Functions of the PcrA Helicase in Bacillus anthracis

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DESCRIPTION (provided by applicant): Bacillus anthracis is an important human pathogen and a potential biological weapon. Drug therapy is available to treat B. anthracis infections since most strains are usually antibiotic sensitive. However, the possibility that bioterrorists may introduce drug resistance plasmids into B. anthracis to generate "super bioterror agents", or such plasmids may transfer naturally into B. anthracis cannot be discounted. Therefore, it is important to identify new drug targets for this organism. PcrA is an essential helicase in Gram-positive bacteria, but its precise function(s) are not known. Gene knock-out experiments will be carried out to confirm that inactivation of the pcrA gene is lethal for B. anthracis. Biochemical studies will be carried out using purified PcrA helicase from B. anthracis to study its role in cellular DNA metabolism. Using substrates that represent DNA molecules that are intermediates in processes such as DNA replication, recombination and repair, we plan to study the DNA binding and helicase activities of PcrA. These studies will reveal whether PcrA is involved in the processing of the Okazaki fragments during DNA replication, and whether it is an editing helicase for the resolution of toxic recombination intermediates. We will carry out experiments to test whether PcrA can block the formation of recombination intermediates such as D-loop and whether it can displace the RecA protein bound to single-stranded DNA. We will also carry out genetic experiments to test whether PcrA is required for the replication of the pXO1 and pXO2 virulence plasmids of B. anthracis. Future development of drugs that specifically inhibit the functions of the PcrA helicase could add to the treatment regimen against B. anthracis and related organisms.