Genotoxic stress activates PARP1 resulting in the post-translational modification of proteins

Genotoxic stress activates PARP1 resulting in the post-translational modification of proteins with poly(ADP-ribose) (PAR). functional consequences in cellular patho-physiology i.e. PARP1\L713F expression brought on 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 APR-246 we demonstrate that this 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. INTRODUCTION Poly(ADP-ribosyl)ation (PARylation) is usually a post-translational modification that plays key roles in cellular physiology and stress response (1). It mainly occurs in the nucleus and to a lesser extent in the cytoplasm. The reaction is carried out by enzymes of the family of poly(ADP-ribose) polymerases (PARPs) which use NAD+ to synthesize poly(ADP-ribose) (PAR) a biopolymer with variable chain length and branching. Of the 17 members of the human gene family at least four have been shown to be true PARPs i.e. these do exhibit PAR-forming capacity while other family members act as mono-ADP-ribosyl transferases or are catalytically inactive. PARP1 is usually a highly abundant chromatin-associated protein that exhibits PARylation activity. Upon binding to DNA damage in APR-246 particular to strand breaks and subsequent conformational APR-246 rearrangements PARP1 is usually catalytically activated and contributes to the bulk of the cellular PAR formation (1). This can happen either in by activation of a single PARP1 molecule (2 3 or in deficient tumors. Recently the PARP inhibitor olaparib has been approved by the HSPC150 EMA and FDA for the use in certain knock-out mouse models and immortalized mouse embryonic fibroblasts (MEFs) derived thereof (28-31) as well as siRNA-based knock-down approaches (32). Strikingly a genetic double knock-out of and resulted in embryonic lethality in the mouse thereby demonstrating a key function of PARylation during development (33). To the best of our knowledge besides a very recent report on a CRISPR/Cas-generated knock-out in HEK cells (34) genetic deletion of in human cancer cell lines has so far not been described. Notably at present no mutations have been directly related to human hereditable diseases – presumably because such mutations lead to embryonic lethality beforehand. Yet several polymorphisms exist that have been associated with an increased risk for cancer development and inflammatory diseases. For example a polymorphism causing the amino acid exchange (aa) V762A (35) leads to reduced enzymatic APR-246 activity of purified recombinant PARP1 protein (36 37 Notably the PARP1\V762A variant is associated with an increased risk for the development of several types of cancers in specific ethnicities (38 39 How the V762A variant and other potentially disease-associated polymorphisms and mutations affect cellular PARP1 activities and functions is so far unknown. Here we report a genetic knock-out of in one of the most widely used human cell systems i.e. HeLa cells via TALEN-mediated gene targeting. We characterized these cells with regards to PARylation metabolism and genotoxic stress resistance. By reconstituting HeLa KO cells with a series of PARP1 variants we then analyzed structure-function relationships of PARP1 variants in a cellular environment without interfering with endogenously expressed WT-PARP1. These variants included sets of artificial mutants and natural variants to illustrate the potential of this system for its wider usage in PARylation research. The first set included two artificial PARP1 mutants that are of high interest to understand the cellular biochemistry of PARylation i.e. a hypomorphic (E988K) and a hypermorphic (L713F) PARP1 mutant. Using a second set of PARP1 variants we then analyzed cellular consequences of naturally occurring PARP1 variants i.e. the PARP1 polymorphism leading to the V762A aa exchange and a newly identified germline PARP1 mutant (F304L).