Functions of the PcrA Helicase in Bacillus anthracis

描述（申请人提供）：炭疽芽孢杆菌是一种重要的人类病原体和潜在的生物武器。药物治疗可用于治疗炭疽杆菌感染，因为大多数菌株通常对抗生素敏感。然而，生物恐怖分子可能会将耐药质粒引入炭疽芽胞杆菌中以产生“超级生物恐怖剂”，或者这些质粒可能会自然转移到炭疽芽胞杆菌中，这种可能性也不能排除。因此，为这种生物寻找新的药物靶点是很重要的。pcr是革兰氏阳性菌必不可少的解旋酶，但其确切功能尚不清楚。将进行基因敲除实验，以证实pcrA基因失活对炭疽杆菌是致命的。生化研究将使用炭疽杆菌纯化的pcr解旋酶来研究其在细胞DNA代谢中的作用。利用代表DNA分子的底物，作为DNA复制、重组和修复过程中的中间体，我们计划研究pcr的DNA结合和解旋酶活性。这些研究将揭示PcrA是否参与DNA复制过程中冈崎片段的加工，以及它是否是一种用于毒性重组中间体分解的编辑解旋酶。我们将进行实验，测试PcrA是否可以阻断D-loop等重组中间体的形成，以及是否可以取代与单链DNA结合的RecA蛋白。我们还将进行基因实验，检验是否需要PcrA来复制炭疽杆菌的pXO1和pXO2毒力质粒。未来开发特异性抑制PcrA解旋酶功能的药物可能会增加针对炭疽杆菌和相关生物的治疗方案。

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.