2.A study of the substitution effect of hOGG1 and hMTH1 in oxidative DNA damage with gene-deficient cell strains.
Yuebin KE ; Shuang WU ; Juan HUANG ; Jianhui YUAN ; Pingjian DENG ; Jinquan CHENG
Chinese Journal of Preventive Medicine 2014;48(3):197-202
OBJECTIVETo investigate the potential substitution effect of hOGG1 and hMTH1 on oxidative DNA damage, based on gene-deficient cell strains models.
METHODShOGG1 and hMTH1 gene deficient cell strains models were established by Human embryonic lung fibroblasts (HFL) cells. After HFL cells being exposed to 100 µmol/L H₂O₂ for 12 h, HPLC-EC detecting technique and RT-PCR method were adopted to analyze the genetic expression level of 8-oxo-dG (7, 8-dihydro-8-oxoguanine).
RESULTSThe gene-deficient cell strains models of hOGG1 and hMTH1 were obtained by infecting target cells with high titer of lentivirus. The mRNA expression level of hOGG1 was 0.09 ± 0.02, 91% lower than it in normal HFL cells, which was 1.00 ± 0.04. As the same, the mRNA expression level of hMTH1 (0.41 ± 0.04) also decreased by 60% compared with it in normal HFL cells (1.02 ± 0.06). After induced by 100 µmol/L H₂O₂ for 12 h, the genetic expression level of hMTH1 in hOGG1 gene-deficient cells (1.26 ± 0.18) increased 25% compared with it in control group (1.01 ± 0.07). Meanwhile, the genetic expression level of hOGG1 in hMTH1 gene-deficient cells (1.54 ± 0.25) also increased by 52%. The DNA 8-oxo-dG levels in hOGG1 gene-deficient cells (2.48 ± 0.54) was 3.1 times compared with it in the control group (0.80 ± 0.16), the difference showed statistical significance (P < 0.01). Whereas the 8-oxo-dG levels in hMTH1 gene-deficient cells (1.84 ± 0.46) was 2.3 times of it in the control group, the difference also showed statistical significance (P < 0.01).
CONCLUSIONBased on gene-deficient HFL cells models, a synergetic substitution effect on DNA damage and repair activity by both hOGG1 and hMTH1 were firstly discovered when induced by oxidation. The substitution effect of hOGG1 were stronger than that of hMTH1.
Cell Line ; DNA Damage ; DNA Glycosylases ; genetics ; DNA Repair ; DNA Repair Enzymes ; genetics ; Fibroblasts ; metabolism ; Humans ; Oxidative Stress ; genetics ; Phosphoric Monoester Hydrolases ; genetics
3.Relationship between genetic polymorphism in hMTH1c.83, hOGG1c.326 and hMYHc.335 and risks of chronic benzene poisoning.
Zhong-bin ZHANG ; Wei-wei LIU ; Shou-yong GU ; Jun-xiang WAN ; Xi-peng JIN ; Zhao-lin XIA
Chinese Journal of Industrial Hygiene and Occupational Diseases 2006;24(3):134-138
OBJECTIVETo explore the relationship between genetic polymorphisms in hMTH1, hOGG1 and hMYH and risks of chronic benzene poisoning (CBP).
METHODSA case control study was conducted. One hundred and fifty-two BP patients and 152 workers occupationally exposed to benzene without poisoning manifestations were investigated. The polymerase chain reaction restrained fragment length polymorphism technique (PCR-RFLP) was applied to detect the single nucleotide polymorphisms (SNPs) on c.83 of hMTH1 gene, c.326 of hOGG1 gene and c.335 of hMYH gene.
RESULTSThere were 2.51 times (OR(adj) = 2.51, 95% CI: 1.14-5.49, P = 0.02) and 2.49 times (OR(adj) = 2.49, 95% CI: 1.52-4.07, P < 0.01) risks of BP for individuals carrying genotypes of hMTH1c.83Val/Met + Met/Met or hOGG1c.326Cys/Cys compared with individuals carrying genotypes of hMTH1c.83Val/Val or hOGG1c.326Ser/Cys + Ser/Ser, respectively. Compared with individuals carrying genotypes of hOGG1c.326Cys/Cy and hMYHc.335 is/His at the same time, there was 0.33 times (OR(adj) = 0.33, 95% CI = 0.15-0.72, P = 0.01) risks of BP for these with genotypes of hOGG1c.326Ser/Cys + Ser/Ser and hMYHc.335His/Gln + Gln/Gln simultaneously. In the smoking group, there was 0.15 times (OR(adj) = 0.15, 95% CI: 0.03-0.68, P = 0.01) risks of BP for subjects carrying genotypes of hMYHc.335His/Gln + Gln/Gln compared with these carrying genotypes of hMYHc.335His/His.
CONCLUSIONPolymorphisms of hMTH1 Val83 Met and hOGG1 Ser326Cys may contribute to altered risks of CBP, and potential interaction may exist among polymorphisms of hOGG1 Ser326Cys and hMYH His335Gln.
Adult ; Benzene ; poisoning ; Case-Control Studies ; Chronic Disease ; DNA Glycosylases ; genetics ; DNA Repair Enzymes ; genetics ; Female ; Humans ; Male ; Middle Aged ; Occupational Exposure ; Phosphoric Monoester Hydrolases ; genetics ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Polymorphism, Restriction Fragment Length
4.DNA mismatch repair enzyme hMSH2 genetic polymorphism in southern Chinese Han population.
Yun HE ; Zhi-xiong ZHUANG ; Chun-hua HE ; Ru-qing LIU
Chinese Journal of Medical Genetics 2003;20(3):256-258
OBJECTIVETo study hMSH2 genetic polymorphism in southern Chinese Han population.
METHODSThe basic materials and blood samples from 163 southern Chinese were collected. The mutations of exon 6 and exon 7 of hMSH2 gene were investigated by PCR-SSCP, followed by DNA sequencing.
RESULTSFragments of 250 bp including exon 6 and fragments of 323 bp including exon 7 of hMSH2 gene were amplified by multiple PCR. The allele frequencies of C18, A82 and B39 type mutations were 0.0184, 0.0031, 0.0031, respectively. The gene frequencies and gene type frequencies of three polymorphism sites in normal population accorded with Hardy-Weinberg equilibrium (P>0.05). The heterozygosity of C18 type mutation (0.0361) was the highest.
CONCLUSIONThere were three polymorphism sites in exon 7 of hMSH2 gene in southern Chinese Han population, among which the genotype frequency of C18 type was the highest, suggesting that C18 type mutation be a useful genetic mark.
Asian Continental Ancestry Group ; genetics ; Base Pair Mismatch ; DNA Ligase ATP ; DNA Ligases ; genetics ; DNA Mismatch Repair ; genetics ; physiology ; DNA Repair Enzymes ; genetics ; Exons ; genetics ; Female ; Humans ; Male ; Microsatellite Repeats ; genetics ; Middle Aged ; Polymorphism, Genetic
6.Ubiquitin-proteasome system and sperm DNA repair: An update.
Guo-Wei ZHANG ; Hong-Cai CAI ; Xue-Jun SHANG
National Journal of Andrology 2016;22(9):834-837
The ubiquitin-proteasome system (UPS) is a proteasome system widely present in the human body, which is composed of ubiquitin (Ub), ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2), ubiquitin protein ligases (E3), 26S proteasome, and deubiquitinating enzymes (DUBs) and involved in cell cycle regulation, immune response, signal transduction, DNA repair as well as protein degradation. Sperm DNA is vulnerable to interference or damage in the progression of chromosome association and homologous recombination. Recent studies show that UPS participates in DNA repair in spermatogenesis by modulating DNA repair enzymes via ubiquitination, assisting in the identification of DNA damage sites, raising damage repair-related proteins, initiating the DNA repair pathway, maintaining chromosome stability, and ensuring the normal process of spermatogenesis.
Cell Cycle Proteins
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physiology
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DNA Damage
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DNA Repair
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physiology
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Humans
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Male
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Proteasome Endopeptidase Complex
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physiology
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Signal Transduction
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physiology
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Spermatogenesis
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physiology
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Spermatozoa
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Ubiquitin
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physiology
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Ubiquitin-Conjugating Enzymes
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physiology
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Ubiquitin-Protein Ligases
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physiology
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Ubiquitination
7.NEDDylation antagonizes ubiquitination of proliferating cell nuclear antigen and regulates the recruitment of polymerase η in response to oxidative DNA damage.
Junhong GUAN ; Shuyu YU ; Xiaofeng ZHENG
Protein & Cell 2018;9(4):365-379
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
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drug effects
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DNA Repair
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genetics
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DNA Replication
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genetics
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DNA-Binding Proteins
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genetics
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DNA-Directed DNA Polymerase
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genetics
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Endopeptidases
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genetics
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Gene Knockout Techniques
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Humans
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Hydrogen Peroxide
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toxicity
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NEDD8 Protein
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genetics
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Oxidative Stress
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genetics
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Proliferating Cell Nuclear Antigen
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genetics
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Ubiquitin-Conjugating Enzymes
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genetics
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Ubiquitin-Protein Ligases
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genetics
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Ubiquitination
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genetics
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Ultraviolet Rays
9.Association of metabolic and DNA repair enzyme gene polymorphisms and DNA damage in coke-oven workers.
Juan CHENG ; Shu-guang LENG ; Yu-fei DAI ; Yong NIU ; Zu-fei PAN ; Bin LI ; Yun HE ; Feng-sheng HE ; Yu-Xin ZHENG
Chinese Journal of Preventive Medicine 2005;39(3):164-167
OBJECTIVETo investigate the association of polymorphisms of metabolic and DNA repair enzyme genes and DNA damage in peripheral blood lymphocytes in coke-oven workers.
METHODSOne hundred and forty-four coke-oven workers and 50 controls were recruited in this study. Urinary 1-hydroxypyrene (1-OHP) levels were measured as the internal dose of polycyclic aromatic hydrocarbons exposure. DNA damage was detected by alkaline comet assay, and the value of 1.74 was used as the cut-off value to determine whether the individual's DNA damage was positive. The genotypes of CYP1A1, CYP2E1, GSTP1, NQO1, mEH and XRCC1 were determined by PCR-based methods. With adjustment for urinary 1-OHP, age, sex, multiple analysis of covariance was used to study the association between genotypes and the ln-transformed olive TM and multiple logistic regression was used to calculate the adjusted OR and the 95% CI for the risk of DNA damage.
RESULTSIn 144 coke-oven workers, with adjustment for urinary 1-OHP, coking history and sex, the olive TM was significantly higher with XRCC1 280His allele than those with Arg allele (5.6 vs. 2.8, P < 0.01). The subjects with XRCC1 280His allele also have significantly higher risk for DNA damage than subjects with Arg allele (adjusted OR = 2.66, 95% CI = 1.00-7.14, P = 0.05) and the subjects with GSTP1 104Val allele have higher risk for DNA damage than subjects with Ile allele (adjusted OR = 1.90, 95% CI = 0.94-3.85, P = 0.07).
CONCLUSIONXRCC1 and GSTP1 polymorphisms might influence the susceptibility of DNA damage in occupational PAH-exposed coke-oven workers.
Adult ; Case-Control Studies ; Coke ; poisoning ; Comet Assay ; Cytochrome P-450 CYP1A1 ; genetics ; Cytochrome P-450 CYP2E1 ; genetics ; DNA Damage ; DNA Ligase ATP ; DNA Ligases ; genetics ; DNA Repair Enzymes ; genetics ; Female ; Genotype ; Glutathione S-Transferase pi ; genetics ; Humans ; Logistic Models ; Male ; Middle Aged ; Multivariate Analysis ; NAD(P)H Dehydrogenase (Quinone) ; genetics ; Occupational Exposure ; adverse effects ; analysis ; Polymorphism, Genetic
10.Construction of eukaryotic expression vector of hMTH1 gene antisense RNA.
Gao-feng JIANG ; Zhi-xiong ZHUANG ; Qi-zhan LIU ; Yun HE ; Liu-tao DU
Chinese Journal of Industrial Hygiene and Occupational Diseases 2003;21(1):57-60
OBJECTIVETo construct pEGFP-C1-T vector, an eukaryotic expression plasmid of hMTH1 gene antisense RNA.
METHODSThe conservative region of hMTH1 gene was amplified by RT-PCR after total RNA being extracted from human embryo lung fibroblast (HLF) and then cloned into pGEM-T vector. After the recombinant plasmid was certified by DNA sequencing, the conservative region of hMTH1 gene was inserted into pEGFP-C1 vector reversedly and pEGFP-C1-T vector was constructed. The efficiency of antisense inhibition was verified by Western blotting after cell transfection.
RESULTS423 bp fragment including conservative region of hMTH1 gene was obtained by RT-PCR. After cloned by pGEM-T vector and certified by DNA sequencing, pEGFP-C1-T vector was successfully constructed by means of recombinant DNA technology. Additionally pEGFP-C1-T vector could efficiently decrease hMTH1 protein level by 46%.
CONCLUSIONThe efficient expression vector of hMTH1 gene antisense RNA, pEGFP-C1-T has been constructed successfully.
DNA Repair Enzymes ; Genetic Vectors ; genetics ; Humans ; Phosphoric Monoester Hydrolases ; genetics ; Plasmids ; RNA, Antisense ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction