Cellular functions of the essential PcrA helicase in Staphylococcus aureus

描述（由申请人提供）：PcrA（质粒拷贝还原A）是存在于革兰氏阳性菌中的必需蛋白，包括重要的人类病原体，如金黄色葡萄球菌、肺炎链球菌、产气荚膜梭菌、单核增生李斯特菌、炭疽芽孢杆菌等。在金黄色葡萄球菌中发现了PcrA作为质粒pT181滚圈（RC）复制所需的解旋酶。PcrA与大肠杆菌UvrD和Rep解旋酶有关，已知是枯草芽孢杆菌紫外线DNA修复所必需的。在枯草芽孢杆菌中，重组基因recFOR中pcrA敲除图谱的抑制子和pcrA的条件敲除子都是高重组基因，这表明pcrA在调控reca介导的DNA重组中发挥了重要作用。我们对金黄色葡萄球菌的pcr蛋白进行了鉴定，发现其具有DNA结合、atp酶、解旋酶和质粒DNA解绕活性。我们还表明，在体外，通过取代与DNA结合的RecA， PcrA抑制RecA介导的DNA链交换。利用PcrA突变体，我们的初步研究表明，PcrA的atp酶和解旋酶活性可能不是其体外对RecA的拮抗作用所必需的。虽然几种革兰氏阳性细菌的PcrA的生化特性已经被表征，并且关于嗜热脂肪芽孢杆菌PcrA （PcrABst）的结构信息已经得到，但对金黄色葡萄球菌和其他革兰氏阳性细菌生长所必需的PcrA的生化活性和功能仍然未知。利用遗传和生化方法，本研究旨在研究使PcrA成为金黄色葡萄球菌必需蛋白的生物学功能。在Aim 1中，我们将根据已知的高度相关的PcrABst的三维结构，纯化出一些位点导向的金黄色葡萄球菌PcrA突变体，并表征它们的生化活性。然后，我们将创建金黄色葡萄球菌pcrA的条件敲除，并使用DNA结合、atp酶、解旋酶和RecA位移/抑制缺陷的pcrA突变体进行互补研究，以确定对金黄色葡萄球菌生长和生存能力以及体内RecA功能调节重要的pcrA活性。在Aim 2中，我们将确定PcrA在UV DNA修复和SOS反应中的可能作用，这是已知的促进金黄色葡萄球菌毒力基因和致病性岛的水平转移。SOS反应是由紫外线和抗生素等药物诱导的，需要RecA细丝的组装。我们将研究紫外DNA修复是否需要PcrA解旋酶活性，以及PcrA水平是否在SOS反应中增加并调节RecA功能。我们的研究应该确定使其成为金黄色葡萄球菌必需蛋白的PcrA的生化活性。由于PcrA是一种保守蛋白，从这些研究中获得的知识可以在未来用于开发针对金黄色葡萄球菌和其他革兰氏阳性人类病原体的药物。

DESCRIPTION (provided by applicant): PcrA (plasmid-copy-reduced A) is an essential protein present in Gram-positive bacteria, including important human pathogens such as Staphylococcus aureus, Streptococcus pneumonia, Clostridium perfringens, Listeria monocytogenes, Bacillus anthracis, etc. PcrA was discovered in S. aureus as a helicase required for the rolling-circle (RC) replication of plasmid pT181. PcrA is related to the Escherichia coli UvrD and Rep helicases and is known to be essential for UV DNA repair in Bacillus subtilis. In B. subtilis, suppressors of pcrA knockout map in the recombination genes recFOR and conditional knockouts of pcrA are hyperrecombinogenic, suggesting an essential role for PcrA in the regulation of RecA-mediated DNA recombination. We have characterized the PcrA protein of S. aureus and shown that it has DNA binding, ATPase, helicase and plasmid DNA unwinding activities. We have also shown that PcrA inhibits RecA-mediated DNA strand exchange in vitro by displacing RecA bound to the DNA. Using PcrA mutants, our preliminary studies suggest that the ATPase and helicase activities of PcrA may not be essential for its antagonistic effects on RecA in vitro. Although the biochemical properties of PcrA from several Gram-positive bacteria have been characterized and structural information is available on Bacillus stearothermophilus PcrA (PcrABst), the biochemical activities and functions of PcrA that are essential for the growth of S. aureus and other Gram-positive bacteria remain unknown. Using genetic and biochemical approaches, this study aims to investigate the biological functions of PcrA that make it an essential protein in S. aureus. In Aim 1, we will puriy a few site-directed mutants of S. aureus PcrA that we have generated based on the known three-dimensional structure of the highly-related PcrABst, and characterize their biochemical activities. We will then create a conditional knockout of S. aureus pcrA and carry out complementation studies using PcrA mutants defective in DNA binding, ATPase, helicase and RecA displacement/inhibition to identify the activities of PcrA important for the growth and viability of S. aureus and the regulation of RecA function in vivo. In Aim 2, we will identify the possible role of PcrA in UV DNA repair and the SOS response, which is known to promote the horizontal transfer of virulence genes and pathogenicity islands in S. aureus. The SOS response is induced by UV light and agents such as antibiotics, and requires the assembly of RecA filaments. We will investigate whether the helicase activity of PcrA is required for UV DNA repair and if PcrA levels increase during the SOS response and regulate RecA functions. Our studies should identify the biochemical activities of PcrA that make it an essential protein in S. aureus. Since PcrA is a conserved protein, the knowledge gained from these studies could be used in the future for the development of drugs against S. aureus and other Gram-positive human pathogens.