Department of Biology, Portland State University, Portland, Oregon, USA.
Department of Biology, Portland State University, Portland, Oregon, USA
J Bacteriol. 2018 Feb 23;200(6). doi: 10.1128/JB.00713-17. Print 2018 Mar 15.
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 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 synthesis and 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. Hydroxyurea 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 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.
在原核生物和真核生物中，羟基脲被认为通过使核糖核苷酸还原酶失活和耗尽脱氧核苷三磷酸池来抑制 DNA 复制。在这项研究中，我们表明，即使在 0.1 M 羟基脲的浓度下， 的复制抑制也是短暂的，并且在其存在下复制会迅速恢复并继续。复制的恢复不需要替代的核糖核苷酸还原酶 NrdEF 和 NrdDG 或跨损伤 DNA 聚合酶 II（Pol II）、Pol IV 和 Pol V。在羟基脲存在下，复制过程中核糖核苷酸的掺入频率更高。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的情况下，并且允许直接损伤 DNA 的副产物积累的情况下，才会观察到羟基脲毒性。结果表明，羟基脲不是 DNA 合成的直接或特异性抑制剂，并且观察到的短暂抑制很可能是由于 0.1 M 羟基脲最初施加时，从酶中普遍耗尽铁辅因子所致。最后，结果支持先前的研究，表明羟基脲毒性主要是通过羟基脲分解产物诱导的直接 DNA 损伤介导的，而不是通过抑制复制或耗尽细胞中的脱氧核苷酸水平介导的。羟基脲通常被认为通过使核糖核苷酸还原酶失活和耗尽脱氧核苷三磷酸池来抑制 DNA 复制。在这里，我们表明，羟基脲仅在 中短暂抑制复制，然后复制在药物存在下迅速恢复并继续。复制的恢复不依赖于替代的核糖核苷酸还原酶、跨损伤合成或 RecA。此外，我们表明，只有在羟基脲分解时积累有毒中间体、损伤 DNA 并诱导致死性时，才会观察到羟基脲毒性。结果表明，羟基脲毒性是通过形成 DNA 损伤间接介导的，而不是通过抑制复制或耗尽细胞中的脱氧核苷酸水平介导的。
