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Buchfellner, A.* ; Yurlova, L.* ; Nueske, S.* ; Scholz, A.M.* ; Bogner, J.* ; Ruf, B.* ; Zolghadr, K.* ; Drexler, S.E.* ; Drexler, G.A.* ; Girst, S.* ; Greubel, C.* ; Reindl, J.* ; Siebenwirth, C.* ; Römer, T.* ; Friedl, A.A. ; Rothbauer, U.*

A new nanobody-based biosensor to study endogenous PARP1 in vitro and in live human cells.

PLoS ONE 11:e0151041 (2016)
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Poly(ADP-ribose) polymerase 1 (PARP1) is a key player in DNA repair, genomic stability and cell survival and it emerges as a highly relevant target for cancer therapies. To deepen our understanding of PARP biology and mechanisms of action of PARP1-targeting anti-cancer compounds, we generated a novel PARP1-affinity reagent, active both in vitro and in live cells. This PARP1-biosensor is based on a PARP1-specific single-domain antibody fragment (similar to 15 kDa), termed nanobody, which recognizes the N-terminus of human PARP1 with nanomolar affinity. In proteomic approaches, immobilized PARP1 nanobody facilitates quantitative immunoprecipitation of functional, endogenous PARP1 from cellular lysates. For cellular studies, we engineered an intracellularly functional PARP1 chromobody by combining the nanobody coding sequence with a fluorescent protein sequence. By following the chromobody signal, we were for the first time able to monitor the recruitment of endogenous PARP1 to DNA damage sites in live cells. Moreover, tracing of the sub-nuclear translocation of the chromobody signal upon treatment of human cells with chemical substances enables real-time profiling of active compounds in high content imaging. Due to its ability to perform as a biosensor at the endogenous level of the PARP1 enzyme, the novel PARP1 nanobody is a unique and versatile tool for basic and applied studies of PARP1 biology and DNA repair.
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Publication type Article: Journal article
Document type Scientific Article
Keywords Human Poly(adp-ribose) Polymerase-1; Transcriptionally Active Nucleoli; Fluorescent 2-hybrid Assay; Dna-damage Response; Actinomycin-d; Rna-synthesis; Living Cells; Chromatin-structure; Antibody Fragments; Mammalian-cells
Language
Publication Year 2016
HGF-reported in Year 2016
ISSN (print) / ISBN 1932-6203
Journal PLoS ONE
Quellenangaben Volume: 11, Issue: 3, Pages: , Article Number: e0151041 Supplement: ,
Publisher Public Library of Science (PLoS)
Publishing Place Lawrence, Kan.
Reviewing status Peer reviewed
Institute(s) Translational Metabolic Oncology (IDC-TMO)
POF-Topic(s) 30504 - Mechanisms of Genetic and Environmental Influences on Health and Disease
Research field(s) Radiation Sciences
PSP Element(s) G-521800-001
DOI 10.1371/journal.pone.0151041
WOS ID WOS:000371990100084
Erfassungsdatum 2016-04-15
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