A novel cell permeable DNA replication and repair marker
A novel cell permeable DNA replication and repair marker
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DOI：
10.4161/nucl.36290
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发表时间：
2014-11-01
期刊：
影响因子：
3.7
通讯作者：
Cardoso, M. Cristina
中科院分区：
文献类型：
--
作者：
Herce, Henry D.;Rajan, Malini;Cardoso, M. Cristina
增殖细胞核抗原是DNA复制和修复过程中的关键蛋白。在活细胞中复制和修复的动力学通常被研究引入翻译融合的增殖细胞核抗原。为了避免需要转染法和绕过难以转染性和/或短命细胞的问题，我们现在开发了一种细胞可穿透性复制和/或修复标记。该标记的设计有三个基本的分子成分：(1)优化的人造增殖细胞核抗原结合肽；(2)来自HIV-1反式转录激活因子(TAT)的细胞穿透性多肽；(3)连接这两个多肽的体内可切割连接物。在加入培养液后的几分钟内，人类、仓鼠和小鼠的细胞就吸收了这种结构。在细胞内，货物与载体多肽分离，并有效结合增殖细胞核抗原。复制和修复位点都可以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渗透性多肽标记物。同时，我们还介绍了一种基于多肽的快速增殖细胞核抗原染色方法，以替代免疫荧光应用中的增殖细胞核抗原抗体。综上所述，我们在这里提出了一个通用的工具，以即时标记修复和复制过程中的固定和活细胞。
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.