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.
