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

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Nucleic Acids Research. 2016, 44(21), pp. 10386-10405. ISSN 0305-1048. eISSN 1362-4962. Available under: doi: 10.1093/nar/gkw859
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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.

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ISO 690RANK, Lisa, Sebastian VEITH, Eva GWOSCH, Janine DEMGENSKI, Magdalena GANZ, Christopher VOGEL, Arthur FISCHBACH, Stefanie BUERGER, Jan M.F. FISCHER, Tabea ZUBEL, Anna STIER, Christina RENNER, Michael SCHMALZ, Sascha BENEKE, Marcus GRÖTTRUP, Alexander BÜRKLE, Elisa FERRANDO-MAY, Aswin MANGERICH, 2016. Analyzing structure–function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells. In: Nucleic Acids Research. 2016, 44(21), pp. 10386-10405. ISSN 0305-1048. eISSN 1362-4962. Available under: doi: 10.1093/nar/gkw859
BibTex
@article{Rank2016-12-01Analy-35469,
  year={2016},
  doi={10.1093/nar/gkw859},
  title={Analyzing structure–function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells},
  number={21},
  volume={44},
  issn={0305-1048},
  journal={Nucleic Acids Research},
  pages={10386--10405},
  author={Rank, Lisa and Veith, Sebastian and Gwosch, Eva and Demgenski, Janine and Ganz, Magdalena and Vogel, Christopher and Fischbach, Arthur and Buerger, Stefanie and Fischer, Jan M.F. and Zubel, Tabea and Stier, Anna and Renner, Christina and Schmalz, Michael and Beneke, Sascha and Gröttrup, Marcus and Bürkle, Alexander and Ferrando-May, Elisa and Mangerich, Aswin}
}
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