title: 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.
authors.affiliation: a Department of Biology , Technische Universität Darmstadt ; Darmstadt , Germany.
authors.forename: Henry D
authors.initials: HD
authors.lastname: Herce
authors.forename: Malini
authors.initials: M
authors.lastname: Rajan
authors.forename: Gisela
authors.initials: G
authors.lastname: Lättig-Tünnemann
authors.forename: Marion
authors.initials: M
authors.lastname: Fillies
authors.forename: M Cristina
authors.initials: MC
authors.lastname: Cardoso
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002453:Cell Cycle / Q000235:genetics; D057846:Cell-Penetrating Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D004260:DNA Repair / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics*; D015497:HIV-1 / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D057846:Cell-Penetrating Peptides; D018809:Proliferating Cell Nuclear Antigen; D054322:tat Gene Products, Human Immunodeficiency Virus
keywords: DNA repair; DNA replication; PCNA; cell cycle; cell-penetrating peptides; proliferating cell nuclear antigen