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A novel cell permeable DNA replication and repair marker

机译：一种新型的细胞渗透性DNA复制和修复标记

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摘要

Proliferating Cell Nuclear Antigen (PCNA) is a key protein in DNA replication and repair. The dynamics of replication and repair in live cells is usually studied introducing translational fusions of PCNA. To obviate the need for transfection and bypass the problem of difficult to transfect and/or short lived cells, we have now developed a cell permeable replication and/or repair marker. The design of this marker has three essential molecular components: (1) an optimized artificial PCNA binding peptide; (2) a cell-penetrating peptide, derived from the HIV-1 Trans Activator of Transcription (TAT); (3) an in vivo cleavable linker, linking the two peptides. The resulting construct was taken up by human, hamster and mouse cells within minutes of addition to the media. Inside the cells, the cargo separated from the vector peptide and bound PCNA effectively. Both replication and repair sites could be directly labeled in live cells making it the first in vivo cell permeable peptide marker for these two fundamental cellular processes. Concurrently, we also introduced a quick peptide based PCNA staining method as an alternative to PCNA antibodies for immunofluorescence applications. In summary, we present here a versatile tool to instantaneously label repair and replication processes in fixed and live cells.
机译：增殖细胞核抗原（PCNA）是DNA复制和修复中的关键蛋白。通常研究活细胞中复制和修复的动力学，引入PCNA的翻译融合。为了消除转染的需要并绕过难以转染和/或寿命短的细胞的问题，我们现已开发出细胞可渗透的复制和/或修复标记。该标记的设计具有三个基本的分子成分：（1）优化的人工PCNA结合肽; （2）源自HIV-1反式转录激活剂（TAT）的穿透细胞的肽; （3）体内可裂解的接头，连接两个肽。加入培养基后数分钟内，人，仓鼠和小鼠细胞吸收了所得的构建体。在细胞内部，货物与载体肽分离并有效结合PCNA。复制和修复位点均可在活细胞中直接标记，使其成为这两个基本细胞过程的第一个体内细胞可渗透肽标记。同时，我们还介绍了一种基于肽的快速PCNA染色方法，作为免疫荧光应用中PCNA抗体的替代方法。总而言之，我们在这里展示了一种多功能工具，可以在固定和活细胞中即时标记修复和复制过程。

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