Fluorescently-tagged repair proteins have been widely used to probe recruitment to micro-irradiation-induced nuclear DNA damage in living cells. Here, we quantify APE1 dynamics after micro-irradiation. Markers of DNA damage are characterized and UV-A laser micro-irradiation energy conditions are selected for formation of oxidatively-induced DNA base damage and single strand breaks, but without detectable double strand breaks. Increased energy of laser micro-irradiation, compared with that used previously in our work, enables study of APE1 dynamics at the lesion site. APE1 shows rapid transient kinetics, with recruitment half-time of less than 1 s and dissociation half-time of less than 15 s. In cells co-transfected with APE1 and PARP1, the recruitment half-time of PARP1 was slower than that of APE1, indicating APE1 is a rapid responder to the damage site. While recruitment of APE1 is unchanged in the presence of co-transfected PARP1, APE1 dissociation is 3-fold slower, revealing PARP1 involvement in APE1 dynamics. Further, we find that APE1 dissociation kinetics are strongly modified in the absence of DNA polymerase β (pol β). After unchanged recruitment to the damage site, dissociation of APE1 became undetectable. This indicates a necessary role for pol β in APE1 release after its recruitment to the damage site. These observations represent an advance in our understanding of in vivo dynamics of base excision repair factors APE1, PARP1 and pol β.

中文翻译：

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揭示活细胞 DNA 中修复中间体的反应。

荧光标记的修复蛋白已被广泛用于探测微辐照诱导的活细胞核 DNA 损伤的募集。在这里，我们量化了微辐照后的 APE1 动力学。对 DNA 损伤的标志物进行了表征，并选择了 UV-A 激光微辐照能量条件以形成氧化诱导的 DNA 碱基损伤和单链断裂，但没有可检测到的双链断裂。与我们之前工作中使用的相比，激光微辐照的能量增加，可以研究病变部位的 APE1 动力学。APE1 表现出快速的瞬态动力学，募集半衰期小于 1 秒，解离半衰期小于 15 秒。在 APE1 和 PARP1 共转染的细胞中，PARP1 的募集半衰期比 APE1 慢，表明 APE1 是对损伤部位的快速反应者。虽然在共转染 PARP1 的情况下 APE1 的募集没有变化，但 APE1 的解离速度要慢 3 倍，这表明 PARP1 参与了 APE1 动力学。此外，我们发现在没有 DNA 聚合酶 β (pol β) 的情况下，APE1 解离动力学被强烈改变。在损伤部位未发生变化后，APE1 的解离变得无法检测到。这表明 pol β 在 APE1 募集到损伤部位后释放中的必要作用。这些观察代表了我们对碱基切除修复因子 APE1、PARP1 和 pol β 的体内动力学理解的进步。我们发现在没有 DNA 聚合酶 β（pol β）的情况下，APE1 解离动力学被强烈改变。在损伤部位未发生变化后，APE1 的解离变得无法检测到。这表明 pol β 在 APE1 募集到损伤部位后释放中的必要作用。这些观察代表了我们对碱基切除修复因子 APE1、PARP1 和 pol β 的体内动力学理解的进步。我们发现在没有 DNA 聚合酶 β（pol β）的情况下，APE1 解离动力学被强烈改变。在损伤部位未发生变化后，APE1 的解离变得无法检测到。这表明 pol β 在 APE1 募集到损伤部位后释放中的必要作用。这些观察代表了我们对碱基切除修复因子 APE1、PARP1 和 pol β 的体内动力学理解的进步。