1. A novel cell permeable DNA replication and repair marker.
- Author
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Herce HD, Rajan M, Lättig-Tünnemann G, Fillies M, and Cardoso MC
- Subjects
- 3T3 Cells, Animals, Cell Cycle genetics, Cell-Penetrating Peptides chemistry, Cell-Penetrating Peptides genetics, Cell-Penetrating Peptides metabolism, Cricetinae, HIV-1 chemistry, HeLa Cells, Humans, Mice, Proliferating Cell Nuclear Antigen chemistry, Proliferating Cell Nuclear Antigen metabolism, Protein Binding, Protein Conformation, tat Gene Products, Human Immunodeficiency Virus chemistry, tat Gene Products, Human Immunodeficiency Virus metabolism, DNA Repair genetics, DNA Replication genetics, Proliferating Cell Nuclear Antigen genetics, tat Gene Products, Human Immunodeficiency Virus genetics
- Abstract
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.
- Published
- 2014
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