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

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 (Polß), 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 Polß 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.

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

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-ß.

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

Temporal dynamics of base excision/single-strand break repair protein complex assembly/disassembly are modulated by the PARP/NAD+/SIRT6 axis.

Assembly and disassembly of DNA repair protein complexes at DNA damage sites are essential for maintaining genomic integrity. Investigating factors coordinating assembly of the base excision repair (BER) proteins DNA polymerase ß (Polß) and XRCC1 to DNA lesion sites identifies a role for Polß in regulating XRCC1 disassembly from DNA repair complexes and, conversely, demonstrates Polß's dependence on XRCC1 for complex assembly. LivePAR, a genetically encoded probe for live-cell imaging of poly(ADP-ribose) (PAR), reveals that Polß and XRCC1 require PAR for repair-complex assembly, with PARP1 and PARP2 playing unique roles in complex dynamics. Further, BER complex assembly is modulated by attenuation/augmentation of NAD+ biosynthesis. Finally, SIRT6 does not modulate PARP1 or PARP2 activation but does regulate XRCC1 recruitment, leading to diminished Polß abundance at sites of DNA damage. These findings highlight coordinated yet independent roles for PARP1, PARP2, and SIRT6 and their regulation by NAD+ bioavailability to facilitate BER.

Comparative study of articaine and lidocaine without palatal injection for maxillary teeth extraction.

OBJECTIVES: There is significant evidence that articaine and lidocaine buccal injections alone are sufficient for painless extraction of maxillary teeth. The aim of this study was to evaluate the extraction of permanent maxillary teeth and to compare pain control between articaine and lidocaine without palatal injection. MATERIALS AND METHODS: Group A received buccal and palatal injections of 2% lidocaine with 0.015 mg/ml epinephrine. Group B received only buccal local anesthetic injection of 2% lidocaine with 0.015 mg/ml epinephrine. Group C received only buccal injection of 4% articaine with 0.012 mg/ml epinephrine. The patients' perception of pain was assessed using visual analogue scale and verbal response scale after the injection and the extraction. RESULTS: Statistical analysis showed that the difference in pain perception of local anesthetic injection was statistically significant between groups A and B and between groups A and C (p < 0.05). CONCLUSION: The extraction of permanent maxillary teeth is possible without palatal injections and there is no difference between articaine and lidocaine. CLINICAL RELEVANCE: Local anesthetic agents are the most frequently administered drugs in dentistry and represent the primary method of pain control for patients undergoing intraoral procedures.