Many DNA replication and DNA repair enzymes have been found to carry [4Fe4S] clusters. The major leading strand polymerase, DNA polymerase ε (Pol ε) from Saccharomyces cerevisiae, was recently reported to have a [4Fe4S] cluster located within the catalytic domain of the largest subunit, Pol2. Here the redox characteristics of the [4Fe4S] cluster in the context of that domain, Pol2_CORE, are explored using DNA electrochemistry, and the effects of oxidation and rereduction on polymerase activity are examined. The exonuclease deficient variant D290A/E292A, Pol2_COREexo^–, was used to limit DNA degradation. While no redox signal is apparent for Pol2_COREexo^– on DNA-modified electrodes, a large cathodic signal centered at −140 mV vs NHE is observed after bulk oxidation. A double cysteine to serine mutant (C665S/C668S) of Pol2_COREexo^–, which lacks the [4Fe4S] cluster, shows no similar redox signal upon oxidation. Significantly, protein oxidation yields a sharp decrease in polymerization, while rereduction restores activity almost to the level of untreated enzyme. Moreover, the addition of reduced EndoIII, a bacterial DNA repair enzyme containing [4Fe4S]²⁺, to oxidized Pol2_COREexo^– bound to its DNA substrate also significantly restores polymerase activity. In contrast, parallel experiments with EndoIII^Y82A, a variant of EndoIII, defective in DNA charge transport (CT), does not show restoration of activity of Pol2_COREexo^–. We propose a model in which EndoIII bound to the DNA duplex may shuttle electrons through DNA to the DNA-bound oxidized Pol2_COREexo^– via DNA CT and that this DNA CT signaling offers a means to modulate the redox state and replication by Pol ε.

中文翻译：

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酵母 DNA 聚合酶 ε 的 [4Fe4S] 簇具有氧化还原活性，可以进行 DNA 介导的信号传导

已发现许多 DNA 复制和 DNA 修复酶携带 [4Fe4S] 簇。最近有报道称，来自酿酒酵母的主要前导链聚合酶 DNA 聚合酶 ε (Pol ε)具有位于最大亚基 Pol2 的催化结构域内的 [4Fe4S] 簇。在这里，使用 DNA 电化学探索了 [4Fe4S] 簇在该域 Pol2 _CORE的背景下的氧化还原特性，并检查了氧化和还原对聚合酶活性的影响。外切核酸酶缺陷变体 D290A/E292A，Pol2 _CORE exo ^–用于限制 DNA 降解。虽然 Pol2 _CORE exo没有明显的氧化还原信号^——在 DNA 修饰电极上，在大量氧化后观察到以 -140 mV vs NHE 为中心的大阴极信号。缺少 [4Fe4S] 簇的 Pol2 _CORE exo的^双半胱氨酸到丝氨酸突变体 (C665S/C668S)在氧化时没有显示出类似的氧化还原信号。重要的是，蛋白质氧化导致聚合急剧下降，而再还原使活性几乎恢复到未处理酶的水平。此外，将还原的 EndoIII（一种含有 [4Fe4S] ²⁺的细菌 DNA 修复酶）添加到与其 DNA 底物结合的氧化 Pol2 _CORE exo^中也显着恢复了聚合酶的活性。相比之下，使用 EndoIII ^{Y82A的平行实验}, EndoIII 的一种变体, DNA 电荷传输 (CT) 缺陷, 不显示 Pol2 _CORE exo的活性恢复^-。我们提出了一个模型，在该模型中，与 DNA 双链体结合的 EndoIII 可以通过 DNA CT 将电子通过 DNA 穿梭到 DNA 结合的氧化 Pol2 _CORE exo ^，并且这种 DNA CT 信号提供了一种通过 Pol ε 调节氧化还原状态和复制的方法。