title: Real-Time Cellular Imaging of Protein Poly(ADP-ribos)ylation.
abstract: Poly(ADP-ribos)ylation (PARylation) is an important posttranslational protein modification, and is involved in major cellular processes such as gene regulation and DNA repair. Its dysregulation has been linked to several diseases, including cancer. Despite its importance, methods to observe PARylation dynamics within cells are rare. By following a chemical biology approach, we developed a fluorescent NAD(+) analogue that proved to be a competitive building block for protein PARylation in vitro and in cells. This allowed us to directly monitor the turnover of PAR in living cells at DNA damage sites after near-infrared (NIR) microirradiation. Additionally, covalent and noncovalent interactions of selected target proteins with PAR chains were visualized in cells by using FLIM-FRET microscopy. Our results open up new opportunities for the study of protein PARylation in real time and in live cells, and will thus contribute to a better understanding of its significance in a cellular context.
authors.affiliation: Department of Chemistry and Center for Applied Photonics, University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany.
authors.forename: Annette
authors.initials: A
authors.lastname: Buntz
authors.affiliation: Department of Chemistry, University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany.
authors.forename: Sarah
authors.initials: S
authors.lastname: Wallrodt
authors.affiliation: Department of Biology, University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany.
authors.forename: Eva
authors.initials: E
authors.lastname: Gwosch
authors.affiliation: Department of Physics and Center for Applied Photonics, University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Schmalz
authors.affiliation: Department of Biology, University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany.
authors.forename: Sascha
authors.initials: S
authors.lastname: Beneke
authors.affiliation: Department of Biology, University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany.
authors.forename: Elisa
authors.initials: E
authors.lastname: Ferrando-May
authors.affiliation: Department of Chemistry, University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany. andreas.marx@uni-konstanz.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Marx
authors.affiliation: Department of Chemistry and Center for Applied Photonics, University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany. andreas.zumbusch@uni-konstanz.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Zumbusch
mesh_terms: D004249:DNA Damage; D005453:Fluorescence; D006367:HeLa Cells; D006657:Histones / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007259:Infrared Rays; D015394:Molecular Structure; D009243:NAD / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry; D061848:Optical Imaging; D011064:Poly Adenosine Diphosphate Ribose / Q000737:chemistry / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors
chemical_list: D006657:Histones; D011506:Proteins; D009243:NAD; D011064:Poly Adenosine Diphosphate Ribose
keywords: DNA repair; FRET; NAD+; poly(ADP-ribose); posttranslational modifications