Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.1.1.37 (DNA methyltransferase)
4,983 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Synthetic oligodeoxyribonucleotide duplexes have been used to study the methylation specificity of M.HpaII, a bacterial DNA methyltransferase. Substrates of four types were compared. A 30-mer containing a Watson-Crick paired CCGG recognition sequence was rapidly methylated at the central cytosine on each strand in the recognition sequence. A 30-mer containing an asymmetrically methylated recognition sequence, of the type transiently produced by DNA replication, was rapidly methylated at the central cytosine on the unmethylated strand. A heteroduplex containing an A.C mispair in the recognition sequence (CCGG/CCAG) was rapidly methylated at the cytosine in the mispair. A heteroduplex containing an A.C and an adjacent C.C mispair in the recognition sequence (CCGG/CCCA) was not methylated at a significant rate. The results show that M.HpaII can tolerate a single mispair at its recognition site in a heteroduplex without loss of activity or specificity.
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PMID:The response of M.HpaII to heteroduplexes. 795 52

O6-Methylguanine DNA methyltransferase (O6-MT) activity and cellular sensitivity to nitrosourea drugs of 10 kinds of tumor cell strains derived from Chinese patients were measured by 3H radioactivity and colony-forming ability, respectively. The results in vitro showed that nimustine (Nim) 25 micrograms.ml-1 and carmustine (Car) 20 micrograms.ml-1 exhibited specific killing effects on Mer-phenotype tumor cells characterized by low O6-MT activity. In vivo both Nim and Car (25 mg.kg-1.wk-1 x 4 wk, ip) had specific curative ability to Mer- tumor cells implanted in nude mice. These findings suggested that assay of O6- MT activity in tumor biopsy could be used as a predictable guide to human tumor chemotherapy with nitrosourea compounds.
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PMID:[Specific curative effects of nitrosourea drugs on tumor cells with Mer--phenotype in Chinese]. 801 61

O6-Methylguanine-DNA methyltransferase (MGMT), an enzyme that repairs adducts at O6 of guanine in DNA, is a major determinant of susceptibility to simple methylating carcinogens or of tumor response to anticancer chloroethylating drugs. To investigate the mechanisms underlying cellular expression of this DNA repair enzyme, we focused on the role of a 59-bp enhancer of the human MGMT gene in the regulation of its expression. By using chloramphenicol acetyltransferase reporter assays, we found that the enhancer activity, which was present in both MGMT-expressing (Mer+) and -deficient (Mer-) cells, correlated with the endogenous MGMT activity in Mer+ cell lines. Band-shift assays and deletion analysis of the 59-bp sequence defined a minimal 9-mer cis element (5'-CTGGGTCGC-3') for specific trans factor binding. The MGMT enhancer binding protein (MEBP), 45 kDa by Southwestern blot analysis, was present in the nuclei of all Mer+ cells tested but was apparently restricted to the cytoplasm of Mer- cells. We conclude that the MEBP-enhancer interaction plays an important role in regulating constitutive MGMT expression in Mer+ cells and that MEBP exclusion from the nucleus may account for the down-regulation of MGMT in Mer- cells.
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PMID:Cytoplasmic sequestration of an O6-methylguanine-DNA methyltransferase enhancer binding protein in DNA repair-deficient human cells. 911 92

Chemically modified phosphorothioate oligodeoxynucleotides (ODNs) have become critical tools for research in the fields of gene expression and experimental therapeutics. Bioanalytical assays were developed that utilized fast anion-exchange high-performance liquid chromatography (HPLC) and capillary gel electrophoresis (CGE) for the determination of 20-mer ODNs in biological fluids (plasma and urine) and tissues. A 20 mer ODN in the antisense orientation directed against DNA methyltransferase (denoted as MT-AS) was studied as the model ODN. The anion-exchange HPLC method employed a short column packed with non-porous polymer support and a ternary gradient elution with 2 M lithium bromide containing 30% formamide. Analysis of the MT-AS is accomplished within 5 min with a detection limit of approximately 3 ng on-column at 267 nm. For plasma and urine, samples were diluted with Nonidet P-40 in 0.9% NaCl and directly injected onto the column, resulting in 100% recovery. For tissue homogenates, a protein kinase K digestion and phenol-chloroform extraction were used, with an average recovery of about 50%. Since the HPLC assay cannot provide one-base separation, biological samples were also processed by an anion-exchange solid-phase extraction and a CGE method to characterize MT-AS and its catabolites of 15-20-mer, species most relevant to biological activity. One base separation, under an electric field of 400 V/cm at room temperature, was achieved for a mixture of 15-20-mer with about 50 pg injected. Assay validation studies revealed that the combined HPLC-CGE methods are accurate, reproducible and specific for the determination of MT-AS and its catabolites in biological fluids and tissue homogenates, and can be used for the pharmacokinetic characterization of MT-AS.
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PMID:Determination of antisense phosphorothioate oligonucleotides and catabolites in biological fluids and tissue extracts using anion-exchange high-performance liquid chromatography and capillary gel electrophoresis. 918 82

The DNA specificity subunit (HsdS) of type I restriction-modification enzymes is composed of two independent target recognition domains and several regions whose amino acid sequence is conserved within an enzyme family. The conserved regions participate in intersubunit interactions with two modification subunits (HsdM) and two restriction subunits (HsdR) to form the complete endonuclease. It has been proposed that the domains of the HsdS subunit have a circular organisation providing the required symmetry for their interaction with the other subunits and with the bipartite DNA target. To test this model, we circularly permuted the HsdS subunit of the type IB R-M enzyme EcoAI at the DNA level by direct linkage of codons for original termini and introduction of new termini elsewhere along the N-terminal and central conserved regions. By analysing the activity of mutant enzymes, two circularly permuted variants of HsdS that had termini located at equivalent positions in the N-terminal and central repeats, respectively, were found to fold into a functional DNA recognition subunit with wild-type specificity, suggesting a close proximity of the N and C termini in the native protein. The wild-type HsdS subunit was purified to homogeneity and shown to form a stable trimeric complex with HsdM, M2S1, which was fully active as a DNA methyltransferase. Gel electrophoretic mobility shift assays revealed that the HsdS protein alone was not able to form a specific complex with a 30-mer oligoduplex containing a single EcoAI recognition site. However, addition of stoichiometric amounts of HsdM to HsdS led to efficient specific DNA binding. Our data provide evidence for the circular organisation of domains of the HsdS subunit. In addition, they suggest a possible role of HsdM subunits in the formation of this structure.
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PMID:The DNA recognition subunit of the type IB restriction-modification enzyme EcoAI tolerates circular permutions of its polypeptide chain. 983 17

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 (alpha and beta) of M. AquI separately. Here we describe the DNA binding properties of M. AquI. The results presented here indicate that the beta 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 beta subunit. The alpha subunit enhances the binding of the beta subunit to DNA specifically and nonspecifically. It is likely that the addition of the alpha subunit to the beta subunit stabilizes the conformation of the beta 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 alpha subunit. These compounds did not have any effect on DNA binding by the beta subunit alone. Using a 30-mer oligodeoxynucleotide substrate containing 5-fluorodeoxycytidine (5-FdC), it was found that the beta 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 alpha subunit to the beta subunit led to the formation of a covalent complex with specific DNA sequence containing 5-FdC.
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PMID:The small subunit of M. AquI is responsible for sequence-specific DNA recognition and binding in the absence of the catalytic domain. 1256 99

DNA methylation is an important cellular mechanism for controlling gene expression. Whereas the mutagenic properties of many DNA adducts, e.g., those arising from polycyclic aromatic hydrocarbons, have been widely studied, little is known about their influence on DNA methylation. We have constructed site-specifically modified 18-mer oligodeoxynucleotide duplexes containing a pair of stereoisomeric adducts derived from a benzo[a]pyrene-derived diol epoxide [(+)- and (-)-r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, or B[a]PDE] bound to the exocyclic amino group of guanine. The adducts, either (+)- or (-)-trans-anti-B[a]P-N(2)-dG (G*), positioned either at the 5'-side or the 3'-side deoxyguanosine residue in the recognition sequence of EcoRII restriction-modification enzymes (5'-...CCA/TGG...) were incorporated into 18-mer oligodeoxynucleotide duplexes. The effects of these lesions on complex formation and the catalytic activity of the EcoRII DNA methyltransferase (M.EcoRII) and EcoRII restriction endonuclease (R.EcoRII) were investigated. The M.EcoRII catalyzes the transfer of a methyl group to the C5 position of the 3'-side cytosine of each strand of the recognition sequence, whereas R.EcoRII catalyzes cleavage of both strands. The binding of R.EcoRII to the oligodeoxynucleotide duplexes and the catalytic cleavage were completely abolished when G was positioned at the 3'-side dG position (5'-...CCTGG*...). When G* was at the 5'-side dG position, binding was moderately diminished, but cleavage was completely blocked. In the case of M.EcoRII, binding is diminished by factors of 5-30 but the catalytic activity was either abolished or reduced 4-80-fold when the adducts were located at either position. Somewhat smaller effects were observed with hemimethylated oligodeoxynucleotide duplexes. These findings suggest that epigenetic effects, in addition to genotoxic effects, need to be considered in chemical carcinogenesis initiated by B[a]PDE, since the inhibition of methylation may allow the expression of genes that promote tumor development.
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PMID:Effects of benzo[a]pyrene-deoxyguanosine lesions on DNA methylation catalyzed by EcoRII DNA methyltransferase and on DNA cleavage effected by EcoRII restriction endonuclease. 1565 62

GEM-231 is an 18-mer hybrid oligonucleotide under development by Hybridon for the potential treatment of cancer. This compound was initially developed for colon cancer [256660], and progressed to phase II trials in October 1998 [301009]. Hybridon initiated a phase I dose-escalation study enrolling up to 25 patients with refractory solid tumors, in January 1998 at the Lombardi Cancer Center, Georgetown University Medical Center [275860]. GEM-231 was well tolerated in multiple, escalating doses, and that high plasma levels could be safely achieved [301009]. In addition to antitumor effects, when used as a single agent in animal tumor models, GEM-231 has also demonstrated potentiation of the effects of certain conventional cytotoxic chemotherapy drugs [230699]. Hybridon is conducting studies of the DNA methyltransferase gene and has identified specific sequences on mRNA as targets for chemically-modified antisense oligonucleotides. Hybridon has synthesized compounds that alter methylation of cultured human cancer cells and inhibit their ability to grow in cell culture and inhibit tumor formation in mice [191303]. The work is being carried out in collaboration with McGill University in Montreal and as part of a joint venture called MethylGene, set up by Hybridon and private investors. GEM-231 and other oligonucleotides were claimed in WO- 09515378. Hybridon has been issued with two US patents, US- 05652355 and US-05562356, claiming chemically advanced, mixed-backbone oligonucleotides. The first claims mixed backbone 'hybrid' oligonucleotides, which are second generation chemistries, comprising an internal segment of modified DNA flanked by segments of modified RNA (2'-O-methyl substituted). The other claims mixed backbone 'inverted hybrid' oligonucleotides, which comprises an internal segment of naturally-linked, 2'-O-substituted RNA flanked by modified DNA segments [257135].
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PMID:GEM-231 (Hybridon). 1604 67

7,8-Dihydro-8-oxoguanine (8-oxoG) is a ubiquitous oxidative DNA lesion resulting from injury to DNA via reactive oxygen species. 8-oxoG lesions may play a role in the formation of aberrant DNA methylation patterns during carcinogenesis. In this study, we assessed the effects of 8-oxoG on methylation and complex formation of nine 30-mer oligodeoxynucleotide duplexes by the catalytic domain of murine Dnmt3a DNA methyltransferase (Dnmt3a-CD). The effects of 8-oxoG on the methylation rate of hemimethylated duplexes varied from a 25-fold decrease to a 1.8-fold increase, depending on the position of the lesion relative to the Dnmt3a-CD recognition site (CpG) and target cytosine (C). The most significant effect was observed when 8-oxoG replaced guanine within the recognition site immediately downstream of the target cytosine. Fluorescence polarization experiments with fluorescein-labeled duplexes revealed that two molecules of Dnmt3a-CD bind per duplex, generating sigmoid binding curves. Duplexes exhibiting the highest apparent binding cooperativity formed the least stable 1:2 complexes with Dnmt3a-CD and were methylated at the lowest rate. Kinetic analyses disclosed the formation of very stable nonproductive enzyme-substrate complexes with hemimethylated duplexes that act as suicide substrates of Dnmt3a-CD. The presence of 8-oxoG within the CpG site downstream of the target cytosine markedly diminished productive versus nonproductive binding. We propose that 8-oxoG located adjacent to the target cytosine interferes with methylation by weakening the affinity of DNA for Dnmt3a-CD, thereby favoring a nonproductive binding mode.
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PMID:Impact of 7,8-dihydro-8-oxoguanine on methylation of the CpG site by Dnmt3a. 1916 Dec 95

(Cytosine-5)-DNA methyltransferase SsoII (M.SsoII) has a long N-terminal region (1-71 residues) preceding the sequence with conservative motifs, which are characteristic for all DNA methyltransferases of such kind. The presence of this region provides M.SsoII capability to act as a transcription regulator in SsoII restriction-modification system. To perform its regulatory function, M.SsoII binds specifically to a 15-mer inverted repeat in the promoter region of SsoII restriction-modification system genes. In the present work, properties of the protein delta(72-379)M.Ecl18kI are studied, which is a deletion mutant of the SsoII-like DNA-methyltransferase M.Ecl18kI and is homologous to M.SsoII N-terminal region. delta(72-379)M.Ecl18kI capability to bind specifically a DNA duplex containing the regulatory site is demonstrated. However, such a binding takes place only in the presence of high protein excess relative to DNA, which could indicate an altered structure in the deletion mutant in comparison with the full-length M.SsoII. Circular dichroism spectroscopy demonstrated that delta(72-379)M.Ecl18kI has a strongly pronounced secondary structure and contains 32% a-helices and 20% beta-sheets. Amino acid sequences alignment of M.SsoII N-terminal region and transcription factors of known spatial structure is made. An assumption is made how alpha-helices and beta-sheets are arranged in M.SsoII N-terminal region.
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PMID:[Secondary structure of SsoII-like (cytosine-5)-DNA methyltransferases N-terminal region determined by circular dichroism spectroscopy]. 2109 Feb 46


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