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首页> 外文期刊>Applied Microbiology >Phosphorothioated DNA Is Shielded from Oxidative Damage
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Phosphorothioated DNA Is Shielded from Oxidative Damage

机译：硫代磷酸化的DNA不受氧化损伤

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DNA is the carrier of genetic information. DNA modifications play a central role in essential physiological processes. Phosphorothioation (PT) modification involves the replacement of an oxygen atom on the DNA backbone with a sulfur atom. PT modification can cause genomic instability in Salmonella enterica under hypochlorous acid stress. This modification restores hydrogen peroxide (H2O2) resistance in the catalase-deficient Escherichia coli Hpx? strain. Here, we report biochemical characterization results for a purified PT modification protein complex (DndCDE) from S. enterica. We observed multiplex oligomeric states of DndCDE by using native PAGE. This protein complex bound avidly to PT-modified DNA. DndCDE with an intact iron-sulfur cluster (DndCDE-FeS) possessed H2O2 decomposition activity, with a Vmax of 10.58?±?0.90 mM min?1 and a half-saturation constant, K0.5S, of 31.03?mM. The Hill coefficient was 2.419?±?0.59 for this activity. The protein’s activity toward H2O2 was observed to be dependent on the intact DndCDE and on the formation of an iron-sulfur (Fe-S) cluster on the DndC subunit. In addition to cysteine residues that mediate the formation of this Fe-S cluster, other cysteine residues play a catalytic role. Finally, catalase activity was also detected in DndCDE from Pseudomonas fluorescens Pf0-1. The data and conclusions presented suggest that DndCDE-FeS is a short-lived catalase. Our experiments also indicate that the complex binds to PT sites, shielding PT DNA from H2O2 damage. This catalase shield might be able to extend from PT sites to the entire bacterial genome.IMPORTANCE DNA phosphorothioation has been reported in many bacteria. These PT-hosting bacteria live in very different environments, such as the human body, soil, or hot springs. The physiological function of DNA PT modification is still elusive. A remarkable property of PT modification is that purified genomic PT DNA is susceptible to oxidative cleavage. Among the oxidants, hypochlorous acid and H2O2 are of physiological relevance for human pathogens since they are generated during the human inflammation response to bacterial infection. However, expression of PT genes in the catalase-deficient E. coli Hpx? strain restores H2O2 resistance. Here, we seek to solve this obvious paradox. We demonstrate that DndCDE-FeS is a short-lived catalase that binds tightly to PT DNA. It is thus possible that by docking to PT sites the catalase activity protects the bacterial genome against H2O2 damage.
机译：DNA是遗传信息的载体。 DNA修饰在基本的生理过程中起着核心作用。硫代磷酸化（PT）修饰涉及用硫原子替换DNA骨架上的氧原子。 PT修饰可在次氯酸胁迫下引起肠沙门氏菌的基因组不稳定。这种修饰可恢复过氧化氢酶缺陷型大肠杆菌Hpx？中过氧化氢（H2O2）的抗性。应变。在这里，我们报告了来自肠球菌的纯化的PT修饰蛋白复​​合物（DndCDE）的生化表征结果。我们通过使用天然PAGE观察到DndCDE的多重寡聚状态。该蛋白质复合物与PT修饰的DNA狂热结合。具有完整的铁硫簇的DndCDE（DndCDE-FeS）具有H2O2分解活性，Vmax为10.58？±？0.90 mM min？1，半饱和常数K0.5S为31.03？mM。该活动的希尔系数为2.419±0.59。观察到该蛋白对H2O2的活性取决于完整的DndCDE以及DndC亚基上铁硫（Fe-S）簇的形成。除了介导该Fe-S簇形成的半胱氨酸残基以外，其他半胱氨酸残基也起催化作用。最后，在荧光假单胞菌Pf0-1的DndCDE中也检测到过氧化氢酶活性。提出的数据和结论表明，DndCDE-FeS是一种短暂的过氧化氢酶。我们的实验还表明，该复合物与PT位点结合，从而保护PT DNA免受H2O2损害。这种过氧化氢酶屏蔽可能能够从PT位点延伸到整个细菌基因组。重要信息据报道，许多细菌中都有DNA硫代磷酸化作用。这些携带PT的细菌生活在非常不同的环境中，例如人体，土壤或温泉。 DNA PT修饰的生理功能仍然难以捉摸。 PT修饰的显着特性是纯化的基因组PT DNA易于氧化裂解。在氧化剂中，次氯酸和过氧化氢对人类病原体具有生理意义，因为它们是在人类对细菌感染的炎症反应过程中产生的。然而，PT基因在过氧化氢酶缺陷型大肠杆菌Hpx1中的表达。应变恢复对H2O2的抵抗力。在这里，我们寻求解决这一明显的悖论。我们证明，DndCDE-FeS是一种短命的过氧化氢酶，与PT DNA紧密结合。因此，通过与PT位点对接，过氧化氢酶活性可以保护细菌基因组免受H2O2损害。

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