Replication Rapidly Recovers and Continues in the Presence of Hydroxyurea in Escherichia coli.
Replication Rapidly Recovers and Continues in the Presence of Hydroxyurea in Escherichia coli.
复制标题
在大肠杆菌中存在羟基脲的情况下，复制迅速恢复并继续。
DOI：
10.1128/jb.00713-17
复制
发表时间：
2018
影响因子：
3.2
通讯作者：
Courcelle,CharmainT
中科院分区：
文献类型：
--
作者：
Nazaretyan,SamvelA;Savic,Neda;Sadek,Michael;Hackert,BrandyJ;Courcelle,Justin;Courcelle,CharmainT
在原核生物和真核生物中，建议通过灭活核糖核苷酸还原酶和消耗脱氧核糖核苷三磷酸池来抑制DNA复制。在这项研究中，我们表明，在大肠杆菌中的复制的抑制是短暂的，即使在0.1 M的浓度和复制迅速恢复，并继续在其存在。复制的恢复不需要替代的核糖核苷酸还原酶NrdEF和NrdDG或translesion DNA聚合酶II（Pol II），Pol IV，和Pol V。核糖核苷酸在复制过程中以更高的频率在存在的羟基脲。然而，它们对观察到的合成或毒性没有显著贡献。只有在羟基脲的稳定性受到损害和已知直接损害DNA的副产物被允许积累的条件下，才观察到羟基脲毒性。结果表明，羟基脲不是体内DNA合成的直接或特异性抑制剂，并且观察到的瞬时抑制最有可能是由于最初应用0.1 M羟基脲时酶中铁辅因子的一般耗尽。最后，结果支持以前的研究表明，羟基脲毒性介导的主要是通过直接的DNA损伤引起的分解产物的羟基脲，而不是通过抑制复制或消耗脱氧核糖核苷酸在cell.IMPORTANCEHydroxyurea水平通常建议通过抑制DNA复制通过灭活核糖核苷酸还原酶和消耗脱氧核糖核苷三磷酸池。在这里，我们表明，在复制迅速恢复并在药物存在下继续之前，羟基脲仅短暂抑制大肠杆菌中的复制。复制的恢复不依赖于替代核糖核苷酸还原酶，translesion合成，或RecA。此外，我们发现，只有在存在毒性中间体的情况下，才能观察到羟基脲的毒性，当羟基脲分解，损伤DNA，并诱导致死性时，这些毒性中间体会积累。结果表明，羟基脲毒性是通过DNA损伤的形成间接介导的，而不是通过抑制细胞中脱氧核糖核苷酸水平的复制或消耗。
In both prokaryotes and eukaryotes, hydroxyurea is suggested to inhibit DNA replication by inactivating ribonucleotide reductase and depleting deoxyribonucleoside triphosphate pools. In this study, we show that the inhibition of replication in Escherichia coli is transient even at concentrations of 0.1 M hydroxyurea and that replication rapidly recovers and continues in its presence. The recovery of replication does not require the alternative ribonucleotide reductases NrdEF and NrdDG or the translesion DNA polymerases II (Pol II), Pol IV, and Pol V. Ribonucleotides are incorporated at higher frequencies during replication in the presence of hydroxyurea. However, they do not contribute significantly to the observed synthesis or toxicity. Hydroxyurea toxicity was observed only under conditions where the stability of hydroxyurea was compromised and by-products known to damage DNA directly were allowed to accumulate. The results demonstrate that hydroxyurea is not a direct or specific inhibitor of DNA synthesisin vivoand that the transient inhibition observed is most likely due to a general depletion of iron cofactors from enzymes when 0.1 M hydroxyurea is initially applied. Finally, the results support previous studies suggesting that hydroxyurea toxicity is mediated primarily through direct DNA damage induced by the breakdown products of hydroxyurea, rather than by inhibition of replication or depletion of deoxyribonucleotide levels in the cell.IMPORTANCEHydroxyurea is commonly suggested to function by inhibiting DNA replication through the inactivation of ribonucleotide reductase and depleting deoxyribonucleoside triphosphate pools. Here, we show that hydroxyurea only transiently inhibits replication in Escherichia coli before replication rapidly recovers and continues in the presence of the drug. The recovery of replication does not depend on alternative ribonucleotide reductases, translesion synthesis, or RecA. Further, we show that hydroxyurea toxicity is observed only in the presence of toxic intermediates that accumulate when hydroxyurea breaks down, damage DNA, and induce lethality. The results demonstrate that hydroxyurea toxicity is mediated indirectly by the formation of DNA damage, rather than by inhibition of replication or depletion of deoxyribonucleotide levels in the cell.