Cellular functions of the essential PcrA helicase in Staphylococcus aureus

金黄色葡萄球菌必需 PcrA 解旋酶的细胞功能

• 批准号: 8284823
• 负责人: SALEEM A. KHAN
• 金额: $ 22.62万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-15 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8284823
• 关键词: ATP phosphohydrolase; Amino Acids; Antibiotics; Bacillus anthracis; Bacillus stearothermophilus; Bacillus subtilis; Binding; Biochemical; Biological Process; Cell physiology; Characteristics; Clostridium perfringens; Complement; DNA; DNA Binding; DNA Repair; Data; Defect; Escherichia coli; Essential Genes; Filament; Frequencies; Future; Genes; Genetic; Genetic Recombination; Gram-Positive Bacteria; Growth; Human; In Vitro; Knock-out; Knowledge; Listeria monocytogenes; Maps; Measures; Mediating; Messenger RNA; Pathogenicity Island; Pharmaceutical Preparations; Plasmids; Property; Proteins; Regulation; Role; SOS Response; Single-Stranded DNA; Site; Staphylococcal Protein A; Staphylococcus aureus; Streptococcus pneumoniae; Testing; Ultraviolet Rays; Virulence; base; cell growth; design; drug development; ds-DNA; helicase; in vivo; mutant; pathogen; plasmid DNA; repaired; research study; three dimensional structure

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. PUBLIC HEALTH RELEVANCE: We plan to study the functions of PcrA which are critical for the growth and viability of S. aureus using genetic and biochemical approaches. The biochemical activities of various PcrA mutants will be correlated with their ability to support the growth of S. aureus and regulate RecA-mediated DNA recombination in vivo. Finally, we will study the possible roles of PcrA in UV DNA repair and the regulation of the SOS response in S. aureus.

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质粒PT181的滚动圆（RC）复制。 PCRA与大肠杆菌和REP解旋酶有关，据说对于枯草芽孢杆菌中的紫外DNA修复至关重要。在枯草芽孢杆菌中，重组基因中PCRA敲除图的抑制剂和PCRA的条件敲除具有过度再生生成性，这表明PCRA在调节RECA介导的DNA重组中的重要作用。我们已经表征了金黄色葡萄球菌的PCRA蛋白，并表明它具有DNA结合，ATPase，Helicase和质粒DNA放松活性。我们还表明，PCRA通过置换与DNA结合的RECA在体外抑制了RECA介导的DNA链交换。使用PCRA突变体，我们的初步研究表明，PCRA的ATPase和Helicase活性对于其对RECA体外的拮抗作用可能不是必不可少的。尽管已经表征了来自几种革兰氏阳性细菌的PCRA的生化特性，并且在芽孢杆菌嗜热菌PCRA（PCRABST）上可以找到结构信息，但PCRA的生化活性和功能对于S. Aureus和其他革雷氏蛋白细菌的生长至关重要。使用遗传和生化方法，本研究旨在研究PCRA的生物学功能，使其成为金黄色葡萄球菌中必不可少的蛋白质。在AIM 1中，我们将根据已知的高度相关PCRABST的已知三维结构生成的几个位置指导的金黄色葡萄球菌PCRA突变体，并表征其生化活性。然后，我们将创建一个有条件的金黄色葡萄球菌PCRA敲除，并使用PCRA突变体在DNA结合，ATPase，Helicase和RecA位移/抑制作用中进行互补研究，以识别PCRA的活性对金黄色葡萄球菌的生长和生长性以及RECA在体内功能的活性很重要。在AIM 2中，我们将确定PCRA在紫外DNA修复和SOS反应中的可能作用，SOS响应已知可以促进金黄色葡萄球菌中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药物诱导的，并且需要组装RECA丝。我们将研究紫外线DNA修复需要PCRA的解旋酶活性以及在SOS响应期间PCRA水平是否增加并调节RECA功能。我们的研究应确定PCRA的生化活性，使其成为金黄色葡萄球菌中必不可少的蛋白质。由于PCRA是一种保守的蛋白质，因此将来可以使用这些研究获得的知识来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。 公共卫生相关性：我们计划研究PCRA的功能，这对于使用遗传和生化方法的金黄色葡萄球菌的生长和生存能力至关重要。各种PCRA突变体的生化活性将与它们的支持能力相关 金黄色葡萄球菌的生长并调节体内再抗介导的DNA重组。最后，我们将研究PCRA在紫外DNA修复中的可能作用，以及金黄色葡萄球菌中SOS反应的调节。