Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America.
PLoS One. 2011 Feb 17;6(2):e17101. doi: 10.1371/journal.pone.0017101.
The strict human pathogen Neisseria gonorrhoeae is the only causative agent of the sexually transmitted infection gonorrhea. The recA gene from N. gonorrhoeae is essential for DNA repair, natural DNA transformation, and pilin antigenic variation, all processes that are important for the pathogenesis and persistence of N. gonorrhoeae in the human population. To understand the biochemical features of N. gonorrhoeae RecA (RecA(Ng)), we overexpressed and purified the RecA(Ng) and SSB(Ng) proteins and compared their activities to those of the well-characterized E. coli RecA and SSB proteins in vitro. We observed that RecA(Ng) promoted more strand exchange at early time points than RecA(Ec) through DNA homologous substrates, and exhibited the highest ATPase activity of any RecA protein characterized to date. Further analysis of this robust ATPase activity revealed that RecA(Ng) is more efficient at displacing SSB from ssDNA and that RecA(Ng) shows higher ATPase activity during strand exchange than RecA(Ec). Using substrates created to mimic the cellular processes of DNA transformation and pilin antigenic variation we observed that RecA(Ec) catalyzed more strand exchange through a 100 bp heterologous insert, but that RecA(Ng) catalyzed more strand exchange through regions of microheterology. Together, these data suggest that the processes of ATP hydrolysis and DNA strand exchange may be coupled differently in RecA(Ng) than in RecA(Ec). This difference may explain the unusually high ATPase activity observed for RecA(Ng) with the strand exchange activity between RecA(Ng) and RecA(Ec) being more similar.
严格的人类病原体淋病奈瑟菌是唯一引起性传播感染淋病的病原体。淋病奈瑟菌的 recA 基因对于 DNA 修复、自然 DNA 转化和菌毛抗原变异至关重要，所有这些过程对于淋病奈瑟菌在人群中的发病机制和持续存在都很重要。为了了解淋病奈瑟菌 RecA（RecA（Ng））的生化特征，我们过表达和纯化了 RecA（Ng）和 SSB（Ng）蛋白，并将它们的活性与经过充分表征的大肠杆菌 RecA 和 SSB 蛋白进行了比较。我们观察到 RecA（Ng）通过同源 DNA 底物在早期促进更多的链交换，并且表现出迄今为止所表征的任何 RecA 蛋白中最高的 ATP 酶活性。对这种强大的 ATP 酶活性的进一步分析表明，RecA（Ng）更有效地从 ssDNA 上置换 SSB，并且 RecA（Ng）在链交换过程中比 RecA（Ec）具有更高的 ATP 酶活性。使用创建的模拟 DNA 转化和菌毛抗原变异的细胞过程的底物，我们观察到 RecA（Ec）通过 100 bp 异源插入催化更多的链交换，但 RecA（Ng）通过微异质性区域催化更多的链交换。总的来说，这些数据表明 ATP 水解和 DNA 链交换的过程在 RecA（Ng）中可能与 RecA（Ec）不同。这种差异可能解释了观察到的 RecA（Ng）异常高的 ATP 酶活性，因为 RecA（Ng）和 RecA（Ec）之间的链交换活性更相似。
