Analyzing structure-function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells

• Published in: Nucleic acids research Vol. 44; no. 21; pp. 10386 - 10405
• Main Authors: Rank, Lisa, Veith, Sebastian, Gwosch, Eva C, Demgenski, Janine, Ganz, Magdalena, Jongmans, Marjolijn C, Vogel, Christopher, Fischbach, Arthur, Buerger, Stefanie, Fischer, Jan M F, Zubel, Tabea, Stier, Anna, Renner, Christina, Schmalz, Michael, Beneke, Sascha, Groettrup, Marcus, Kuiper, Roland P, Bürkle, Alexander, Ferrando-May, Elisa, Mangerich, Aswin
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01-12-2016
• Subjects: Animals; Cell Line; DNA Damage; Gene Knockout Techniques; Gene Targeting; Genetic Variation; HeLa Cells; Humans; NAD - metabolism; Nucleic Acid Enzymes; Poly (ADP-Ribose) Polymerase-1 - chemistry; Poly (ADP-Ribose) Polymerase-1 - genetics; Poly (ADP-Ribose) Polymerase-1 - metabolism; Protein Binding; Protein Conformation; Recombinant Proteins; Sequence Deletion; Structure-Activity Relationship; Transcription Activator-Like Effector Nucleases - chemistry; Transcription Activator-Like Effector Nucleases - metabolism
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkw859

Genotoxic stress activates PARP1, resulting in the post-translational modification of proteins with poly(ADP-ribose) (PAR). We genetically deleted PARP1 in one of the most widely used human cell systems, i.e. HeLa cells, via TALEN-mediated gene targeting. After comprehensive characterization of these cells during genotoxic stress, we analyzed structure-function relationships of PARP1 by reconstituting PARP1 KO cells with a series of PARP1 variants. Firstly, we verified that the PARP1\E988K mutant exhibits mono-ADP-ribosylation activity and we demonstrate that the PARP1\L713F mutant is constitutively active in cells. Secondly, both mutants exhibit distinct recruitment kinetics to sites of laser-induced DNA damage, which can potentially be attributed to non-covalent PARP1-PAR interaction via several PAR binding motifs. Thirdly, both mutants had distinct functional consequences in cellular patho-physiology, i.e. PARP1\L713F expression triggered apoptosis, whereas PARP1\E988K reconstitution caused a DNA-damage-induced G2 arrest. Importantly, both effects could be rescued by PARP inhibitor treatment, indicating distinct cellular consequences of constitutive PARylation and mono(ADP-ribosyl)ation. Finally, we demonstrate that the cancer-associated PARP1 SNP variant (V762A) as well as a newly identified inherited PARP1 mutation (F304L\V762A) present in a patient with pediatric colorectal carcinoma exhibit altered biochemical and cellular properties, thereby potentially supporting human carcinogenesis. Together, we establish a novel cellular model for PARylation research, by revealing strong structure-function relationships of natural and artificial PARP1 variants.