Herce Henry D, Rajan Malini, Lättig-Tünnemann Gisela, Fillies Marion, Cardoso M Cristina
a Department of Biology , Technische Universität Darmstadt ; Darmstadt , Germany.
Nucleus. 2014;5(6):590-600. doi: 10.4161/nucl.36290. Epub 2014 Oct 31.
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.
增殖细胞核抗原（PCNA）是DNA复制和修复过程中的关键蛋白。活细胞中复制和修复的动态过程通常通过引入PCNA的翻译融合体来研究。为了避免转染的必要性并绕过难以转染和/或寿命短暂的细胞问题，我们现在开发了一种细胞可渗透的复制和/或修复标记物。该标记物的设计有三个基本分子成分：（1）一个优化的人工PCNA结合肽；（2）一个源自HIV-1转录激活因子（TAT）的细胞穿透肽；（3）一个连接这两种肽的体内可裂解连接子。将所得构建体添加到培养基中几分钟内，人、仓鼠和小鼠细胞就会摄取它。在细胞内，货物与载体肽分离并有效地结合PCNA。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首个体内细胞可渗透肽标记物。同时，我们还引入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。总之，我们在此展示了一种通用工具，可即时标记固定细胞和活细胞中的修复和复制过程。
