Analyzing structure–function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLaPARP1knock-out cells

Abstract

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.

Increased PARP1‐DNA binding due to autoPARylation inhibition of PARP1 on DNA rather than PARP1‐DNA trapping is correlated with PARP1 inhibitor's cytotoxicity

“…On the one hand, mutations that impair PARP1 polymerase activity have been reported to increase the risk of different cancers. For instance, V762A leads to about 40% decrease of PARP1 polymerase activity, 41 which is associated with the increased risk of cancers such as esophageal, lung, gastric, prostate 41 and colorectal 34 cancer. On the other hand, the mutations reducing PARP1 polymerase activity have been found to cause cellular and clinical resistance to PARPis.…”

“…4a, Supporting Information Table 1). 21,22,24,30,31,[33][34][35][36] Then we separately transfected them into RD-ES/KO2 cells and obtained their variants that stably expressed corresponding mutated proteins of PARP1 (Fig. 4b).…”

Comprehensive ADP‐ribosylome analysis identifies tyrosine as an ADP‐ribose acceptor site

“…Our group and others have shown that genotoxic stress conditions specifically induce the ADP-ribosylation of hundreds of proteins. These studies identified ARTD1/PARP1 and histones to be the main acceptors of ADPr under these conditions [11][12][13][14]. ARTD1 and ARTD2/PARP2 are considered the two main enzymes that "write" nuclear PARylation [2,3,9].…”

The Enigmatic Function of PARP1: From PARylation Activity to PAR Readers

“…The PARP1-E988K and PARP1-L713F mutant proteins were investigated through their overexpression in the TALEN-generated PARP1 knock-out HeLa cells. PARP1-E988K delayed its recruitment and persisted longer at the site of DNA lesions and induced G2 arrest in the cell cycle [63]. The gain-of-function mutant PARP1-L713F promoted cell apoptosis, even in the absence of DNA damage induction [63].…”