A novel cell permeable DNA replication and repair marker

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.

Primary cilia found on HeLa and other cancer cells

“…To further support this conclusion, we stained HeLa cells for Arl13b as well as PCNA (proliferating cell nuclear antigen). PCNA is a DNA clamp that promotes polymerase δ interaction with DNA during the S-phase of the cell cycle to increase processivity (Herce et al, 2014). While PCNA is found in the nucleus at all stages of the cell cycle, it becomes concentrated and localized in foci of active DNA synthesis during S-phase forming a specific, punctate staining pattern within the nucleus (Herce et al 2014).…”

“…PCNA is a DNA clamp that promotes polymerase δ interaction with DNA during the S-phase of the cell cycle to increase processivity (Herce et al, 2014). While PCNA is found in the nucleus at all stages of the cell cycle, it becomes concentrated and localized in foci of active DNA synthesis during S-phase forming a specific, punctate staining pattern within the nucleus (Herce et al 2014). As shown in Figure 2B, HeLa cells that exhibited the distinctive PCNA S-phase staining pattern (Figure 2B, depicted with arrow) in addition stained positive for Arl13b (Figure 2B, depicted with arrowhead), further suggesting the presence of primary cilia in cells that are in the S-Phase of the cell cycle.…”

Long non-coding RNA EPB41L4A-AS2 inhibited non-small cell lung cancer proliferation and invasion and promoted cell apoptosis

“…Images of PCNA nuclear distribution can be used to differentiate the early, mid and late cell-cycle S phase [39]. Moreover, research on DNA replication and restoration in living cells can be enhanced by introducing PCNA translational fusions which would offer the excellent advantage of application in in situ living tissues, and it especially distinguishes between cells in the process of replication and cells undergoing repair [40]. One study also proved that PCNA is a potential therapeutic target in cancer therapy [41] and we confirmed that PCNA decreased in the EPB41L4A-AS2 over-expressing group.…”

The Influence of Diet Containing Wheat Gluten Supplemented with Dipeptides or Amino Acids on the Morphology of White Muscle of Yellow Perch (Perca flavescens)

“…The histological analysis conducted in the present study showed the positive effect of FAA and DP diets on white muscle morphology, more precisely, on the diameter of white muscles and the number of the PCNA positive myonuclei, which reflect the ability of muscle cells to proliferate. It was confirmed that the PCNA protein indicated the replication or repair processes of myonuclei [31]. Similarly, common carp feeding with feeds based on WG supplemented with free Lys and Gly amino acids had a beneficial effect on the proliferation of muscle cells, whereas fish feeding the WG-based diet supplemented with Lys-Gly dipeptide stimulated the development of muscle tissue by the increase of TFN [14].…”