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

作者：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; Buerkle Alexander; Ferrando May Elisa; Mangerich Aswin

来源：Nucleic Acids Research, 2016, 44(21): 10386-10405.

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.

• 出版日期2016-12