Genome-Wide Screening with Hydroxyurea Reveals a Link between Nonessential Ribosomal Proteins and Reactive Oxygen Species Production
Genome-Wide Screening with Hydroxyurea Reveals a Link between Nonessential Ribosomal Proteins and Reactive Oxygen Species Production
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DOI：
10.1128/jb.02145-12
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发表时间：
2013-03-01
影响因子：
3.2
通讯作者：
Mori, Hirotada
中科院分区：
文献类型：
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作者：
Nakayashiki, Toru;Mori, Hirotada
我们进行了筛选的羟基脲（HU）敏感的突变体，使用单基因缺失突变体收集在大肠杆菌。HU抑制核糖核苷酸还原酶（RNR），导致复制叉停滞。令人惊讶的是，野生型对HU的抗性低于Keio Collection的平均水平。呼吸缺陷型突变体对HU的抗性显著更强，这表明活性氧（ROS）的产生有助于细胞死亡。高通量筛选显示，15个突变体在含有7.5 mM HU的平板上完全敏感。出乎意料的是，基于COG分类的突变体是最丰富的，其中三个是非必需核糖体蛋白的缺失突变体（L1，L32和L36）。我们发现，在这些突变体中，引起了膜应激反应的增加，导致ROS产生增加。添加OH自由基清除剂硫脲拯救了这些突变体的HU敏感性，表明ROS的产生是细胞死亡的直接原因。相反，rpsF的缺失和rimK的缺失（分别编码S6和S6修饰酶）均显示出HU抗性表型。这些突变体增加了基于p15A的质粒的拷贝数，并表现出SOS反应的基础水平降低。这些数据表明，非必需蛋白质间接影响DNA损伤过程。
We performed a screening of hydroxyurea (HU)-sensitive mutants using a single-gene-deletion mutant collection in Escherichia coli. HU inhibits ribonucleotide reductase (RNR), which leads to arrest of the replication fork. Surprisingly, the wild-type was less resistant to HU than the average for the Keio Collection. Respiration-defective mutants were significantly more resistant to HU, suggesting that the generation of reactive oxygen species (ROS) contributes to cell death. High-throughput screening revealed that 15 mutants were completely sensitive on plates containing 7.5 mM HU. Unexpectedly, translation-related mutants based on COG categorization were the most enriched, and three of them were deletion mutants of nonessential ribosomal proteins (L1, L32, and L36). We found that, in these mutants, an increased membrane stress response was provoked, resulting in increased ROS generation. The addition of OH radical scavenger thiourea rescued the HU sensitivity of these mutants, suggesting that ROS generation is the direct cause of cell death. Conversely, both the deletion of rpsF and the deletion of rimK, which encode S6 and S6 modification enzymes, respectively, showed an HU-resistant phenotype. These mutants increased the copy number of the p15A-based plasmid and exhibited reduced basal levels of SOS response. The data suggest that nonessential proteins indirectly affect the DNA-damaging process.