Levels of base excision repair proteins in CD4+ T cells in patients with systemic lupus erythematosus / 中南大学学报(医学版)

OBJECTIVES@#Systemic lupus erythematosus (SLE) is a multi-systemic disease with the unknown pathogenic mechanism. DNA demethylation is involved in SLE pathogenesis. Growth arrest and DNA damage inducible 45 alpha (Gadd45a) takes part in the process of DNA demethylation. Gadd45a is a DNA repair-related protein. This study aims to investigate the expressions of some proteins [including activation-induced cytidine deaminase (AID), thymine DNA glycosylase (TDG), and methyl-CpG-binding domain protein 4 (MBD4)] involving in base excision repair (BER) process in CD4+ T cells in patients with SLE, and to analyze the correlations between the above BER proteins and lupus disease.@*METHODS@#From January 2019 to September 2020, 12 SLE patients and 12 healthy controls were recruited from Second Xiangya Hospital of Central South University. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Hypaque density gradient centrifugation and then CD4+ T cells were isolated via positive selection using Miltenyi beads. We measured the messenger RNA (mRNA) and protein expressions of AID, TDG, and MBD4 by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively.@*RESULTS@#In contrast to controls, in SLE CD4+ T cells, the mRNA and protein expressions of AID were elevated (P=0.003, P=0.022, respectively); TDG protein expression was increased (P=0.017); and MBD4 protein level was reduced (P<0.001). No visible distinctions was found in the mRNA expressions of either TDG or MBD4 between the 2 groups (both P>0.05). The mRNA and protein expressions of AID and the protein levels of TDG were positively correlated with SLE disease activity index (SLEDAI). And the mRNA and protein expressions of MBD4 were negatively correlated with SLEDAI.@*CONCLUSIONS@#In SLE CD4+ T cells, the increased expressions of AID and TDG and the decreased MBD4 expression may participate in SLE pathogenic mechanism.

DNA alkylation lesion repair: outcomes and implications in cancer chemotherapy / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Alkylated DNA lesions, induced by both exogenous chemical agents and endogenous metabolites, represent a major form of DNA damage in cells. The repair of alkylation damage is critical in all cells because such damage is cytotoxic and potentially mutagenic. Alkylation chemotherapy is a major therapeutic modality for many tumors, underscoring the importance of the repair pathways in cancer cells. Several different pathways exist for alkylation repair, including base excision and nucleotide excision repair, direct reversal by methyl-guanine methyltransferase (MGMT), and dealkylation by the AlkB homolog (ALKBH) protein family. However, maintaining a proper balance between these pathways is crucial for the favorable response of an organism to alkylating agents. Here, we summarize the progress in the field of DNA alkylation lesion repair and describe the implications for cancer chemotherapy.

Alterations of DNA damage response pathway: Biomarker and therapeutic strategy for cancer immunotherapy

Genomic instability remains an enabling feature of cancer and promotes malignant transformation. Alterations of DNA damage response (DDR) pathways allow genomic instability, generate neoantigens, upregulate the expression of programmed death ligand 1 (PD-L1) and interact with signaling such as cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signaling. Here, we review the basic knowledge of DDR pathways, mechanisms of genomic instability induced by DDR alterations, impacts of DDR alterations on immune system, and the potential applications of DDR alterations as biomarkers and therapeutic targets in cancer immunotherapy.

Association of Base Excision Repair Gene Polymorphisms with the Response to Chemotherapy in Advanced Non-Small Cell Lung Cancer / 中华医学杂志(英文版)

Background@#Base excision repair (BER) plays an important role in the maintenance of genome integrity and anticancer drug resistance. This study aimed to explore the role of BER gene polymorphisms in response to chemotherapy for advanced non-small cell lung cancer (NSCLC) patients treated with platinum-based chemotherapy.@*Methods@#During the period from November 2009 to January 2016, a total of 152 patients diagnosed with NSCLC Stage IIIB and IV in the First Hospital of Jilin University were admitted into this study. The XRCC1 G28152A, MUTYH G972C, HOGG1 C1245G, and PARP1 T2444C polymorphisms of all the patients were detected by mass spectrometry. The logistic regression was used for statictical analysis. All tests were bilateral test, and a P < 0.05 was considered statistically significant.@*Results@#The logistic regression model showed that the response rate of chemotherapy of the PARP1 T2444C polymorphisms, CC genotype (odds ratio [OR]: 5.216, 95% confidence interval [CI]: 1.568-17.352, P = 0.007), TC genotype (OR: 2.692, 95% CI: 1.007-7.198, P = 0.048), as well as the genotype of TC together with CC (OR: 3.178, 95% CI: 1.229-8.219, P = 0.017) were significantly higher than those of TT wild type. There was no relationship between the MUTYH G972C, XRCC1 G28152A, and HOGG1 C1245G gene polymorphisms and chemosensitivity.@*Conclusions@#The PARP1 2444 mutation allele C might be associated with the decreased sensitivity to platinum-based chemotherapy in advanced NSCLC. These findings may be helpful in designing individualized cancer treatment.

Protective effect of Wuzi Yanzong recipe on testicular DNA oxidative damage in natural ageing rats / 中国中药杂志

To study the protective effects of Wuzi Yanzong recipe on DNA oxidative damage of testis germ cells in natural ageing rats based on Nrf2/HO-1 signaling pathway and base excision repair (BER). In the study, 16-month-old SPF grade male SD rats were randomly divided into three groups, namely ageing model group, and low and high-dose Wuzi Yanzong recipe groups (WZ, 1, 4 g·kg⁻¹). In addition, 2-month-old SD rats were used as adult control group (10 rats in each group). The ageing model group and the adult control group were fed with normal diet for 4 months. WZ groups were given medicated feed for 4 months. After fasting for 12 hours, the rats were put to death. Then, the testes were immediately removed. The vitality of superoxide dismutase (SOD) and malondialdehyde (MDA) content in testis were detected by xanthine oxidase method and thiobarbituric acid (TBA) method. The levels of Nrf2 and 8-OHdG were detected by immunofluorescence. The protein expression levels of Nrf2, HO-1, NQO1, APE1, OGG1 and XRCC1 were detected by Western blot. Compared with the ageing model group, WZ significantly increased the SOD vitality and decreased MDA content of testis. In addition, immunofluorescence results showed that WZ significantly attenuated testicular DNA oxidative damage and improved antioxidant capacity. Such changes were accompanied by the down-regulation of DNA oxidative damage response protein 8-OHdG levels and the up-regulation of Nrf2 levels. Moreover, Western blot results showed that WZ significantly increased the protein expression levels of Nrf2, HO-1 and NQO1 of the testis germ cells, when compared with ageing model group. In parallel, the protein expression levels of APE1, OGG1 and XRCC1 were significantly decreased. In conclusion, WZ improves ageing-related DNA oxidative damage via Nrf2/HO-1 and BER pathways.

Protective effect of Wuzi Yanzong Prescription on testicular DNA damage in natural ageing rats based on P53/P21 and BER signal pathway / 中草药

Objective: To study the protective effect of Wuzi Yanzong Prescription (WYP) on DNA damage in testis of natural ageing rats based on P53/P21 signaling pathway and base excision repair (BER). Methods: SPF grade 16-month-old male SD rats were randomly divided into three groups with eight rats in each group: ageing model group, low and high dose of WYP groups (1 and 4 g/kg). In addition, 2-month-old SD male rats were used as adult control group. The ageing model group and the adult control group were fed with normal diet for four months. Rats in the WYP groups were given the medicated feed for four months. After fasting for 12 h, the rats were sacrificed. Then, the testes were immediately removed from rats. The expression and localization of DNA damage-related protein γH2AX and BER-related proteins OGG1, APE1, and XRCC1 were detected by immunofluorescence, and the content of 8-OHdG in testis was detected by ELISA. Also, the protein expression levels of p-P53 and P21 were detected by Western blotting. Results: Compared with the ageing model group, immunofluorescence results showed WYP significantly decreased the expression levels of DNA damage-related protein γH2AX and BER-related proteins OGG1, APE1, and XRCC1 in testis of natural ageing rats. ELISA results showed that WYP significantly downregulated 8-OHdG levels, compared with the ageing model group. Moreover, Western blotting results showed that WYP significantly decreased the protein expression levels of p-P53 and P21 of the testis when compared with the ageing model group. Conclusion: WYP reduced the DNA damage of testes in ageing related rats via P53/P21 and BER pathways.

Role of H4K20me1 and base excision repair-related genes in repairing DNA damage induced by arsenic pollution through coal burning / 中华地方病学杂志

Objective To investigate the global level of histone 4 lysine 20 monomethylation (H4K20me1) and expression of base excision repair related mRNA in coal-burning-borne endemic arsenism patients and to analyze its relationship with DNA damage,in order to provide a scientific basis in deepening the interpretation of the role of arsenic in inhibiting repair of DNA damage.Methods In 2014,47 hair samples,blood samples and skin tissue samples of the cases in Xingren County Guizhou Province were collected from the voluntary surgical treatment patients with endemic arsenism (15 general pathological change cases,14 precancerous cases and 18 cancerous cases) and 12 controls.The hair arsenic content was tested via the inductively coupled plasma-mass spectrometry method.The expression of histone H4K20me1 in skin tissues was detected via the immunohistochemistry method;quantitative real-time polymerase chain reaction was used to detect the mRNA levels of poly (ADP-ribose) polymerase (PARP1),N-methylation of purine-DNA-glycosylation (MPG) and X-ray repair cross complementary gene 1 (XRCC1);and DNA damage in peripheral blood was detected by single cell gel electrophoresis test,the level of H4K20me1 in peripheral blood cells was detected by using a sandwich enzyme-linked immunosorbent assay.Results Compared with the control group [median (25 ～ 75 percentile):0.15 (0.07-0.23) μg/L],the hair arsenic content in the case group [0.34 (0.17-0.51) μg/L] was significantly increased (Z =6.037,P ＜ 0.05).Compared with the control group (0.32 ± 0.13,0.17 ± 0.12),the modification level of H4K20me1 in peripheral blood with cancerous group (0.62 ± 0.11) was significantly increased,the modification levels of H4K20me1 (0.54 ± 0.20,0.83 ± 0.10) in skin tissues were increased in the precancerous group and cancerous group (P ＜ 0.05).Compared with the control group [0.95 (0.50-1.49),1.12 (0.98-1.48),0.96 (0.67-1.17)],the mRNA expression levels of PARP1 and MPG in cancerous group [0.37 (0.30-0.44),0.38 (0.15-0.48)] were significantly decreased;the mRNA expression levels of XRCC1 [0.48 (0.38-0.89),0.32 (0.20-0.55)] were significantly decreased in the precancerous group and cancerous group (P ＜ 0.05).Compared with the control group (1.19 ± 0.55,1.27 ± 0.51),Tail DNA％ and Tail moment were significantly increased in the general pathological change group (6.49 ± 0.98,6.60 ± 1.11),the precancerous lesion group (11.22 ± 1.40,10.07 ± 1.11),and the cancerous group (20.38 ± 1.72,27.01 ± 1.78,P ＜ 0.05).There was a positive correlation between the degree of skin lesion and modification level of H4K20me1 in peripheral blood and skin tissues and DNA damage levels (TailDNA％,OTM,r =0.885,0.855,0.806,0.883,P ＜ 0.05).There was a positive correlation between modification level of H4K20me1 in peripheral blood and level of DNA damage (TailDNA％,OTM),the level of H4K20me1 protein expression in skin (r =0.535,0.804,0.754,P ＜ 0.05),and a negative correlation with mRNA expression of MPG,XRCC1 and PARP1 (r =-0.563,-0.514,-0.550,P ＜ 0.05).There was a positive correlation between the modification level of H4K20me1 in skin and DNA damage levels (TailDNA％,OTM,r =0.602,0.875,P ＜ 0.05),and a negative correlation with mRNA expression of MPG,XRCC1 and PARP1 (r =-0.492,-0.502,-0.552,P ＜ 0.05).Conclusion The arsenic pollution of coal burning may affect the level of H4K20me1 modification,inhibit mRNA transcriptional expression of PARP1,MPG and XRCC1 genes related with base excision repair,which may lead to increased DNA damage and participate in the occurrence and development of arsenic poisoning skin lesions.

Base excision repair and targeted therapy strategies / 国际肿瘤学杂志

Base excision repair (BER) is an important pathway for DNA oxidative damage repair,and mutations in this pathway cause gene instability and increase cancer risk.A large number of studies have shown that abnormal expression of key proteins in the BER pathway in a variety of tumors,such as DNA glycosylase,apurinic/apyrimidinic endonuclease 1,DNA polymerase and so on.Single nucleotide polymorphisms of these proteins affect the repair activity of tumor cells,and are expected to be important indicators for the diagnosis,treatment and evaluation of prognosis.

Updates on OGG1 gene / 国际儿科学杂志

OGG1 gene is widely expressed in eukaryotic cells as a housekeeping gene,which can identi-fy 8-oxoG,oxidative damage product,and remove it via the base excision repair pathway to repair DNA and pro-tect it from mutation. Increasing attention is paid to investigate the OGG1 gene,which is found to play an impor-tant role in tumorigenesis,aging,degenetative diseases,cardiomyopathy,and allergic diseases. Insight of the ex-pression of OGG1 and its regulatory mechanism can bring us a new way to treat these diseases.

Updates on OGG1 gene / 国际儿科学杂志

OGG1 gene is widely expressed in eukaryotic cells as a housekeeping gene,which can identi-fy 8-oxoG,oxidative damage product,and remove it via the base excision repair pathway to repair DNA and pro-tect it from mutation. Increasing attention is paid to investigate the OGG1 gene,which is found to play an impor-tant role in tumorigenesis,aging,degenetative diseases,cardiomyopathy,and allergic diseases. Insight of the ex-pression of OGG1 and its regulatory mechanism can bring us a new way to treat these diseases.

Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents

During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins.

The role of polymerase chain reaction with confronting two-pair primers in SNP genotyping of DNA base excision repair genes / 重庆医学

Objective It is important to precisely determinate the single nucleotide polymorphisms (SNPs) in many genes in‐cluding genes related with base excision repair (BER) pathway .This research is conducted to evaluate the role of polymerase chain reaction with confronting two‐pair primers (PCR‐CTPP) in analyzing the SNPs of BER pathway .Methods Four common SNPs of BER pathway (OGG1 Ser326Cys ,XRCC1 Arg399Gln ,APE1 Asp148Glu and‐141T/G in the promoter region) was detected with PCR‐CTPP .10 of the products were sent for genotype sequencing .Compare the results of PCR and sequencing to evaluate the accu‐racy of PCR‐CTPP .Results The genotypes were exactly the same as the sequencing .Conclusion The PCR‐CTPP was a reliable and rapid detective technology for SNPs genotyping .Its broadest application would be great help for gene variant analysis .

Studying on BER pathways ofXRCC1 site SNP and laryngeal cancer susceptibility of different nations in Xinjiang / 中国癌症杂志

Background and purpose:Major repair genes that affect the tumor genetic susceptibility exists in repair pathways base excision repair (BER) approach, X-ray repair cross complementing group 1(XRCC1) gene, respectively is the core of BER pathway. At home and abroad in recent years, has carried out many studies of genetic polymorphism and laryngeal cancer susceptibility. Researching on the base excision repair (BER) pathway of DNA repair geneXRCC1 bases mononuclear nucleotide polymorphism and the relationship between different ethnic groups laryngeal cancer susceptibility in xinjiang.Methods:A case-control study was performed on 58 patient with laryngeal squamous cell carcinoma and 120 random healthy control group. Multiplex SNaPshot technic was used to detect DNA base excision repair geneXRCC1 Gln632Gln (rs3547), Arg399Gln (rs25487), Arg280His (rs25489), Arg194Trp (rs1799782) loci single nucleotide polymorphism distribution in the case group and normal control group.Results:Three sites of the rest of the cases ofXRCC1 Gln632Gln (rs3547) C/T (hybrid) and T/T (mutant) genotype, Arg399Gln (rs25487) C/T (hybrid) and T/T (mutant) genotype, Arg194Trp (rs1799782) G/A (hybrid) and A/A (mutant) genotype is notably higher than that of control group (P<0.01). Gln632Gln (rs3547) C/T (hybrid) and T/T (mutant) genotype, Arg399Gln (rs25487) C/T (hybrid) and T/T (mutant) genotype, Arg194Trp (rs1799782) G/A (hybrid) and A/A (mutant) genotype ratio is signiifcantly higher than control group (P<0.05) in cases of Han,Uygur and Kazakh nations, carrying (rs3547) C/T and T/T genotype, (rs25487) C/T and T/T genotype, (rs1799782) G/A and A/A genotype individual risk of laryngeal squamous cell carcinoma are added to the 0.96, 1.74 and 1.39 times; 1.47, 1.32 and 0.77 times; 1.49, 1.51 and 1.56 times thanXRCC1 (rs3547) C/C genotype, (rs25487) C/C genotype, (rs1799782) G/G genotype.Conclusion:In the 3 nations,XRCC1 Gln632Gln, Arg399Gln, Arg280His and Arg194Trp loci polymorphism may be associated with laryngeal cancer genetic and there are differences,XRCC1 Gln632Gln, Arg399Gln, Arg194Trp locus mutation will lead to an elevated risk of throat cancer.XRCC1 Arg280His locus mutation has no statistically signiifcant difference with the onset of throat cancer, may have nothing to do with the onset of laryngeal cancer on the site of mutation.

APE1/Ref-1 as an emerging therapeutic target for various human diseases: phytochemical modulation of its functions

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional enzyme involved in the base excision repair (BER) pathway, which repairs oxidative base damage caused by endogenous and exogenous agents. APE1 acts as a reductive activator of many transcription factors (TFs) and has also been named redox effector factor 1, Ref-1. For example, APE1 activates activator protein-1, nuclear factor kappa B, hypoxia-inducible factor 1alpha, paired box gene 8, signal transducer activator of transcription 3 and p53, which are involved in apoptosis, inflammation, angiogenesis and survival pathways. APE1/Ref-1 maintains cellular homeostasis (redox) via the activation of TFs that regulate various physiological processes and that crosstalk with redox balancing agents (for example, thioredoxin, catalase and superoxide dismutase) by controlling levels of reactive oxygen and nitrogen species. The efficiency of APE1/Ref-1's function(s) depends on pairwise interaction with participant protein(s), the functions regulated by APE1/Ref-1 include the BER pathway, TFs, energy metabolism, cytoskeletal elements and stress-dependent responses. Thus, APE1/Ref-1 acts as a 'hub-protein' that controls pathways that are important for cell survival. In this review, we will discuss APE1/Ref-1's versatile nature in various human etiologies, including neurodegeneration, cancer, cardiovascular and other diseases that have been linked with alterations in the expression, subcellular localization and activities of APE/Ref-1. APE1/Ref-1 can be targeted for therapeutic intervention using natural plant products that modulate the expression and functions of APE1/Ref-1. In addition, studies focusing on translational applications based on APE1/Ref-1-mediated therapeutic interventions are discussed.

Base excision repair and antineoplastic drug resistance / 肿瘤

Chemotherapy is one of the main methods to treat malignant tumors in clinical practice. Resistance to antineoplastic agents is one of the important reasons for treatment failure. The antineoplastic mechanism of various chemotherapeutic agents is to cause DNA damage, then result in apoptosis of tumor cells. It is suggested that the function of DNA repair is directly associated with the efficacy of antineoplastic agents. Current studies suggest that there are four major DNA repair pathways including BER (base excision repair), NER (nucleotide excision repair), MMR (mismatch repair) and DSBR (double strand break repair). Of these four pathways, BER is one of the main mechanisms of DNA repair and its malfunction is closely related to the resistance to antineoplastic agents. Recently, many kinds of agents and strategies targeting BER have been developed to reverse chemoresistance. This review summarizes the progress in research in this area and discusses the mechanism of resistance to antineoplastic agents and the potential preventive and therapeutic strategies. Copyright © 2013 by TUMOR.

Distribution of XRCC1 genotypes in north Indian population.

Background & objectives: XRCC1, a major DNA repair gene, acts as a scaffold of different activities involved in repair by interacting with components of base excision repair (BER) at the site of damage. Polymorphisms in this gene are associated with variations in the repair efficiency which might predispose an individual to cancer risk. To associate a gene polymorphism with disease risk, it is imperative to have the data for its genotype distribution in normal population. The present study was therefore carried out to find distribution of XRCC1 polymorphisms (codons 194, 280 and 399) in normal north Indian population. Methods: Healthy volunteers hailing from north India (150) were enrolled in the study. DNA was isolated from blood samples and genotyping of codons 194, 280 and 399 of XRCC1 gene was done by PCRrestriction fragment length polymorphism (RFLP), using specific primers. Results: The frequencies obtained for heterozygous genotype of codons 194 and 399 were 45 and 49 per cent respectively and were higher than wild and variant genotypes. For codon 280, the highest frequency (59%) was obtained for the wild genotype. Frequencies of the variant genotypes of codons 194 and 399 were higher in males and females respectively. The allele frequencies also followed the similar trends. Interpretation & conclusions: A significant distribution of variant and heterozygous XRCC1 genotypes was noticed that warrants further studies on the association between these genotypes and disease risk in our study population.

Participação de enzimas de reparo por excisão de bases na restauração de lesões induzidas pela associação da radiação ultravioleta A e cloreto estanoso em Escherichia coli / Participation of repair enzyme for base excision in lesions restoration induced by the combination of A ultraviolet radiation and stannous chloride in Escherichia coli

O cloreto estanoso (SnCI2) e a radiação ultravioleta A (UVA) são agentes que lesam diversas estruturas celulares, inclusive o DNA, principalmente pela geração de espécies reativas de oxigênio. O objetivo deste trabalho foi estudar a mutagênese e o reparo das lesões produzidas pela combinação do UVA, na condição de pré-iluminação, com o SnCI2. Avaliou-se a ação de enzimas do reparo por excisão de bases (BER), em Escherichia coli (E. coli), por eletroforese em gel alcalino de agarose e sobrevivência bacteriana. Também se estudou a indução do sistema SoxRS pelo cromoteste, e a mutagênese pelo teste de Ames. De acordo com os resultados: i)o UVA induziu quebras no DNA das cepas testadas e os mutantes fpg-nfo e fpg apresentaram maior retardo no reparo das lesões; ii) o SnCI2 induziu mais quebras que o UVA e os mutantes nfo e fpg mostraram maior dificuldade em reparar as lesões; iii) o UVA+SnCI2 provocou mais quebras que o SnCI2 e os mutantes nfo e fpg também apresentaram maior lentidão no reparo das lesões; iv) o UVA não inativou as cepas testadas; v) as cepas nfo e fpg foram as mais sensíveis ao SnCI2; vi) o UVA+SnCI2 provocou maior letalidade em todas as cepas testadas, em relação ao SnCI2, e os mutantes nfo e fpg também foram os mais sensíveis ao tratamento com ambos os agentes; vii) a transformação dos mutantes nfo com o plasmídio pBW21 (nfo+) e dos mutantes fpg com o plasmídio pFPG (fpg+) aumentou a sobrevivência das cepas aos tratamentos com SnCI2 e UVA+SnCI2; viii) o SnCI2 induziu o sistema SoxRS; ix) o SnCI2, UVA e UVA+SnCI2 não induziram mutagênese; x) o reparo das lesões parece ser preferencialmente realizado pelas proteínas Fpg e Nfo.

Stannous chloride (SnCI2) and ultraviolet radiation A (UVA) are able to induce lesions in different cellular structures, including DNA, manly through ROS generation. The aim of this work was to study the mutagenesis and repair of lesions induced by the association of UVA (pre treatment) with SnCI2. It was evaluated the action of base excision repair (BER) enzymes in Escherichia coli (E. coli) by alkaline gel electrophoresis and bacterial survival. It was also evaluated the SoxRS system induction by chromotest and mutagenesis through the Ames test. According to the results: i) UVA induced DNA strand breaks in all strains and fpg-nfo and fpg mutants showed greater delay in the repair of lesions; ii) SnCI2 induced more breaks than UVA and nfo and fpg mutants showed more difficult to repair the damage; iii) UVA + SnCI2 caused more breaks than the SnCI2 and nfo and fpg mutants also showed a slowest repair of injuries; iv) UVA did not inactivate any bacterial strains tested; v) nfo and fpg strains were more sensitive to SnCI2; vi) UVA + SnCI2 caused higher mortality in all strains tested, when compared to SnCI2, and, again, nfo and fpg mutants were the most sensitives to the treatment with both agents; vii) the transformation of nfo mutant with the plasmid pBW21 (nfo+) and fpg mutants with plasmid pFPG (fpg+) increased the survival of the strains to SnCI2 and UVA + SnCI2 treatments; viii) SnCI2 was able to induce SoxRS system; ix) SnCI2, UVA + SnCI2 and UVA did not induce mutagenesis; x) damage repair seems to be preferentially performed by Fpg and Nfo proteins.

Alkaline gel electrophoresis assay to detect DNA strand breaks and repair mechanisms in Escherichia coli

Reactive oxygen species (ROS) can induce lesions in different cellular targets, including DNA. Stannous chloride (SnCl2) is a ROS generator, leading to lethality in Escherichia coli (E. coli), with the base excision repair (BER) mechanism playing a role in this process. Many techniques have been developed to detect genotoxicity, as comet assay, in eukaryotic cells, and plasmid DNA agarose gel electrophoresis. In this study, an adaptation of the alkaline gel electrophoresis method was carried out to ascertain the induction of strand breaks by SnCl2 in bacterial DNA, from E. coli BER mutants, and its repair pathway. Results obtained show that SnCl2 was able to induce DNA strand breaks in all strains tested. Moreover, endonuclease IV and exonuclease III play a role in DNA repair. On the whole, data has shown that the alkaline gel electrophoresis assay could be used both for studying DNA strand breaks induction and for associated repair mechanisms.

Espécies reativas de oxigênio (ERO) podem induzir lesões em diferentes alvos celulares, incluindo o DNA. O cloreto estanoso (SnCl2) é um gerador de ERO que induz letalidade em E. coli, sendo o reparo por excisão de bases (BER) um mecanismo importante neste processo. Técnicas como o ensaio cometa (em eucariotos) e a eletroforese de DNA plasmidial em gel de agarose têm sido utilizadas para detectar genotoxicidade. No presente estudo, uma adaptação do método de eletroforese em gel alcalino de agarose foi usada para verificar a indução de quebras, pelo SnCl2, no DNA de E. coli, bem como a participação de enzimas do BER na restauração das lesões. Os resultados mostraram que o SnCl2 induziu quebras no DNA de todas as cepas testadas. Além disso, endonuclease IV e exonuclease III estão envolvidas na reparação dos danos. Em resumo, os dados obtidos indicam que a metodologia de eletroforese em gel alcalino de agarose pode ser empregada tanto para o estudo de quebras no DNA, quanto para avaliação dos mecanismos de reparação associados.

Role of polymerase ? in DNA metabolism and genomic instability / 中国病理生理杂志

The major role of DNA polymerase ? was thought to be limited in its involvement in short patch base excision repair by removing 5'-deoxyribose phosphate and base insertion. However, the recent researches indicate that polymerase ? might take part in a wide spectrum of DNA metabolism reactions, including long patch base excision repair, DNA replication, recombination, meiosis and transleisional DNA synthesis. Because of its wide and important cellular function, an inappropriate intracellular polymerase ? level might be associated with genomic instability. Down-regulation or mutation of polymerase ? is mutagenic due to deficient in DNA repair, while overexpression of this error-prone ? polymerase might perturb the normal function of other accurate polymerases and cause genomic instability as well.