RecQ helicases and type IA topoisomerases are highly conserved from bacteria to humans. Extensive studies have supported that RecQ helicases in concert with type IA topoisomerases are involved in the maintenance of genome stability;however, its molecular basis hasn't been clearly established. Unlike many single cellular organisms, such as Escherichia coli, Saccharomyces cerevisiae, and Saccharomyces pombe, which possess one or two type IA topoisomerases and single RecQ helicase, some Gram-positive bacteria such as Bacillus cereus and Bacillus anthracis, the etiologic agent of anthrax, encode multiple chromosomal copies of type IA topoisomerase (annotated as bcTopo I, bcTopo III? and bcTopo III? in Bacillus cereus) and two chromosomal copies of RecQ helicase (annotated as bcRecQS and bcRecQL in Bacillus cereus). In Bacillus cereus, a physical interaction between bcTopo III? and bcRecQL has been identified. Characterization of the bcTopo III? and bcRecQL protein complex using highly purified active enzymes and a feasible genetic system will reveal the biochemical and biological functions of Bacillus cereus type IA topoisomerase and RecQ helicase in genomic stability, as well as other bacterial specific DNA metabolic processes, such as transposon integration, plasmid maintenance and segregation, and DNA conjugational mobilization. Furthermore, insights gained from these studies may shed light on how bacteria cope with severe environmental stress such as antibiotics, chemicals, UV and free radicals. The following specific aims have been proposed to elucidate the functions of bcTopo III? and bcRecQL protein complex.
Specific Aims are: 1) To isolate the bcTopo III? and bcRecQL protein complex and characterize its biochemical properties. 2) To map and characterize domains of bcTopo III? and bcRecQL that are essential for the interaction. 3) To study the interaction between bcTopo III? and bcRecQL in vivo and investigate its biological consequences. Insights gained from this project may shed light on understating how Gram-positive pathogens cope with severe environmental stress and develop antibiotic resistance. These studies may also benefit antibiotic design targeting bacterial type IA topoisomerase and RecQ helicase.