Cellular functions of the essential PcrA helicase in Staphylococcus aureus

描述(由申请人提供)：PcrA(质粒拷贝减少A)是存在于革兰氏阳性细菌中的一种重要蛋白质，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、单核细胞增生性李斯特菌、炭疽杆菌等。PcrA是在金黄色葡萄球菌中发现的一种解旋酶，需要对质粒pT181进行滚环复制。PcrA与大肠杆菌UvrD和Rep解旋酶相关，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基因敲除图的抑制子和PcrA的条件性敲除是高度重组的，这表明PcrA在调节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PcrA蛋白进行了鉴定，结果表明它具有DNA结合、ATPase、解旋酶和质粒DNA解旋活性。我们还表明，在体外，PcrA通过取代与DNA结合的RecA来抑制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ATPase和解旋酶活性在体外对RecA的拮抗作用可能不是必需的。尽管一些革兰氏阳性菌的PcrA的生化性质已经被鉴定，并且已经有关于脂肪嗜热芽孢杆菌PcrA(PcrABst)的结构信息，但对于金黄色葡萄球菌和其他革兰氏阳性菌生长所必需的PcrA的生化活性和功能仍不清楚。本研究旨在利用遗传学和生物化学方法研究PcrA的生物学功能，使其成为金黄色葡萄球菌的必需蛋白质。在目标1中，我们将根据已知的高度相关的PcrABst的三维结构，提纯我们所产生的几个金黄色葡萄球菌PcrA的定点突变，并对它们的生化活性进行表征。然后，我们将建立金黄色葡萄球菌PcrA的条件敲除，并使用在DNA结合、ATPase、解旋酶和RecA置换/抑制方面存在缺陷的PcrA突变体进行互补研究，以确定PcrA对金黄色葡萄球菌的生长和存活以及体内RecA功能的调节至关重要。在目标2中，我们将确定PcrA在紫外线DNA修复和SOS反应中的可能作用，SOS反应是已知促进金黄色葡萄球菌毒力基因和致病岛的水平转移。SOS反应是由紫外线和抗生素等试剂诱导的，需要RecA细丝的组装。我们将研究PcrA的解旋酶活性是否是UV DNA修复所必需的，以及PcrA水平是否在SOS反应过程中增加并调节RecA的功能。我们的研究应该确定PcrA的生化活性，使其成为金黄色葡萄球菌的必需蛋白质。由于PcrA是一种保守的蛋白质，从这些研究中获得的知识可以在未来用于开发治疗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.