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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.
Disclosure of Potential Conflicts of Interest
No potential conflict of interest was disclosed.
Acknowledgments
We thank Petra Domaing and Anne Lehmkuhl for excellent technical assistance. We are indebted to Roger Y Tsien for the gift of mRFP and mOrange cDNAs.
Funding
This work was supported by grants of the German Research Council (DFG GRK 1657 and DFG CA198/7) and the National Institutes of Health (NIH R01-GM086801). This work used the Extreme Science and Engineering Discovery Environment (XSEDE), request number: MCB130086 and MCB140075.
Supplemental Materials
Supplemental materials may be found on the publisher's website: http://www.tandfonline.com/kncl