Devigne Alice, Guérin Philippe, Lisboa Johnny, Quevillon-Cheruel Sophie, Armengaud Jean, Sommer Suzanne, Bouthier de la Tour Claire, Servant Pascale
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette cedex, France.
CEA, DSV, IBiTec-S, SPI, Li2D, Laboratory Innovative Technologies for Detection and Diagnostics, Bagnols-sur-Cèze, France.
mSphere. 2016 Jan 13;1(1). doi: 10.1128/mSphere.00036-15. eCollection 2016 Jan-Feb.
PprA, a radiation-induced Deinococcus-specific protein, was previously shown to be required for cell survival and accurate chromosome segregation after exposure to ionizing radiation. Here, we used an in vivo approach to determine, by shotgun proteomics, putative PprA partners coimmunoprecipitating with PprA when cells were exposed to gamma rays. Among them, we found the two subunits of DNA gyrase and, thus, chose to focus our work on characterizing the activities of the deinococcal DNA gyrase in the presence or absence of PprA. Loss of PprA rendered cells hypersensitive to novobiocin, an inhibitor of the B subunit of DNA gyrase. We showed that treatment of bacteria with novobiocin resulted in induction of the radiation desiccation response (RDR) regulon and in defects in chromosome segregation that were aggravated by the absence of PprA. In vitro, the deinococcal DNA gyrase, like other bacterial DNA gyrases, possesses DNA negative supercoiling and decatenation activities. These two activities are inhibited in vitro by novobiocin and nalidixic acid, whereas PprA specifically stimulates the decatenation activity of DNA gyrase. Together, these results suggest that PprA plays a major role in chromosome decatenation via its interaction with the deinococcal DNA gyrase when D. radiodurans cells are recovering from exposure to ionizing radiation. IMPORTANCE D. radiodurans is one of the most radiation-resistant organisms known. This bacterium is able to cope with high levels of DNA lesions generated by exposure to extreme doses of ionizing radiation and to reconstruct a functional genome from hundreds of radiation-induced chromosomal fragments. Here, we identified partners of PprA, a radiation-induced Deinococcus-specific protein, previously shown to be required for radioresistance. Our study leads to three main findings: (i) PprA interacts with DNA gyrase after irradiation, (ii) treatment of cells with novobiocin results in defects in chromosome segregation that are aggravated by the absence of PprA, and (iii) PprA stimulates the decatenation activity of DNA gyrase. Our results extend the knowledge of how D. radiodurans cells survive exposure to extreme doses of gamma irradiation and point out the link between DNA repair, chromosome segregation, and DNA gyrase activities in the radioresistant D. radiodurans bacterium.
PprA是一种辐射诱导产生的、仅存在于耐辐射球菌属中的蛋白质，先前的研究表明，它是细胞在受到电离辐射后存活以及准确进行染色体分离所必需的。在此，我们采用体内实验方法，通过鸟枪法蛋白质组学来确定在细胞受到γ射线照射时，与PprA共同免疫沉淀的假定PprA相互作用蛋白。在这些蛋白中，我们发现了DNA促旋酶的两个亚基，因此选择将工作重点放在表征耐辐射球菌属DNA促旋酶在有或没有PprA存在时的活性上。PprA的缺失使细胞对新生霉素（一种DNA促旋酶B亚基的抑制剂）高度敏感。我们发现用新生霉素处理细菌会导致辐射干燥应答（RDR）调节子的诱导以及染色体分离缺陷，而PprA的缺失会加剧这些缺陷。在体外，耐辐射球菌属DNA促旋酶与其他细菌的DNA促旋酶一样，具有DNA负超螺旋和解连环活性。这两种活性在体外会被新生霉素和萘啶酸抑制，而PprA能特异性地刺激DNA促旋酶的解连环活性。总之，这些结果表明，当耐辐射奇异球菌细胞从电离辐射暴露中恢复时，PprA通过与耐辐射球菌属DNA促旋酶相互作用，在染色体解连环过程中发挥主要作用。重要性耐辐射奇异球菌是已知的最耐辐射的生物之一。这种细菌能够应对因暴露于极高剂量电离辐射而产生的高水平DNA损伤，并能从数百个辐射诱导的染色体片段中重建功能基因组。在此，我们鉴定了PprA（一种辐射诱导产生的、仅存在于耐辐射球菌属中的蛋白质，先前已证明它是抗辐射所必需的）的相互作用蛋白。我们的研究得出了三个主要发现：（i）照射后PprA与DNA促旋酶相互作用；（ii）用新生霉素处理细胞会导致染色体分离缺陷，而PprA的缺失会加剧这些缺陷；（iii）PprA刺激DNA促旋酶的解连环活性。我们的结果扩展了对耐辐射奇异球菌细胞如何在极高剂量γ射线照射下存活的认识，并指出了耐辐射奇异球菌中DNA修复、染色体分离和DNA促旋酶活性之间的联系。
