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

Lisa Rank, Sebastian Veith, Eva C Gwosch, Janine Demgenski, Magdalena Ganz, Marjolijn C Jongmans, Christopher Vogel, Arthur Fischbach, Stefanie Buerger, Jan M F Fischer, Tabea Zubel, Anna Stier, Christina Renner, Michael Schmalz, Sascha Beneke, Marcus Groettrup, Roland P Kuiper, Alexander Bürkle, Elisa Ferrando-May, Aswin Mangerich

Onderzoeksoutput: Bijdrage aan tijdschriftArtikelpeer review

Samenvatting

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.

Originele taal-2Engels
Pagina's (van-tot)10386-10405
Aantal pagina's20
TijdschriftNucleic acids research
Volume44
Nummer van het tijdschrift21
DOI's
StatusGepubliceerd - 1 dec. 2016
Extern gepubliceerdJa

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