B中的维护。burgdorferi。 B的几个端粒。burgdorferi 线性质粒已被克隆和测序。 的核苷酸 序列、预测结构和某些序列基序的定相 表明蛋白质与端粒结合。 这些假定的反式作用 因子包括小DNA结合蛋白HBb。HBB将是 纯化至均一，其与端粒的相互作用将被 研究了 此外，其他DNA结合蛋白将通过 分析它们识别端粒序列或结构的能力。 的 这些蛋白质的DNA结合特性将被表征， 他们的基因将被克隆。 16 kb线性质粒上的序列， 复制和隔离所需的数据将由 操纵重组质粒。 这些质粒将 构建使用香豆霉素A1抗性gryB，唯一可用的 B中的抗生素抗性标记。burgdorferi。 的顺式作用序列 参与了环状质粒的复制， 基因组也将被分析。 B的噬菌体。伯格多费里将是 为了研究DNA复制的工具。

Lyme disease, caused by infection with the spirochete Borrelia burgdorferi, is the most common arthropod-borne disease in the United States. The genome of B. burgdorferi is unusual for a bacterium in that it consists predominantly of linear DNA molecules. Telomeres, the physical ends of linear DNA molecules, are essential for the stability of these molecules. Distinct proteins and DNA sequences are required for replicating and maintaining telomeres. The mechanisms of linear DNA replication and telomere maintenance in bacteria remain largely unknown. The replication and stability of prokaryotic linear DNA with hairpin telomeres has not been previously investigated. This proposal focuses on trans-acting proteins and cis- acting DNA sequences required for DNA replication and telomere maintenance in B. burgdorferi. Several telomeres from B. burgdorferi linear plasmids have been cloned and sequenced. The nucleotide sequence, predicted structure, and phasing of certain sequence motifs suggest that proteins bind at the telomeres. These putative trans-acting factors include the small DNA-binding protein HBb. HBb will be purified to homogeneity and its interaction with telomeres will be studied. In addition, other DNA-binding proteins will be purified by assaying their ability to recognize telomere sequence or structure. The DNA-binding properties of these proteins will be characterized and their genes will be cloned. Sequences on the 16kb linear plasmid that are required for replication and segregation will be analyzed by manipulating recombinant plasmids. These plasmids will be constructed using coumermycin A1-resistant gryB, the only available antibiotic resistant marker in B. burgdorferi. The cis-acting sequences involved in the replication of circular plasmids from this unusual genome will also be analyzed. A phage of B. burgdorferi will be characterized in order to fashion a tool for studying DNA replication.