Transcriptional Stress Induces Chromatin Relocation of the Nucleotide Excision Repair Factor XPG.

Endonuclease XPG participates in nucleotide excision repair (NER), in basal transcription, and in the processing of RNA/DNA hybrids (R-loops): the malfunction of these processes may cause genome instability. Here, we investigate the chromatin association of XPG during basal transcription and after transcriptional stress. The inhibition of RNA polymerase II with 5,6-dichloro-l-β-D-ribofuranosyl benzimidazole (DRB), or actinomycin D (AD), and of topoisomerase I with camptothecin (CPT) resulted in an increase in chromatin-bound XPG, with concomitant relocation by forming nuclear clusters. The cotranscriptional activators p300 and CREB-binding protein (CREBBP), endowed with lysine acetyl transferase (KAT) activity, interact with and acetylate XPG. Depletion of both KATs by RNA interference, or chemical inhibition with C646, significantly reduced XPG acetylation. However, the loss of KAT activity also resulted in increased chromatin association and the relocation of XPG, indicating that these processes were induced by transcriptional stress and not by reduced acetylation. Transcription inhibitors, including C646, triggered the R-loop formation and phosphorylation of histone H2AX (γ-H2AX). Proximity ligation assay (PLA) showed that XPG colocalized with R-loops, indicating the recruitment of the protein to these structures. These results suggest that transcriptional stress-induced XPG relocation may represent recruitment to sites of R-loop processing

In Situ Analysis of DNA-Protein Complex Formation upon Radiation-Induced DNA Damage

The importance of determining at the cellular level the formation of DNA–protein complexes after radiation-induced lesions to DNA is outlined by the evidence that such interactions represent one of the first steps of the cellular response to DNA damage. These complexes are formed through recruitment at the sites of the lesion, of proteins deputed to signal the presence of DNA damage, and of DNA repair factors necessary to remove it. Investigating the formation of such complexes has provided, and will probably continue to, relevant information about molecular mechanisms and spatiotemporal dynamics of the processes that constitute the first barrier of cell defense against genome instability and related diseases. In this review, we will summarize and discuss the use of in situ procedures to detect the formation of DNA-protein complexes after radiation-induced DNA damage. This type of analysis provides important information on the spatial localization and temporal resolution of the formation of such complexes, at the single-cell level, allowing the study of heterogeneous cell populations

Studio del ruolo dell' interazione p21-PCNA nel processo di riparazione del DNA

The cyclin-dependent kinase inhibitor p21CDK1NA is a protein involved in various cellular processes, such as cell cycle arrest, transcription regulation, apoptosis and cell motility. p21 was discovered to actively participate also to DNA repair process, such as nucleotide excision repair (NER), through the interaction with Proliferative Cell Nuclear Antigen (PCNA). For this study, we used plasmids driving the expression of three different mutated form of p21 with a fluorescent tag (GFP) fused to their C-term: p21DD-GFP (T148D), p21AAA-GFP (KRR154-156AAA) and p212KQ-GFP (K161,163Q). These mutants, reported in literature to be resistant to degradation, have been used to test whether the PCNA-interacting partners dynamics at the DNA damage sites (e.g., the recruitment and subsequent release from the repair site) is influenced by the p21 susceptibility to degradation, while the interaction with PCNA is conserved. We initially performed fluorescence microscopy analysis and immunoprecipitation assays on transfected HeLa cells to characterize the p21-GFP mutants. The results showed that, although both p21DD-GFP and p212KQ-GFP mutants have a clear nuclear localization, unexpectedly p21AAA-GFP did not interact with PCNA inhibiting its recruitment to DNA damage sites. Subsequently, we confirmed the enhanced stability of p21AAA and p212KQ mutants, while p21DD showed a poor resistance to UVC-induced degradation. To test whether p21 could influence the PCNA-interacting partners turnover, we compared the persistence of p212KQ with p21WT at the DNA damage sites performing an immunofluorescence assay on HeLa cells transfected with HA-p21WT and HA-p212KQ. The results showed a longer retention of DNA repair factors (e.g., DNA polymerase δ) at the DNA lesions in the presence of p21 mutant, suggesting a consequent delay in the DNA repair process. Then, we also performed live cell imaging confocal analyses using HeLa cell line stably expressing mCherry-PCNA co-transfected with two plasmids driving the expression of fluorescent-tagged (miRFP) DNA Ligase 1 and either p21WT-GFP or p212KQ-GFP, respectively. PCNA and DNA Lig 1 release kinetics in the absence p21 and in the presence of p212KQ resulted to be remarkably slower as compared to p21WT. Therefore, the data suggest that not only the persistence of p21 at the DNA damage sites, but even the absence of p21 could deregulate the DNA repair process. Along with these results, a significant reduction of NER efficiency was detected in HeLa cells transfected with p212KQ-GFP plasmid and in the absence of p21. Consistently, we observed a similar reduction in DNA re-synthesis after UV-C irradiation in human fibroblasts lacking p21. In conclusion, these results suggest a possible mechanism through which p21 influences the PCNA partner dynamics by coupling to the degradation process, which is fundamental to finely tune p21 cellular levels

Revisiting the Function of p21CDKN1A in DNA Repair: The Influence of Protein Interactions and Stability

The p21CDKN1A protein is an important player in the maintenance of genome stability through its function as a cyclin-dependent kinase inhibitor, leading to cell-cycle arrest after genotoxic damage. In the DNA damage response, p21 interacts with specific proteins to integrate cell-cycle arrest with processes such as transcription, apoptosis, DNA repair, and cell motility. By associating with Proliferating Cell Nuclear Antigen (PCNA), the master of DNA replication, p21 is able to inhibit DNA synthesis. However, to avoid conflicts with this process, p21 protein levels are finely regulated by pathways of proteasomal degradation during the S phase, and in all the phases of the cell cycle, after DNA damage. Several lines of evidence have indicated that p21 is required for the efficient repair of different types of genotoxic lesions and, more recently, that p21 regulates DNA replication fork speed. Therefore, whether p21 is an inhibitor, or rather a regulator, of DNA replication and repair needs to be re-evaluated in light of these findings. In this review, we will discuss the lines of evidence describing how p21 is involved in DNA repair and will focus on the influence of protein interactions and p21 stability on the efficiency of DNA repair mechanisms

Single Cell Determination of7,8-dihydro-8-oxo-2′-deoxyguanosine by FluorescenceTechniques: Antibody vs. Avidin Labeling

An important biomarker of oxidative damage in cellular DNA is the formation of 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG). Although several methods are available for the bio-chemical analysis of this molecule, its determination at the single cell level may provide significantadvantages when investigating the influence of cell heterogeneity and cell type in the DNA damageresponse. to. For this purpose, antibodies recognizing 8-oxodG are available; however, detectionwith the glycoprotein avidin has also been proposed because of a structural similarity between itsnatural ligand biotin and 8-oxodG. Whether the two procedures are equivalent in terms of reliabilityand sensitivity is not clear. In this study, we compared the immunofluorescence determination of8-oxodG in cellular DNA using the monoclonal antibody N45.1 and labeling using avidin conjugatedwith the fluorochrome Alexa Fluor488 (AF488). Oxidative DNA damage was induced in different celltypes by treatment with potassium bromate (KBrO3), a chemical inducer of reactive oxygen species(ROS). By using increasing concentrations of KBrO3, as well as different reaction conditions, ourresults indicate that the monoclonal antibody N45.1 provides a specificity of 8-oxodG labeling greaterthan that attained with avidin-AF488. These findings suggest that immunofluorescence techniquesare best suited to the in situ analysis of 8-oxodG as a biomarker of oxidative DNA damage

In Vitro Neurotoxicity of Flumethrin Pyrethroid on SH-SY5Y Neuroblastoma Cells: Apoptosis Associated with Oxidative Stress

Pyrethroids are neurotoxicants for animals, showing a pattern of toxic action on the nervous system. Flumethrin, a synthetic pyrethroid, is used against ectoparasites in domestic animals, plants, and for public health. This compound has been shown to be highly toxic to bees, while its effects on other animals have been less investigated. However, in vitro studies to evaluate cytotoxicity are scarce, and the mechanisms associated with this effect at the molecular level are still unknown. This study aimed to investigate the oxidative stress and cell death induction in SH-SY5Y neuroblastoma cells in response to flumethrin exposure (1–1000 µM). Flumethrin induced a significant cytotoxic effect, as evaluated by MTT and LDH leakage assays, and produced an increase in the biomarkers of oxidative stress as reactive oxygen species and nitric oxide (ROS and NO) generation, malondialdehyde (MDA) concentration, and caspase-3 activity. In addition, flumethrin significantly increased apoptosis-related gene expressions (Bax, Casp-3, BNIP3, APAF1, and AKT1) and oxidative stress and antioxidative (NFκB and SOD2) mediators. The results demonstrated, by biochemical and gene expression assays, that flumethrin induces oxidative stress and apoptosis, which could cause DNA damage. Detailed knowledge obtained about these molecular changes could provide the basis for elucidating the molecular mechanisms of flumethrin-induced neurotoxicity

Mutations in CREBBP and EP300 genes affect DNA repair of oxidative damage in Rubinstein-Taybi syndrome cells

Rubinstein-Taybi syndrome (RSTS) is an autosomal-dominant disorder characterized by intellectual disability, skeletal abnormalities, growth deficiency, and an increased risk of tumors. RSTS is predominantly caused by mutations in CREBBP or EP300 genes encoding for CBP and p300 proteins, two lysine acetyl-transferases (KAT) playing a key role in transcription, cell proliferation and DNA repair. However, the efficiency of these processes in RSTS cells is still largely unknown. Here we have investigated whether pathways involved in the maintenance of genome stability are affected in lymphoblastoid cell lines (LCLs) obtained from RSTS patients with mutations in CREBBP or in EP300 genes. We report that RSTS LCLs with mutations affecting CBP or p300 protein levels or KAT activity, are more sensitive to oxidative DNA damage and exhibit defective base excision repair (BER). We have found reduced OGG1 DNA glycosylase activity in RSTS compared to control cell extracts, and concomitant lower OGG1 acetylation levels, thereby impairing the initiation of the BER process. In addition, we report reduced acetylation of other BER factors, such as DNA polymerase β and PCNA, together with acetylation of histone H3. We also show that complementation of CBP or p300 partially reversed RSTS cell sensitivity to DNA damage. These results disclose a mechanism of defective DNA repair as a source of genome instability in RSTS cells