[Show abstract][Hide abstract] ABSTRACT: MEH IS A CRITICAL BIOTRANSFORMATION ENZYME THAT CATALYZES THE CONVERSION OF XENOBIOTIC EPOXIDE SUBSTRATES INTO MORE POLAR DIOL METABOLITES: it is also capable of inactivating a large number of structurally different molecules. Two polymorphisms affecting enzyme activity have been described in the exon 3 and 4 of the mEH gene. The hypothesis of this study is that inherent genetic susceptibility to a primary brain tumor is associated with mEH gene polymorphisms. The polymorphisms of the mEH gene were determined with PCR-RFLP techniques and 255 Turkish individuals. Our results indicate that the frequency of the mEH exon 4 polymorphism (in controls) is significantly higher than that of primary brain tumor patients (OR = 1.8, 95% CI = 1.0-3.4). This report, however, failed to demonstrate a significant association between mEH exon 3 polymorphism and primary brain tumor susceptibility in this population. Analysis of patients by both histological types of primary brain tumor and gene variants showed no association, although analysis of family history of cancer between cases and controls showed a statistically significant association (χ (2) = 7.0, P = 0.01). Our results marginally support the hypothesis that genetic susceptibility to brain tumors may be associated with mEPHX gene polymorphisms.
[Show abstract][Hide abstract] ABSTRACT: Sulfotransferase 1A1 (SULT1A1) is a member of the sulfotransferase family that plays an important role in the biotransformation of numerous carcinogenic and mutagenic compounds through sulfation. A transition, G to A at position 638, in the SULT1A1 gene, results in the Arg(213)His change. This single nucleotide polymorphism reduces the activity and thermostability of the SULT1A1 enzyme. In the present study, the relationship between the SULT1A1 Arg(213)His polymorphism and prostate cancer was investigated using PCR-RFLP. No significant difference in genotype and allele distribution was noted between the prostate cancer and control populations (P=0.072; P=0.099, respectively). The risk of prostate cancer in individuals carrying the SULT1A1(*)2 allele (His(213) allele) was determined by combining the SULT1A1(*)1/SULT1A1(*)2 (Arg/His(213)) and SULT1A1(*)2/SULT1A1(*)2 (His/His(213)) genotypes. No association was observed between SULT1A1 Arg(213)His polymorphism and prostate cancer incidence (P=0.24; OR, 1.36; 95% CI, 0.84-2.25). However, the His(213) allele was found to increase the risk of prostate cancer by 1.36-fold. In smoker and non-smoker populations, no significant relationship was determined between the prostate cancer and control population (P=0.45; P=0.34, respectively).
Preview · Article · Nov 2011 · Experimental and therapeutic medicine
[Show abstract][Hide abstract] ABSTRACT: Myeloperoxidase (MPO) is a phase I enzyme that can bioactivate many specific procarcinogens, including polycyclic aromatic hydrocarbons and aromatic amines. The MPO gene contains a common single nucleotide polymorphism, for which the -463G>A substitution within the promoter region has been shown to reduce MPO expression and activity. We investigated the association between the MPO -463G>A polymorphism and lung and prostate cancer in a Turkish population. MPO genotypes in the study populations were determined using polymerase chain reaction-based restriction fragment length polymorphism assay. The allelic frequency was significantly different between the cases and controls for lung cancer (p=0.02), but not prostate cancer (p=0.30). No significant difference was noted between the lung and prostate cancer cases and control populations in terms of genotype distribution (p=0.07, p=0.53, respectively). Control groups of lung and prostate cancer were in Hardy-Weinberg equilibrium (p=0.87 and p=0.41, respectively). To determine the protective effect against lung cancer among individuals with the -463A allele, G/A and A/A genotypes were combined. Comparison of the G/G and G/A + A/A genotypes between the lung cancer cases and control groups showed a statistically significant relationship (p=0.032, OR=0.60, 95% CI 0.38-0.95). No gender-specific difference was found in terms of genotype distribution between the lung cancer patients and the controls (female, p=0.20; male, p=0.34). In the case of smokers, a difference in genotype distribution between the lung cancer patients and the controls was statistically significant (p=0.02), although this difference was not statistically significant for non-smokers (p=0.90). Overall, no statistically significant difference was found between the prostate cancer cases and the controls in terms of genotype combination (p=0.46, OR=0.83, 95% CI 0.51-1.36). Additionally, in smokers and non-smokers, no significant relationship was determined between the prostate cancer patients and the control population (p=0.21, p=0.91, respectively). These results suggest that the MPO -463A allele significantly contributes to a protective effect overall and in smokers against lung cancer.
Preview · Article · Jan 2011 · Molecular Medicine Reports
[Show abstract][Hide abstract] ABSTRACT: Polymorphism of AXIN2, a component of Wnt signaling, has been shown to play a role in tumorigenesis and dysregulated in cancer cells. In order to find out if AXIN2 polymorphism is a risk factor for prostate cancer, we analyzed eight polymorphic regions of this gene in 84 patients with prostate cancer and compared the results with 100 healthy controls in a Turkish population using PCR-RFLP methods. The genotype frequencies and risk factors of prostate cancer and control groups were analyzed by Chi-square test. We found a statistically significant result between prostate cancer risk and AXIN2 Intron2-956+16A/G (rs35285779) SNP. The frequency of the homozygous G/G (0%) and heterozygous A/G (18%) genotypes was significantly less in patients with prostate cancer than in healthy controls (7 and 32%, respectively) (P<0.05) for this SNP. When compared with the wild-type A/A genotype of the controls, prostate cancer patients with the A/G and G/G genotype showed reduced risk of cancer; the adjusted odds ratio (OR) for patients with the homozygous G/G genotype was 0.87 (95% CI: 0.81-0.95) and for heterozygous A/G genotype was 0.42 (95% CI: 0.20-0.85). We found no statistically significant association between controls and prostate cancer for other seven SNPs of AXIN2 including Exon1-148 C/T (rs2240308), Exon1-432 T/C (rs2240308), Exon5-1365 G/A (rs9915936), Exon5-1386 C/T (rs1133683), Intron5-1712+19 T/G, Exon7-2062 C/T, and Intron7-2141+73 G/A (rs4072245) (P>0.05). These results suggest that the AXIN2 Intron2 rs35285779 SNP is associated with development of prostate cancer as a protective SNP, while an association between other seven SNPs of the AXIN2 and risk of prostate cancer was not observed.
No preview · Article · Nov 2010 · Medical Oncology
[Show abstract][Hide abstract] ABSTRACT: There is an overwhelming abundance of genetic association studies available in the literature, which can often be collectively difficult to interpret. To address this issue, the Venice interim guidelines were established for determining the credibility of the cumulative evidence. The objective of this report is to evaluate the literature on the association of common glutathione S-transferase (GST) variants (GSTM1 null, GSTT1 null and GSTP1 Ile105Val polymorphism) and lung cancer, and to assess the credibility of the associations using the newly proposed cumulative evidence guidelines.
Information from the literature was enriched with an updated meta-analysis and a pooled analysis using data from the Genetic Susceptibility to Environmental Carcinogens database.
There was a significant association between GSTM1 null and lung cancer for the meta-analysis (meta odds ratio=1.17, 95% confidence interval: 1.10-1.25) and pooled analysis (adjusted odds ratio=1.10, 95% confidence interval: 1.04-1.16), although substantial heterogeneity was present. No overall association between lung cancer and GSTT1 null or GSTP1 Ile105Val was found. When the Venice criteria was applied, cumulative evidence for all associations were considered 'weak', with the exception of East Asian carriers of the G allele of GSTP1 Ile105Val, which was graded as 'moderate' evidence.
Despite the large amounts of studies, and several statistically significant summary estimates produced by meta-analyses, the application of the Venice criteria suggests extensive heterogeneity and susceptibility to bias for the studies on association of common genetic polymorphisms, such as with GST variants and lung cancer.
Full-text · Article · Oct 2010 · Pharmacogenetics and Genomics
[Show abstract][Hide abstract] ABSTRACT: The product of AXIN2, a component of Wnt signalling, plays a role in tumorigenesis and is dysregulated in cancer cells. In order to determine whether the AXIN2 polymorphism is a risk factor for astrocytoma, we analysed eight polymorphic regions of this gene in 100 astrocytoma patients compared to 100 healthy controls in a Turkish population using PCR-RFLP methods. For the Exon1-148 T/C, Exon1-432 C/T, Exon5-1365 G/A, Intron5-1712+19G/T, Exon7-2062 C/T and Intron7-2141+73 G/A SNPs of AXIN2, no significant association between controls and astrocytoma patients was found. For the Exon5-1386 C/T SNP, a statistically significant association between controls and patients was found (p<0.05). For this astrocytoma, patients with the TT genotype showed an increased risk with an OR of 2.92 (adjusted for age, gender and smoking status) (95% CI 1.14-7.47) as compared to the controls with the CC genotype. Our results suggest that AXIN2 SNPs may be associated with astrocytoma.
Preview · Article · Jul 2010 · Molecular Medicine Reports
[Show abstract][Hide abstract] ABSTRACT: Microsomal epoxide hydrolase plays a dual role in the activation and detoxification of carcinogenic compounds. Two polymorphic sites have been described in exons 3 and 4 of the microsomal epoxide hydrolase gene that change tyrosine residue 113 to histidine (Tyr113His) and histidine 139 to arginine (His139Arg), respectively. The exon 3 polymorphism reduces enzyme activity by approximately 50%, whereas the exon 4 polymorphism causes a 25% increase in activity. In the present study, the distribution of these polymorphisms in a Turkish population including 625 unrelated healthy individuals was examined using a PCR-RFLP method. The observed genotype frequencies of microsomal epoxide hydrolase exon 3 were 54, 38 and 8% for Tyr113Tyr, Tyr113His and His113His, respectively. Exon 4 genotype frequencies were found to be 69, 29 and 2% for His139His, His139Arg and Arg139Arg, respectively.
No preview · Article · Jul 2010 · Molecular Medicine Reports
[Show abstract][Hide abstract] ABSTRACT: Polymorphisms in the glutathione S-transferase (GST) family may be associated with increased risk of lung cancer, somatic changes in lung tumour tissue, and survival. We evaluated survival according to GST polymorphism in lung cancer patients.
We studied DNA polymorphisms of 81 primary lung cancer patients at 2 glutathione-related loci: GSTM1, and GSTT1 that encode glutathione S-transferase-mu, and glutathione S-transferase- square. The presences of the GSTM1 and GSTT1 genes were assayed by PCR. Kaplan-Meier with log rank tests, and Cox regression models were applied in the analysis.
The median age of 75 males and 6 females was 60 years. Median survival of the whole population was 8 months. In the first presentation, none of the patients with GSTT1 null genotype but 30 percent of the patients with GSTT1-positive genotype had liver metastasis (p < 0.01) but GSTT1 genotype was not associated with survival. Sputum (p < 0.01) was more common in patients with GSTM1 null genotype. Subjects with the GSTM1-null genotype had shorter survival. Using a Cox proportional hazard model, GSTM1, tumor (T) factor and thoracic irradiation status were identified as independent prognostic factors.
Our preliminary results showed that GSTM1-null genotype was associated with shorter survival.
No preview · Article · Jun 2009 · Cancer Investigation
[Show abstract][Hide abstract] ABSTRACT: Human sulfotransferase 1A1 (SULT1A1), the most expressed isoform of the phenol SULT1 subfamily, is an important member of sulfotransferase superfamily. A transition, G to A at position 638, in SULT1A1 gene, results in Arg(213)His change. This single nucleotide polymorphism reduces the activity and thermostability of SULT1A1 enzyme. Thus, in the present study the relationship between SULT1A1 Arg(213)His polymorphism and lung cancer was investigated. One hundred and six case and 271 control samples were studied using PCR-RFLP. There was no significant difference in genotype and allele distribution between lung cancer and control populations (p = 0.07; p = 0.06, respectively). Compared with the SULT1A1*1/SULT1A1*1 genotype the variant SULT1A1 genotype (SULT1A1*1/SULT1A1*2 or SULT1A1*2/SULT1A1*2) was associated with a significantly increased lung cancer risk in cases (p = 0.027). In male populations, there was no significant difference between case and controls (p = 0.313). In female populations, however, this difference was found to be significant (p = 0.04). In smoker and non-smoker populations, no significant relationship was evident between lung cancer and control population (p = 0.170, p = 0.065, respectively). Statistical analyses of histological types of lung cancer in comparison with the control individuals indicated a significant difference between SULT1A1 Arg(213)His polymorphism and SCC (p = 0.027) and other types of cancer (p = 0.037), except SMCC (p = 0.854).
No preview · Article · Jun 2009 · Cell Biochemistry and Function
[Show abstract][Hide abstract] ABSTRACT: Accumulated evidence suggests that alterations due to mutations or genetic polymorphisms in the AXIN2 tumor suppressor gene, a component of the Wnt signaling pathway, contributes to carcinogenesis. The effect of the AXIN2 exon 1 148 C↷T polymorphism was recently investigated in a Japanese population, but has not been investigated in other populations. Additionally, other common polymorphisms of this gene have not been studied. In the present study, 8 polymorphisms of the AXIN2 gene, including exon 1 148 C↷T, were investigated in a Turkish population of 100 lung cancer patients using PCR-RFLP methods. For the exon 1 432 C↷T, exon 5 1365 G↷A, exon 5 1386 C↷T, intron 5 1712+19 G↷T, exon 7 2062 C↷T and intron 7 2141+73 G↷A single nucleotide polymorphisms of AXIN2, no significant association was found between the controls and the lung cancer patients. For exon 1 148 C↷T, a statistically significant association between the controls and lung cancer patients was found. For this region, lung cancer patients with the TT genotype showed a decreased risk [odds ratio (ORTT) 0.33, 95% confidence interval (CI) 0.12-0.89; p=0.032 (adjusted for age, gender and smoking status)] as compared with the controls with the CC genotype. Concerning histological tumor type, it has been found that exon 1 148 C↷T SNP is associated with a significant decreased risk in squamous cell carcinoma patients (ORTT 0.16; 95% CI 0.03-0.79; p=0.014). Male (ORTT 0.19; 95% CI 0.04-0.77; p=0.015) and smoker (ORTT 0.11; 95% CI 0.01-0.71; p=0.019) lung cancer patients with the TT genotype showed a decreased risk for the same region. Our results suggest that the risk of lung cancer in a Turkish population is related to polymorphisms of the AXIN2 gene.
No preview · Article · Nov 2008 · Molecular Medicine Reports
[Show abstract][Hide abstract] ABSTRACT: To evaluate the roles of CYP1A1 polymorphisms [Ile 462Val and T 6235C (MspI)] and deletion of GSTM1 and GSTT1 in lung cancer development in Asian populations, a pooled analysis was conducted on 13 existing studies included in Genetic Susceptibility to Environmental Carcinogenesis database. This pooled analysis included 1,971 cases and 2,130 controls. Lung cancer risk was estimated as odds ratios (OR) and 95% confidence intervals (95% CI) using unconditional logistic regression model adjusting for age, sex, and pack-year. The CYP1A1 6235C variant was associated with squamous cell lung cancer (TC versus TT: OR, 1.42; 95% CI, 0.96-2.09; CC versus TT: OR, 1.97; 95% CI, 1.26-3.07; P trend = 0.003). In haplotype analysis, 462Val-6235T and Ile-C haplotypes were associated with lung cancer risk with reference to the Ile-T haplotype (OR, 3.41; 95% CI, 1.78-6.53 and OR, 1.39; 95% CI, 1.12-1.71, respectively). The GSTM1-null genotype increased squamous cell lung cancer risk (OR, 1.36; 95% CI, 1.05-1.77). When the interaction was evaluated with smoking, increasing trend of lung cancer risk as pack-year increased was stronger among those with the CYP1A1 6235 TC/CC genotype compared with those with TT genotype (P interaction = 0.001) and with the GSTM1-null genotype compared with the present type (Pinteraction = 0.08, when no genotype effect with no exposure was assumed). These results suggest that genetic polymorphisms in CYP1A1 and GSTM1 are associated with lung cancer risk in Asian populations. However, further investigation is warranted considering the relatively small sample size when subgroup analyses were done and the lack of environmental exposure data other than smoking.
Full-text · Article · Jun 2008 · Cancer Epidemiology Biomarkers & Prevention
[Show abstract][Hide abstract] ABSTRACT: Prostate cancer is the most common cancer among men in many countries. Although the etiology of prostate cancer largely is unknown, both genetic and environmental factors may be involved. Advanced age, androgen metabolism, and heredity-race have been reported to be possible risk factors. On the other hand, several studies indicate that genetic polymorphisms in biotransformation enzymes play a role in prostate cancer development. In this study, association of the prostate cancer risk with genotype frequencies of the Phase I (CYP1A1) and Phase II (GSTM1 and GSTT1) biotransformation enzymes was investigated in 321 Turkish individuals (152 prostate cancer patients and 169 age-matched male controls). The presence or absences of the GSTM1 and GSTT1 genes were determined by a PCR-based method. Genotypes of CYP1A1 were determined by MspI-RFLP. The prevalence of GSTM1 null genotype in the cases was 64 percent, compared to 31 percent in the control group, indicating a strong association (OR = 4.08, 95%CI = 2.50-6.69). No association was observed between either GSTT1 null genotype or CYP1A1 polymorphism and prostate cancer incidence. No statistically significant association was observed between smoking status of the patients and any of the polymorphisms studied. In conclusion, results of this study indicate that only the GSTM1 null genotype may play an important role as a risk factor for prostate cancer development in Turkish population.
Full-text · Article · Mar 2006 · Cancer Investigation
[Show abstract][Hide abstract] ABSTRACT: There is substantial evidence that genetic factors play a role in pre-eclampsia. The aim of this study was to determine whether genetic variability in the encoding of genes for glutathione S-transferase M1 (GSTM1) and glutathione S-transferase T1 (GSTT1) contributes to individual differences in susceptibility to pre-eclampsia, eclampsia, or hemolysis, elevated liver enzymes, and low platelets (HELLP syndrome).
A total of 221 women with pre-eclampsia, eclampsia and HELLP syndrome and 147 healthy female controls were genotyped for GSTM1 and GSTT1 polymorphisms by polymerase chain reaction (PCR). Statistical evaluation of differences in polymorphic rates was carried out using chi(2) analysis.
This study included 140 pre-eclamptic, 33 eclamptic and 48 HELLP syndrome cases and 147 healthy controls. The frequencies for the GSTM1 null genotype were 58%, 45%, and 60% for pre-eclampsia, eclampsia, and HELLP syndrome, respectively, and in controls it was 55%. The distribution of the GSTT1 null genotype was 22%, 21%, and 27% for pre-eclampsia, eclampsia, and HELLP syndrome, respectively, and in controls it was 22%. There was no significant association between GSTM1 and GSTT1 polymorphisms and pre-eclampsia, eclampsia, and HELLP syndrome.
Our data do not support a role for polymorphisms of the GSTM1 and GSTT1 genes in the pathogenesis of pre-eclampsia, eclampsia and HELLP syndrome.
No preview · Article · Jul 2005 · Journal of Obstetrics and Gynaecology Research
[Show abstract][Hide abstract] ABSTRACT: Glutathione S-transferases GSTM1, GSTT1, and GSTP1 are phase II biotransformation enzymes that function on detoxification of a wide range of exogenous agents including carcinogens. It has been shown that genetic variations in these genes play an important role in determining the response of an individual to environmental carcinogens. Some studies revealed a statistically significant association between the polymorphisms in the genes encoding GST enzymes and some cancers, although contrary reports exist. In this study, the association between polymorphisms in these genes and primary brain tumor incidence was investigated in 228 Turkish individuals (75 patients with primary brain tumor and 153 controls). The prevalence of GSTM1 null genotype in the case group was 43%, compared to 24% in the control group, giving an odds ratio (OR) of 2.33 (95% confidence interval CI=1.24-4.39). No association was observed between the GSTT1 or GSTP1 Ile105Val polymorphism and brain tumor incidence. Polymorphisms in GSTM1, GSTT1, and GSTP1 did not show association with histopathologic type of brain tumor (glioma or meningioma). Analysis of the polymorphisms in the studied genes and smoking status of the brain tumor patients revealed no statistically significant association. The presented data clearly suggest a relation between brain tumor incidence with GSTM1 null genotype but not with GSTT1 or GSTP1 gene variants.
No preview · Article · Feb 2005 · Cancer Genetics and Cytogenetics
[Show abstract][Hide abstract] ABSTRACT: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major causes of nosocomial infections in our hospital. Therefore, we aimed to characterize MRSA isolates phenotypically from patients with nosocomial infections at Cumhuriyet University Hospital between December, 1999, and June, 2001, in Sivas by analysis of antibiotic patterns and genotypically using pulsed-field gel electrophoresis (PFGE) and repetitive element sequence-based polymerase chain reaction (rep-PCR). Forty-three nosocomial isolates were collected from various wards. All isolates were resistant to penicillin, tetracycline, oxacillin, and gentamicin. By rep-PCR and by separation of SmaI fragments of genomic DNA using PFGE, one major type (eight subtypes with PFGE) was identified among the strains. This clone was found to be different than some clones such as Iberian, Brazilian, and a major clone that was found in another Turkish University Hospital in Ankara. According to our results, there is a major MRSA clone with a potential to spread in our hospital. Infection control measures should be directed toward restricting the further spread of this clone. Therefore, in accordance with these findings, a surveillance culturing program should be established.
No preview · Article · Feb 2004 · Microbial Drug Resistance
[Show abstract][Hide abstract] ABSTRACT: Glutathione S-transferases are possibly related to the detoxification of many xenobiotics involved in the etiology of cancer. To investigate the role of the glutathione S-transferase M1 deletion (GSTM1-null) in lung cancer, the polymerase chain reaction was used to determine the GSTM1 genotypes of lung cancer patients (n=101) and hospital (n=206) in a Turkish population. The prevalence of the GSTM1-null genotype in the case group was 48%, compared to 18% in the control group, giving an odds ratio (OR) of 4.14 (95% confidence interval [CI]=2.36-7.27). The analysis of patients by histologic type of lung cancer (10% adenocarcinoma, 43% squamous cell carcinoma, 26% small cell carcinoma, and 11% large cell carcinoma) showed no association between histopathologic type of lung cancer and GSTM1-null genotype. When the interaction between the GSTM1-null genotype and smoking status was analyzed, among the 67 smokers, the GSTM1-null genotype was found in 37 (55%) with an OR of 2.58 (95% CI=1.00-6.73) indicating a significant association. However, no association was found between smoking exposure (<30 and > or =30 packs/year) and GSTM1-null genotype. We conclude that, in this study the null GSTM1 genotype is an independent risk factor for the development of lung cancer for Turkish population.
No preview · Article · Oct 2003 · Cancer Genetics and Cytogenetics
[Show abstract][Hide abstract] ABSTRACT: AquI DNA methyltransferase (M · AquI) catalyzes the transfer of a methyl group from S-adenosyl-l-methionine to the C5 position of the outermost deoxycytidine base in the DNA sequence 5′-CCCGGG-3′. M · AquI is a heterodimer in which the polypeptide chain is separated at the junction between the two equivalent structural domains
in the related enzyme M · HhaI. Recently, we reported the subcloning, overexpression, and purification of the subunits (α and β) of M · AquI separately. Here we describe the DNA binding properties of M · AquI. The results presented here indicate that the β subunit alone contains all of the information for sequence-specific DNA
recognition and binding. The first step in the sequence-specific recognition of DNA by M · AquI involves the formation of binary complex with the target recognition domain in conjunction with conserved sequence motifs
IX and X, found in all known C5 DNA methyltransferases, contained in the β subunit. The α subunit enhances the binding of
the β subunit to DNA specifically and nonspecifically. It is likely that the addition of the α subunit to the β subunit stabilizes
the conformation of the β subunit and thereby enhances its affinity for DNA indirectly. Addition of S-adenosyl-l-methionine and its analogues S-adenosyl-l-homocysteine and sinefungin enhances binding, but only in the presence of the α subunit. These compounds did not have any
effect on DNA binding by the β subunit alone. Using a 30-mer oligodeoxynucleotide substrate containing 5-fluorodeoxycytidine
(5-FdC), it was found that the β subunit alone did not form a covalent complex with its specific sequence in the absence or
presence of S-adenosyl-l-methionine. However, the addition of the α subunit to the β subunit led to the formation of a covalent complex with specific
DNA sequence containing 5-FdC.
Full-text · Article · Mar 2003 · Journal of Bacteriology
[Show abstract][Hide abstract] ABSTRACT: AquI DNA methyltransferase, M.AquI, catalyses the transfer of a methyl group from S-adenosyl-L-methionine to the C5 position of the outermost deoxycytidine base in the DNA sequence 5'CYCGRG3'. M.AquI is encoded by two overlapping ORFs (termed alpha and beta) instead of the single ORF that is customary for Class II methyltransferase genes. The structural organization of the M.AquI protein sequence is quite similar to that of other bacterial C5-DNA methyltransferases. Ten conserved motifs are also present in the correct order, but only on two polypeptides. We separately subcloned the genes that encode the alpha and beta subunits of M.AquI into expression vectors. The overexpressed His-fusion alpha and beta subunits of the enzyme were purified to homogeneity in a single step by Nickel-chelate affinity chromatography. The purified recombinant proteins were assayed for biological activity by an in vitro DNA tritium transfer assay. The alpha and beta subunits of M.AquI alone have no DNA methyltransferase activity, but when both subunits are included in the assay, an active enzyme that catalyses the transfer of the methyl group from S-adenosyl-Lmethionine to DNA is reconstituted. We also showed that the beta subunit alone contains all of the information that is required to generate recognition of specific DNA duplexes in the absence of the alpha subunit
No preview · Article · Jun 2002 · Journal of biochemistry and molecular biology
[Show abstract][Hide abstract] ABSTRACT: AquI DNA methyltransferase, M.AquI, catalyses the transfer of a methyl group from S-adenosyl-L-methionine to the C5 position of the outermost deoxycytidine base in the DNA sequence 5'CYCGRG3'. M.AquI is encoded by two overlapping ORFs (termed and ) instead of the single ORF that is customary for Class II methyltransferase genes. The structural organization of the M.AquI protein sequence is quite similar to that of other bacterial C5-DNA methyltransferases. Ten conserved motifs are also present in the correct order, but only on two polypeptides. We separately subcloned the genes that encode the and subunits of M.AquI into expression vectors. The overexpressed His-fusion and subunits of the enzyme were purified to homogeneity in a single step by Nickel-chelate affinity chromatography. The purified recombinant proteins were assayed for biological activity by an in vitro DNA tritium transfer assay. The and subunits of M.AquI alone have no DNA methyltransferase activity, but when both subunits are included in the assay, an active enzyme that catalyses the transfer of the methyl group from S-adenosyl-L-methionine to DNA is reconstituted. We also showed that the subunit alone contains all of the information that is required to generate recognition of specific DNA duplexes in the absence of the subunit.
Full-text · Article · May 2002 · Journal of biochemistry and molecular biology
[Show abstract][Hide abstract] ABSTRACT: Enzyme-catalysed DNA methylation provides an opportunity for the modulation of protein-DNA recognition in biological systems. Recently we have demonstrated that the smaller of the two subunits of the heterodimeric, cytosine-specific DNA methyltransferase, M. AquI, is largely responsible for sequence-specific DNA recognition. Here we present evidence from a series of NMR, fluorescence and circular dichroism spectroscopy experiments that the DNA binding subunit of M. AquI has the characteristics of a molten globule in the absence of the catalytic machinery. In this metastable state this subunit retains its ability to bind DNA in a sequence-specific manner. We believe this finding offers an insight into the structural flexibility which underpins the mechanism of action of these enzymes, and may provide a possible biological role for molten globules in protein function.