Abstract
Poly(ADP-ribosyl)ation (PARylation) is a complex and reversible posttranslational modification catalyzed by poly(ADP-ribose)polymerases (PARPs), which orchestrates protein function and subcellular localization. The function of PARP1 in genotoxic stress response upon induction of oxidative DNA lesions and strand breaks is firmly established, but its role in the response to chemical-induced, bulky DNA adducts is understood incompletely. To address the role of PARP1 in the response to bulky DNA adducts, we treated human cancer cells with benzo[a]pyrene 7,8-dihydrodiol-9,10-epoxide (BPDE), which represents the active metabolite of the environmental carcinogen benzo[a]pyrene [B(a)P], in nanomolar to low micromolar concentrations. Using a highly sensitive LC-MS/MS method, we revealed that BPDE induces cellular PAR formation in a time- and dose-dependent manner. Consistently, PARP1 activity significantly contributed to BPDE-induced genotoxic stress response. On one hand, PARP1 ablation rescued BPDE-induced NAD+ depletion and protected cells from BPDE-induced short-term toxicity. On the other hand, strong sensitization effects of PARP inhibition and PARP1 ablation were observed in long-term clonogenic survival assays. Furthermore, PARP1 ablation significantly affected BPDE-induced S- and G2-phase transitions. Together, these results point towards unresolved BPDE-DNA lesions triggering replicative stress. In line with this, BPDE exposure resulted in enhanced formation and persistence of DNA double-strand breaks in PARP1-deficient cells as evaluated by microscopic co-localization studies of 53BP1 and γH2A.X foci. Consistently, an HPRT mutation assay revealed that PARP inhibition potentiated the mutagenicity of BPDE. In conclusion, this study demonstrates a profound role of PARylation in BPDE-induced genotoxic stress response with significant functional consequences and potential relevance with regard to B[a]P-induced cancer risks.
Highlights
Thousands of DNA lesions occur in every cell during a day by the encounter with genotoxic agents form endogenous and exogenous sources
Figure 1), we tested if BPDE can induce PARylation in HeLa cells using a highly sensitive bioanalytical method based on isotope dilution mass spectrometry (LC-MS/MS) (Martello et al 2013; Zubel et al 2017) (Fig. 1)
Having observed increased levels of γH2A.X in PARylation-deficient cells compared to Wt cells after BPDE exposure (8–24 h) in Western blot analyses (Fig. 6a), a double staining of γH2A.X and 53BP1 was performed to analyze the impact of PARylation on replication stress-induced DNA double-strand breaks (DSBs) (Fig. 7). 24 h after treatment with 150-nM BPDE, a pronounced co-localization of γH2A.X and 53BP1 was observed, which is indicative of DNA double-strand break formation. When these experiments were performed with a PARP1 KO cell line, even after low-dose treatment with 50-nM BPDE, a significant increase in γH2A.X/53BP1 was evident (Fig. 7b), which further increased to an average of ~ 37 foci per cell when cells were treated with 150-nM BPDE (Fig. 7c). These results demonstrate that BPDE treatment in the absence of PARP activity led to DNA double-strand breaks in S-phase cells, which are indicative of collapsed replication forks
Summary
Thousands of DNA lesions occur in every cell during a day by the encounter with genotoxic agents form endogenous and exogenous sources. Various DNA repair mechanisms have evolved during evolution to ensure genomic integrity (Ciccia and Elledge 2010). One of those mechanisms is nucleotide excision repair (NER)—a versatile molecular machinery involved in the removal of bulky and helix distorting DNA adducts (Marteijn et al 2014). NER is unique among DNA repair mechanisms, since it detects a wide spectrum of DNA lesions only with a small set of common damage recognition and repair initiators. The underlying feature of all these structurally different DNA lesions is the varying degree of DNA kinking and helix distortion.
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