Author(s):
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Rank, L.; Veith, S.; Gwosch, E.C.; Demgenski, J.; Ganz, M.;
Jongmans, M.C.J.
; Vogel, C.; Fischbach, A.; Buerger, S.; Fischer, J.M.; Zubel, T.; Stier, A.; Renner, C.; Schmalz, M.; Beneke, S.; Groettrup, M.;
Kuiper, R.P.
; Burkle, A.; Ferrando-May, E.; Mangerich, A.
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Subject:
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Radboudumc 14: Tumours of the digestive tract RIMLS: Radboud Institute for Molecular Life Sciences Radboudumc 9: Rare cancers RIHS: Radboud Institute for Health Sciences |
Abstract:
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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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