NEDDylation has been shown to participate in the DNA damage pathway, but the substrates of neural precursor cell expressed developmentally downregulated 8 (NEDD8) and the roles of NEDDylation involved in the DNA damage response (DDR) are largely unknown. Translesion synthesis (TLS) is a damage-tolerance mechanism, in which RAD18/RAD6-mediated monoubiquitinated proliferating cell nuclear antigen (PCNA) promotes recruitment of polymerase η (polη) to bypass lesions. Here we identify PCNA as a substrate of NEDD8, and show that E3 ligase RAD18-catalyzed PCNA NEDDylation antagonizes its ubiquitination. In addition, NEDP1 acts as the deNEDDylase of PCNA, and NEDP1 deletion enhances PCNA NEDDylation but reduces its ubiquitination. In response to HO stimulation, NEDP1 disassociates from PCNA and RAD18-dependent PCNA NEDDylation increases markedly after its ubiquitination. Impairment of NEDDylation by Ubc12 knockout enhances PCNA ubiquitination and promotes PCNA-polη interaction, while up-regulation of NEDDylation by NEDD8 overexpression or NEDP1 deletion reduces the excessive accumulation of ubiquitinated PCNA, thus inhibits PCNA-polη interaction and blocks polη foci formation. Moreover, Ubc12 knockout decreases cell sensitivity to HO-induced oxidative stress, but NEDP1 deletion aggravates this sensitivity. Collectively, our study elucidates the important role of NEDDylation in the DDR as a modulator of PCNA monoubiquitination and polη recruitment.
DNA Damage ; drug effects ; DNA Repair ; genetics ; DNA Replication ; genetics ; DNA-Binding Proteins ; genetics ; DNA-Directed DNA Polymerase ; genetics ; Endopeptidases ; genetics ; Gene Knockout Techniques ; Humans ; Hydrogen Peroxide ; toxicity ; NEDD8 Protein ; genetics ; Oxidative Stress ; genetics ; Proliferating Cell Nuclear Antigen ; genetics ; Ubiquitin-Conjugating Enzymes ; genetics ; Ubiquitin-Protein Ligases ; genetics ; Ubiquitination ; genetics ; Ultraviolet Rays

Werner syndrome (WS) is a premature aging disorder that mainly affects tissues derived from mesoderm. We have recently developed a novel human WS model using WRN-deficient human mesenchymal stem cells (MSCs). This model recapitulates many phenotypic features of WS. Based on a screen of a number of chemicals, here we found that Vitamin C exerts most efficient rescue for many features in premature aging as shown in WRN-deficient MSCs, including cell growth arrest, increased reactive oxygen species levels, telomere attrition, excessive secretion of inflammatory factors, as well as disorganization of nuclear lamina and heterochromatin. Moreover, Vitamin C restores in vivo viability of MSCs in a mouse model. RNA sequencing analysis indicates that Vitamin C alters the expression of a series of genes involved in chromatin condensation, cell cycle regulation, DNA replication, and DNA damage repair pathways in WRN-deficient MSCs. Our results identify Vitamin C as a rejuvenating factor for WS MSCs, which holds the potential of being applied as a novel type of treatment of WS.
Animals ; Ascorbic Acid ; pharmacology ; Cell Cycle Checkpoints ; drug effects ; Cell Line ; Cellular Senescence ; drug effects ; DNA Damage ; DNA Repair ; drug effects ; DNA Replication ; drug effects ; Disease Models, Animal ; Heterochromatin ; metabolism ; pathology ; Humans ; Mesenchymal Stem Cells ; metabolism ; pathology ; Mice ; Nuclear Lamina ; metabolism ; pathology ; Reactive Oxygen Species ; metabolism ; Telomere Homeostasis ; drug effects ; Werner Syndrome ; drug therapy ; genetics ; metabolism

Our study explored the dynamic changes in and the relationship between the DNA damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) and the DNA repair marker 8-hydroxyguanine DNA glycosidase 1 (hOGG1) according to the length of occupational employment in nickel smelting workers. One hundred forty nickel-exposed smelting workers and 140 age-matched unexposed office workers were selected from the Jinchang cohort. The 8-OHdG levels in smelting workers was significantly higher than in office workers (Z=-8.688, P<0.05) and the 8-OHdG levels among nickel smelting workers in the 10-14 y employment length category was significantly higher than among all peers. The hOGG1 levels among smelting workers were significantly lower than those of non-exposed workers (Z=-8.948, P<0.05). There were significant differences between employment length and hOGG1 levels, with subjects employed in nickel smelting for 10-14 y showing the highest levels of hOGG1. Correlation analysis showed positive correlations between 8-OHdG and hOGG1 levels (r=0.413; P<0.01). DNA damage was increased with employment length among nickel smelting workers and was related to the inhibition of hOGG1 repair capacity.
Biomarkers ; Case-Control Studies ; Cohort Studies ; DNA Damage ; drug effects ; DNA Glycosylases ; blood ; DNA Repair ; Deoxyadenosines ; blood ; Humans ; Male ; Metallurgy ; Nickel ; toxicity ; urine ; Occupational Exposure ; adverse effects ; Time Factors

OBJECTIVETo study the role of poly (ADP-ribose) polymerase-l (PARP-1) in formaldehyde-induced DNA damage response in human bronchial epithelial (HBE) cells and to investigate the mechanism of formaldehyde carcinogenicity.

METHODSThe protein levels were measured by Western blot. The interaction between different proteins was determined by co-immunoprecipitation assay. The chemical inhibitor was used to confirm the relationship between PARP-1 and DNA damage repair.

RESULTSAfter being exposed to different concentrations of formaldehyde for 4 h, HBE cells showed no significant changes in cell viability. Cell viability was significantly reduced after 24-h exposure to 80 and 160 µmol/L formaldehyde (P < 0.05). The 10 µmol/L formaldehyde resulted in significant increases in the protein levels of PARP-1 and XRCC-1. However, 80 µmol/L formaldehyde led to a significant decrease in the protein level of PARP-1 of 124 KD molecular weight but a significant increase in the protein level of PARP-1 of 89 KD molecular weight; there was no significant change in the protein level of XRCC-1. The co-immunoprecipitation assay showed that 10 µmol/L formaldehyde induced increased binding between PARP-1 and XRCC-1, but 80 µmol/L formaldehyde led to no significant change in binding between PARP-1 and XRCC-1. Here, we confirmed the role of 10 µmol/L formaldehyde in strand breaks by comet assay which showed an increase in the tail DNA content of HBE cells after 4-h formaldehyde exposure. No significant difference was observed in tail DNA content between treated HBE cells and control cells at 2 h after formaldehyde was removed. Moreover, compared with control, inhibition of PARP-1 induced a significant increase in tail DNA content, and a significant difference was observed in tail DNA content between inhibited HBE cells and control cells at 2 h after formaldehyde was removed. Inhibition of PARP-1 significantly reduced DNA repair capacity.

CONCLUSIONPARP-1 mediated the repair of DNA damage induced by low-concentration formaldehyde through recruiting XRCC-1 protein, and may be involved in the regulation of cell apoptosis induced by high-concentration formaldehyde.

Apoptosis ; drug effects ; Cells, Cultured ; DNA Damage ; drug effects ; DNA Repair ; drug effects ; DNA-Binding Proteins ; metabolism ; Epithelial Cells ; drug effects ; metabolism ; pathology ; Formaldehyde ; toxicity ; Humans ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; metabolism ; X-ray Repair Cross Complementing Protein 1

Benzene is an established leukotoxin and leukemogen in humans. We have previously reported that exposure of workers to benzene and to benzene metabolite hydroquinone in cultured cells induced DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to mediate the cellular response to DNA double strand break (DSB) caused by DNA-damaging metabolites. In this study, we used a new, small molecule, a selective inhibitor of DNA-PKcs, 2-(morpholin-4-yl)-benzo[h]chomen-4-one (NU7026), as a probe to analyze the molecular events and pathways in hydroquinone-induced DNA DSB repair and apoptosis. Inhibition of DNA-PKcs by NU7026 markedly potentiated the apoptotic and growth inhibitory effects of hydroquinone in proerythroid leukemic K562 cells in a dose-dependent manner. Treatment with NU7026 did not alter the production of reactive oxygen species and oxidative stress by hydroquinone but repressed the protein level of DNA-PKcs and blocked the induction of the kinase mRNA and protein expression by hydroquinone. Moreover, hydroquinone increased the phosphorylation of Akt to activate Akt, whereas co-treatment with NU7026 prevented the activation of Akt by hydroquinone. Lastly, hydroquinone and NU7026 exhibited synergistic effects on promoting apoptosis by increasing the protein levels of pro-apoptotic proteins Bax and caspase-3 but decreasing the protein expression of anti-apoptotic protein Bcl-2. Taken together, the findings reveal a central role of DNA-PKcs in hydroquinone-induced hematotoxicity in which it coordinates DNA DSB repair, cell cycle progression, and apoptosis to regulate the response to hydroquinone-induced DNA damage.
Apoptosis ; drug effects ; physiology ; Benzene ; toxicity ; Catalysis ; Chromones ; pharmacology ; DNA Damage ; drug effects ; genetics ; DNA Repair ; drug effects ; physiology ; DNA-Activated Protein Kinase ; antagonists & inhibitors ; metabolism ; Humans ; K562 Cells ; Morpholines ; pharmacology ; Protein Subunits

Development of controllable hypermutable cells can greatly benefit understanding and harnessing microbial evolution. However, there have not been any similar systems developed for Clostridium, an important bacterial genus. Here we report a novel two-step strategy for developing controllable hypermutable cells of Clostridium acetobutylicum, an important and representative industrial strain. Firstly, the mutS/L operon essential for methyldirected mismatch repair (MMR) activity was inactivated from the genome of C. acetobutylicum to generate hypermutable cells with over 250-fold increased mutation rates. Secondly, a proofreading control system carrying an inducibly expressed mutS/L operon was constructed. The hypermutable cells and the proofreading control system were integrated to form a controllable hypermutable system SMBMutC, of which the mutation rates can be regulated by the concentration of anhydrotetracycline (aTc). Duplication of the miniPthl-tetR module of the proofreading control system further significantly expanded the regulatory space of the mutation rates, demonstrating hypermutable Clostridium cells with controllable mutation rates are generated. The developed C. acetobutylicum strain SMBMutC2 showed higher survival capacities than the control strain facing butanol-stress, indicating greatly increased evolvability and adaptability of the controllable hypermutable cells under environmental challenges.
Butanols ; pharmacology ; Cell Engineering ; methods ; Clostridium acetobutylicum ; cytology ; drug effects ; genetics ; physiology ; DNA Methylation ; genetics ; DNA Mismatch Repair ; genetics ; Evolution, Molecular ; Genome, Bacterial ; genetics ; MutS DNA Mismatch-Binding Protein ; genetics ; Mutation ; Operon ; genetics ; Stress, Physiological ; drug effects ; genetics

Sapacitabine is an orally bioavailable prodrug of the nucleoside analog 2'-C-cyano-2'-deoxy-1-β-D-arabino-pentofuranosylcytosine (CNDAC). Both the prodrug and active metabolite are in clinical trials for hematologic malignancies and/or solid tumors. CNDAC has a unique mechanism of action: after incorporation into DNA, it induces single-strand breaks (SSBs) that are converted into double-strand breaks (DSBs) when cells go through a second S phase. In our previous studies, we demonstrated that CNDAC-induced SSBs can be repaired by the transcription-coupled nucleotide excision repair pathway, whereas lethal DSBs are mainly repaired through homologous recombination. In the current work, we used clonogenic assays to compare the DNA damage repair mechanism of CNDAC with two other deoxycytidine analogs: cytarabine, which is used in hematologic malignacies, and gemcitabine, which shows activity in solid tumors. Deficiency in two Rad51 paralogs, Rad51D and XRCC3, greatly sensitized cells to CNDAC, but not to cytarabine or gemcitabine, indicating that homologous recombination is not a major mechanism for repairing damage caused by the latter two analogs. This study further suggests clinical activity and application of sapacitabine that is distinct from that of cytarabine or gemcitabine.
Animals ; Antimetabolites, Antineoplastic ; pharmacology ; Arabinonucleosides ; pharmacology ; CHO Cells ; Cricetinae ; Cricetulus ; Cytarabine ; analogs & derivatives ; pharmacology ; Cytosine ; analogs & derivatives ; pharmacology ; DNA Breaks, Double-Stranded ; drug effects ; DNA Repair ; drug effects ; DNA-Binding Proteins ; deficiency ; Deoxycytidine ; analogs & derivatives ; pharmacology ; Homologous Recombination ; genetics ; Inhibitory Concentration 50 ; Prodrugs

OBJECTIVETo explore the relationship between the polymorphisms of DNA repair gene (XRCC1 194, 280 and 399) and the chromosomal damage induced by benzene.

METHODSThe chromosomal damage of the peripheral lymphocytes in 459 workers occupationally exposed to benzene and 88 non-exposed controls were detected with cytokinesis-block micronucleus (CBMN) assay. PCR-RFLP technique was used to measure polymorphisms in XRCC1 194, 280 and 399.

RESULTSIt was found that the MN frequency (2.12‰ ± 1.88‰) of the exposed group was significantly higher than that (1.19‰ ± 1.68‰) of the control group (P < 0.05), in the exposed group, the MN frequency (3.00‰ ± 2.76‰) of older workers (> 35 years) was significantly higher than that (2.02‰ ± 1.71‰) of younger workers (≤ 35 years) (P < 0.05). The effect of genetic polymorphisms of XRCC1 on CBMN was not found. The haplotypes AAA/BAA, AAB/AAB, ABA/ABA, ABB/ABB could associated with the increased frequencies of total micronucleus (P < 0.05).

CONCLUSIONBenzene exposure could result in chromosome damage. Age of workers and diplotypes of XRCC1 could associated with chromosomal damage induced by benzene.

Adult ; Benzene ; adverse effects ; DNA Damage ; drug effects ; genetics ; DNA-Binding Proteins ; genetics ; Humans ; Micronuclei, Chromosome-Defective ; Micronucleus Tests ; Occupational Exposure ; Polymorphism, Single Nucleotide ; X-ray Repair Cross Complementing Protein 1 ; Young Adult

Tumors often have DNA repair defects, suggesting additional inhibition of other DNA repair pathways in tumors may lead to synthetic lethality. Accumulating data demonstrate that DNA repair-defective tumors, in particular homologous recombination (HR), are highly sensitive to DNA-damaging agents. Thus, HR-defective tumors exhibit potential vulnerability to the synthetic lethality approach, which may lead to new therapeutic strategies. It is well known that poly (adenosine diphosphate (ADP)-ribose) polymerase (PARP) inhibitors show the synthetically lethal effect in tumors defective in BRCA1 or BRCA2 genes encoded proteins that are required for efficient HR. In this review, we summarize the strategies of targeting DNA repair pathways and other DNA metabolic functions to cause synthetic lethality in HR-defective tumor cells.
Animals ; Antineoplastic Agents ; pharmacology ; Breast Neoplasms ; genetics ; DNA Repair ; drug effects ; genetics ; Gene Expression Regulation, Neoplastic ; drug effects ; Genes, Lethal ; genetics ; Genes, Tumor Suppressor ; drug effects ; Genes, cdc ; drug effects ; Humans ; Mutagenesis ; Poly(ADP-ribose) Polymerase Inhibitors ; Rad52 DNA Repair and Recombination Protein ; antagonists & inhibitors ; Recombination, Genetic ; drug effects ; genetics

Antineoplastic Agents, Alkylating ; pharmacology ; Apoptosis ; drug effects ; Benzimidazoles ; pharmacology ; DNA Repair ; Dacarbazine ; analogs & derivatives ; pharmacology ; Drug Resistance, Neoplasm ; drug effects ; physiology ; Humans ; Membrane Proteins ; metabolism ; O(6)-Methylguanine-DNA Methyltransferase ; antagonists & inhibitors ; metabolism ; Poly(ADP-ribose) Polymerase Inhibitors ; Poly(ADP-ribose) Polymerases ; metabolism ; Proto-Oncogene Proteins ; metabolism ; Purines ; pharmacology ; Pyrimidines ; pharmacology ; Tumor Suppressor Protein p53 ; metabolism