visualization-quantitative-analysis-genotoxin-induced-parp1parp2

Visualization and Quantitative Analysis of Genotoxin-Induced PARP1/PARP2 Activation in Cells Using a Fluorescent Fusion Protein-Based Reporter

Poly(ADP-ribose) (PAR) is a polymer of ADP-ribose synthesized by four members of the ADP-ribose polymerase family of enzymes&#8212;PARP1, PARP2, PARP5a, ...

Brown University

Poly(ADP-ribose) (PAR), catalyzed by members of the poly(ADP-ribose) polymerase family of enzymes, is a posttranslational modification with a critical role in most mechanisms of DNA repair. Upon activation of poly(ADP-ribose) polymerase isoforms 1 and 2 (PARP-1 and PARP-2), the proteins of the base excision repair (BER) and single-strand break repair (SSBR) pathways form DNA lesion-dependent, transient complexes to facilitate repair. PAR is central to the temporal dynamics of BER/SSBR complex assembly and disassembly. To enhance cellular PAR analysis, we developed LivePAR, a fluorescently tagged PAR-binding fusion protein and genetically encoded imaging probe for live cell, quantitative analysis of PAR in mammalian cells. LivePAR has the advantage that it enables real-time imaging of PAR formation in cells and significantly overcomes limitations of immunocytochemistry for PAR analysis. This chapter describes the protocols needed to develop cells expressing LivePAR or EGFP-tagged BER proteins and to evaluate laser-induced formation of PAR and comparison to the assembly of the BER proteins XRCC1 and DNA polymerase-β.

Quantitative Analysis of Nuclear Poly(ADP-Ribose) Dynamics in Response to Laser-Induced DNA Damage.

Protein poly-ADP-ribosylation (PARylation) is a post-translational modification formed by transfer of successive units of ADP-ribose to target proteins to form poly-ADP-ribose (PAR) chains. PAR plays a critical role in the DNA damage response (DDR) by acting as a signaling platform to promote the recruitment of DNA repair factors to the sites of DNA damage that bind via their PAR-binding domains (PBDs). Several classes of PBD families have been recognized, which identify distinct parts of the PAR chain. Proteins encoding PBDs play an essential role in conveying the PAR-mediated signal through their interaction with PAR chains, which mediates many cellular functions, including the DDR. The WWE domain identifies the iso-ADP-ribose moiety of the PAR chain. We recently described the WWE domain of RNF146 as a robust genetically encoded probe, when fused to EGFP, for detection of PAR in live cells. Here, we evaluated other PBD candidates as molecular PAR probes in live cells, including several other WWE domains and an engineered macrodomain. In addition, we demonstrate unique PAR dynamics when tracked by different PAR binding domains, a finding that that can be exploited for modulation of the PAR-dependent DNA damage response.

Overexpression of the WWE domain of RNF146 modulates poly-(ADP)-ribose dynamics at sites of DNA damage.

Mutations in isocitrate dehydrogenase isoform 1 (IDH1) are primarily found in secondary glioblastoma (GBM) and low-grade glioma but are rare in primary GBM. The standard treatment for GBM includes radiation combined with temozolomide, an alkylating agent. Fortunately, IDH1 mutant gliomas are sensitive to this treatment, resulting in a more favorable prognosis. However, it's estimated that up to 75 % of IDH1 mutant gliomas will progress to WHO grade IV over time and develop resistance to alkylating agents. Therefore, understanding the mechanism(s) by which IDH1 mutant gliomas confer sensitivity to alkylating agents is crucial for developing targeted chemotherapeutic approaches. The base excision repair (BER) pathway is responsible for repairing most base damage induced by alkylating agents. Defects in this pathway can lead to hypersensitivity to these agents due to unresolved DNA damage. The coordinated assembly and disassembly of BER protein complexes are essential for cell survival and for maintaining genomic integrity following alkylating agent exposure. These complexes rely on poly-ADP-ribose formation, an NAD-dependent post-translational modification synthesized by PARP1 and PARP2 during the BER process. At the lesion site, poly-ADP-ribose facilitates the recruitment of XRCC1. This scaffold protein helps assemble BER proteins like DNA polymerase beta (Polβ), a bifunctional DNA polymerase containing both DNA synthesis and 5'-deoxyribose-phosphate lyase (5'dRP lyase) activity. Here, we confirm that IDH1 mutant glioma cells have defective NAD metabolism, but still produce sufficient nuclear NAD for robust PARP1 activation and BER complex formation in response to DNA damage. However, the overproduction of 2-hydroxyglutarate, an oncometabolite produced by the IDH1 R132H mutant protein, suppresses BER capacity by reducing Polβ protein levels. This defines a novel mechanism by which the IDH1 mutation in gliomas confers cellular sensitivity to alkylating agents and to inhibitors of the poly-ADP-ribose glycohydrolase, PARG.

Oncometabolite 2-hydroxyglutarate suppresses basal protein levels of DNA polymerase beta that enhances alkylating agent and PARG inhibition induced cytotoxicity.

ADP-ribosylation is a reversible post-translational modification that plays a role as a signaling mechanism in various cellular processes. This modification is characterized by its structural diversity, highly dynamic nature, and short half-life. Hence, it is tightly regulated at many levels by cellular factors that fine-tune its formation, downstream signaling, and degradation that together impacts cellular outcomes. Poly-ADP-ribosylation is an essential signaling mechanism in the DNA damage response that mediates the recruitment of DNA repair factors to sites of DNA damage via their poly-ADP-ribose (PAR)-binding domains (PBDs). PAR readers, encoding PBDs, convey the PAR signal to mediate cellular outcomes that in some cases can be dictated by PAR structural diversity. Several PBD families have been identified, each with variable PAR-binding affinity and specificity, that also recognize and bind to distinct parts of the PAR chain. PARylation signaling has emerged as an attractive target for the treatment of specific cancer types, as the inhibition of PAR formation or degradation can selectively eliminate cancer cells with specific DNA repair defects and can enhance radiation or chemotherapy response. In this review, we summarize the key players of poly-ADP-ribosylation and its regulation and highlight PBDs as tools for studying PARylation dynamics and the expanding potential to target PARylation signaling in cancer treatment.

Poly-ADP-ribosylation dynamics, signaling, and analysis.

Protein poly-ADP-ribosylation (PARylation) is a post-translational modification formed by transferring successive units of ADP-ribose to target proteins to form poly-ADP-ribose (PAR) chains. PAR plays a critical role in the DNA damage response (DDR) by acting as a signaling platform to promote the recruitment of DNA repair factors to the sites of DNA damage that bind via their PAR-binding domains (PBDs). Several classes of PBD families have been identified, which recognize distinct parts of the PAR chain. Proteins encoding PBDs play an essential role in conveying the PAR-mediated signal through their interaction with PAR chains, which mediates many cellular functions, including the DDR. The WWE domain, encoded in 12 human proteins, identifies the iso-ADP-ribose moiety of the PAR chain. PARylation is a heterogeneous structure that is highly dynamic in cells. Capturing the dynamics of PARylation is essential to understanding its role in the DDR, which can be achieved by expanding the tool kit for PAR detection and tracking mediated by the unique binding capability of various sensors. We recently described the WWE domain of RNF146 as a robust genetically encoded probe, when fused to EGFP, for the detection of PAR in live cells. Expanding on this, we used structural prediction tools to evaluate all of the WWE domains encoded in human proteins, evaluating each as molecular PAR probes in live cells. We demonstrate unique PAR dynamics when tracked by WWE-encoded PAR binding domains, in addition to an engineered macrodomain, that can be exploited for modulation of the PAR-dependent DNA damage response.

Poly(ADP-ribose) polymerases 1 and 2 (PARP1/PARP2), and poly(ADP-ribose) glycohydrolase (PARG), modulate the level of poly(ADP-ribose) (PAR), a post-translational protein modification, in response to DNA damage or replication stress. Here, we find that replication-dependent and PARP1/PARP2-mediated PARylation recruits the base excision repair (BER)/single-strand break repair (SSBR) scaffold protein XRCC1 and the associated factors DNA polymerase β (POLB), aprataxin (APTX), and DNA ligase isoform 3 (LIG3). Further, these BER/SSBR proteins promote resistance to inhibitors of PARP1/PARP2 and PARG, as loss of these proteins sensitizes glioblastoma and ovarian cancer cells to each. In addition, depletion of these replication-associated BER/SSBR factors leads to enhanced PAR levels and PARG inhibitor-induced activation of the ATR/CHK1 S-phase checkpoint kinases. Both PARG inhibition and ATR inhibition lead to elevated ATM- and DNA-PK-dependent KAP1 phosphorylation. In turn, inhibition of either ATR or CHK1 enhances the cellular response to PARG inhibitors. Finally, inhibition of the ATR regulators PRMT1 or PRMT5 synergizes with PARG inhibition, implicating replication-associated BER/SSBR and PARylation in the activation of the PRMT1/PRMT5/ATR axis. This study highlights the role of BER/SSBR in protecting the cell during S-phase to suppress PARylation-induced checkpoint activation, which may suggest a potential intervention strategy for PARG inhibitor-resistant tumors.

Article	Year
Quantitative Analysis of Nuclear Poly(ADP-Ribose) Dynamics in Response to Laser-Induced DNA Damage. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 36515828	2023
Overexpression of the WWE domain of RNF146 modulates poly-(ADP)-ribose dynamics at sites of DNA damage. bioRxiv : the preprint server for biology\| PubMed ID: 38234836	2023
Oncometabolite 2-hydroxyglutarate suppresses basal protein levels of DNA polymerase beta that enhances alkylating agent and PARG inhibition induced cytotoxicity. DNA repair\| PubMed ID: 38897003	2024
Poly-ADP-ribosylation dynamics, signaling, and analysis. Environmental and molecular mutagenesis\| PubMed ID: 39221603	2024
Overexpression of the WWE domain of RNF146 modulates poly-(ADP)-ribose dynamics at sites of DNA damage. DNA repair\| PubMed ID: 40403420	2025
Replication-associated base excision repair/single-strand break repair regulates PARG inhibitor response via the PRMT1/PRMT5/ATR axis. NAR cancer\| PubMed ID: 41480640	2025

Rasha Q. Al-Rahahleh

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