Free radical attack on C1' of deoxyribose forms the oxidized abasic (AP) site 2-deoxyribonolactone (dL). In vitro, dL traps the major base excision DNA repair enzyme DNA polymerase beta (Polβ) in covalent DNA-protein crosslinks (DPC) via the enzyme's N-terminal lyase activity acting on 5'-deoxyribose-5-phosphate residues. We previously demonstrated formation of Polβ-DPC in cells challenged with oxidants generating significant levels of dL. Proteasome inhibition under 1,10-copper-ortho-phenanthroline (CuOP) treatment significantly increased Polβ-DPC accumulation and trapped ubiquitin in the DPC, with Polβ accounting for 60-70 % of the total ubiquitin signal. However, the identity of the remaining oxidative ubiquityl-DPC remained unknown. In this report, we surveyed whether additional AP lyases are trapped in oxidative DPC in mammalian cells in culture. Poly(ADP-ribose) polymerase 1 (PARP1), Ku proteins, DNA polymerase λ (Polλ), and the bifunctional 8-oxoguanine DNA glycosylase 1 (OGG1), were all trapped in oxidative DPC in mammalian cells. We also observed significant trapping of Polλ, PARP1, and OGG1 in cells treated with the alkylating agent methylmethane sulfonate (MMS), in addition to dL-inducing agents. Ku proteins, in contrast, followed a pattern of trapping similar to that for Polβ: MMS failed to produce Ku-DPC, while treatment with CuOP or (less effectively) H2O2 gave rise to significant Ku-DPC. Unexpectedly, NEIL1 and NEIL3 were trapped following H2O2 treatment, but not detectably in cells exposed to CuOP. The half-life of all the AP lyase-DPC ranged from 15-60 min, consistent with their active repair. Accordingly, CuOP treatment under proteasome inhibition significantly increased the observed levels of DPC in cultured mammalian cells containing PARP1, Ku protein, Polλ, and OGG1 proteins. As seen for Polβ, blocking the proteasome led to the accumulation of DPC containing ubiquitin. Thus, the ubiquitin-dependent proteolytic mechanisms that control Polβ-DPC removal may also apply to a broad array of oxidative AP lyase-DPC, preventing their toxic accumulation in cells.

中文翻译：

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通过参与DNA修复的无碱基位点裂解酶在哺乳动物细胞中形成的氧化DNA-蛋白质交联。

自由基对脱氧核糖C1'的攻击形成了氧化的无碱基（AP）位点2-脱氧核糖内酯（dL）。在体外，dL通过作用于5'-脱氧核糖-5-磷酸残基的N末端裂解酶活性，在共价DNA-蛋白质交联（DPC）中捕获主要的碱基切除DNA修复酶DNA聚合酶beta（Polβ）。我们先前证明了在用氧化剂挑战的细胞中会形成Polβ-DPC，从而产生显着水平的dL。蛋白酶体在1,10-铜-邻-菲咯啉（CuOP）处理下的抑制作用显着增加了Polβ-DPC的积累并在DPC中捕获了泛素，其中Polβ占泛素总信号的60-70％。但是，剩余的氧化泛素-DPC的身份仍然未知。在这份报告中 我们调查了是否有其他AP裂解酶被捕获在培养的哺乳动物细胞中的氧化DPC中。聚（ADP-核糖）聚合酶1（PARP1），Ku蛋白，DNA聚合酶λ（Polλ）和双功能8-氧鸟嘌呤DNA糖基化酶1（OGG1）均被困在哺乳动物细胞的氧化DPC中。我们还观察到除dL诱导剂外，在用烷基化剂甲基甲烷磺酸盐（MMS）处理的细胞中，Polλ，PARP1和OGG1的大量捕获。相反，Ku蛋白遵循类似于Polβ的捕获模式：MMS无法产生Ku-DPC，而用CuOP或（效果较差）H2O2处理则产生了显着的Ku-DPC。出乎意料的是，在H2O2处理后，NEIL1和NEIL3被捕获，但是在暴露于CuOP的细胞中却无法检测到。所有AP裂解酶DPC的半衰期为15-60分钟，与其积极维修相符。因此，在蛋白酶体抑制下的CuOP处理显着增加了在培养的含有PARP1，Ku蛋白，Polλ和OGG1蛋白的哺乳动物细胞中DPC的水平。如对于Polβ所见，阻断蛋白酶体导致含有泛素的DPC的积累。因此，控制Polβ-DPC去除的泛素依赖性蛋白水解机制也可能适用于各种氧化性AP裂解酶-DPC，从而防止其在细胞中的毒性积累。