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Several reports suggest that CmCWGG methylation tends not to co-exist with mCG methylation in human cells. We have asked whether or not methylation at CCWGG sites can influence CG methylation. DNA from
cells expressing an M.EcoRII–GFP fusion was actively methylated at CCWGG sites. CG methylation as measured by R.HpaII/R.MspI
ratios was unchanged in...
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... R.BstNI- cleaved DNA was cloned as described in Materials and Methods, and multiple transformants were analyzed by sequencing. Characteristics of representative sequences cloned from the R.EcoRII protected DNA area are depicted in Table 1. Only two clones were obtained from the unprotected DNA from the GFP expressors, in agreement with the low level of R.EcoRII protected DNA shown in Figure 5. ...
Similar publications
The prokaryotic DNA(cytosine-5)methyltransferase M.SssI shares the specificity of eukaryotic DNA methyltransferases (CG) and is an important model and experimental tool in the study of eukaryotic DNA methylation. Previously, M.SssI was shown to be able to catalyze deamination of the target cytosine to uracil if the methyl donor S-adenosyl-methionin...
The goal of this study is to elucidate the mechanism of sequence specific DNA recognition and base extrusion by DNA cytosine-5 methyltransferase from Haemophilns aegyptius M.HaeIII. We have solved the crystal structure of the C71S mutant of M.HaeIII in complex with the substrate DNA at 2.4.Å resolution (InC below for brevity). For the first time an...
DNA methyltransferases (DNMTs) represent promising targets for the development of unique anticancer drugs. However, all DNMT inhibitors currently in clinical use are nonselective cytosine analogs with significant cytotoxic side-effects. Several natural products, covering diverse chemical classes, have indicated DNMT inhibitory activity, but these e...
Deamination of 5-methylcytosine in DNA results in T/G mismatches. If unrepaired, these mismatches can lead to C-to-T transition mutations. The very short patch (VSP) repair process in Escherichia coli counteracts the mutagenic process by repairing the mismatches in favor of the G-containing strand. Previously we have shown that a plasmid containing...
In mammals, faithful inheritance of genomic methylation patterns ensures proper gene regulation and cell behaviour, impacting normal development and fertility. Following establishment, genomic methylation patterns are transmitted through S-phase by the maintenance methyltransferase Dnmt1. Using a protein interaction screen, we identify Microprocess...
Citations
... Cell culture and DNA isolation. Human prostate cancer cells (PC3) were cultured as previously described (30). Human prostate stromal cells (HPS-19I) were a gift of Prof. David R. Rowley Baylor University College of Medicine, Houston, TX, USA. ...
... A scanned profile of the molecular weight distribution of purified PC3 DNA separated by capillary electrophoresis was obtained by using a DNA 7500 microfluidic chip (Agilent Technologies). The number average molecular weight of the DNA preparation was determined from the scan as previously described (30). ...
... We estimated the probability that clones could originate from the same location by random shear as follows. Using the method also described in (30) we determined that spontaneous cleavage frequency for the PC3 DNA preparation to be 1/8800bp from the scanned molecular weight profile for purified PC3 DNA used in the preparation of the PC3 DNA library (not shown). ...
Background:
Replication impediments can produce helicase-polymerase uncoupling allowing lagging strand synthesis to continue for as much as 6 kb from the site of the impediment.
Materials and methods:
We developed a cloning procedure designed to recover fragments from lagging strand near the helicase halt site.
Results:
A total of 62% of clones from a p53-deficient tumor cell line (PC3) and 33% of the clones from a primary cell line (HPS-19I) were within 5 kb of a G-quadruplex forming sequence. Analyses of a RACK7 gene sequence, that was cloned multiple times from the PC3 line, revealed multiple deletions in region about 1 kb from the cloned region that was present in a non-B conformation. Sequences from the region formed G-quadruplex and i-motif structures under physiological conditions.
Conclusion:
Defects in components of non-B structure suppression systems (e.g. p53 helicase targeting) promote replication-linked damage selectively targeted to sequences prone to G-quadruplex and i-motif formation.
... Artificial extension of the valuable properties of site-specific DNA methyltransferases may result in the use of new experimental methods and models and are especially prospective for the study of protein-DNA interactions. Up to now only M•EcoRII-GFP fusion protein with CpHpG-type recognition motif (H = A, T, or C) is described and its application for visualization in the nuclei of transformed human cells was reported [9]. ...
Design of the transcriptionally-fused protein M.HhaI-EGFP, composed of bacterial DNA-methyltransferase M.HhaI and enhanced green fluorescent protein (GFP) is described. The mentioned M.HhaI-EGFP was expressed in E. coli ER1821 and purified by affinity chromatography on Ni-NTA agarose. According to expectations M.HhaI-EGFP fused protein retained significant features of corresponding original proteins: the ability to transfer methyl group to the C5 carbon atom of internal cytosine in CGCG site and absorption-emission spectral characteristics. The created transcriptionally-fused protein M.HhaI-EGFP could be used in various experiments in molecular biology.
... Then 4 ml of the PCR reaction was cloned using the TOPO-TA cloning kit (Invitrogen) following the manufacturer's instructions. Plasmids with inserts of the appropriate size were sequenced at the City of Hope DNA Sequencing Lab as previously described (30). ...
... The resulting colonies were inoculated into liquid cultures and plasmid DNA was isolated from each. Plasmids with inserts of the appropriate size were sequenced at the City of Hope DNA Sequencing Lab as previously described (30). ...
5-Aza-2′-deoxycytidine (5azaC-dR) has been employed as an inhibitor of DNA methylation, a chemotherapeutic agent, a clastogen,
a mutagen, an inducer of fragile sites and a carcinogen. However, its effects are difficult to quantify because it rapidly
breaks down in aqueous solution to the stable compound 2′-deoxyriboguanylurea (GuaUre-dR). Here, we used a phosphoramidite
that permits the introduction of GuaUre-dR at defined positions in synthetic oligodeoxynucleotides to demonstrate that it
is a potent inhibitor of human DNA methyltransferase 1 (hDNMT1) and the bacterial DNA methyltransferase (M.EcoRII) and that it is a mutagen that can form productive base pairs with either Guanine or Cytosine. Pure GuaUre-dR was found
to be an effective demethylating agent and was able to induce 5azaC-dR type fragile sites FRA1J and FRA9E in human cells.
Moreover, we report that demethylation associated with C:G → G:C transversion and C:G → T:A transition mutations was observed
in human cells exposed to pure GuaUre-dR. The data suggest that most of the effects attributed to 5azaC-dR are exhibited by
its stable primary breakdown product.
... cytosine extension assay [8] with minor modifications [9] was used to assess global DNA methylation. DNA was extracted from mononuclear cells [10] and genomic DNA (0.75 μg) was digested with an excess of methylation-sensitive HpaII restriction endonuclease (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. ...
DNA methylation is an epigenetic feature that is associated with X chromosome inactivation, genomic imprinting, transcriptional silencing of genes and genomic stability. Folate provides a labile source of methyl groups which may be used for cellular methylation reactions including DNA methylation. The methylenetetrahydrofolate reductase (MTHFR) 677C-->T variant is an important determinant of folate nutriture and may influence DNA methylation. This study sought to assess the influence of the MTHFR C677T genotype on global leukocyte DNA methylation in young (18-45y) Mexican American women (n=43; 14 CC, 12 CT and 17 TT). Subjects consumed a folate restricted diet (135 mug DFE/d) for 7 wk followed by folate treatment with 400 or 800 mug DFE/d for 7 wk. Global leukocyte DNA methylation was assessed via the cytosine extension assay at week 0, week 7 (after folate restriction) and week 14 (after folate treatment). No main effects of MTHFR C677T genotype or folate intake were detected at any time point during the study. However, at the end of folate treatment (wk 14), DNA methylation was lower (P<0.05) in women with the MTHFR 677TT genotype relative to the CT or CC genotype. Because it is unlikely that folate treatment would result in methyl group loss, we suggest that there was a delay in DNA methylation response to folate intake. Overall, these data suggest that the MTHFR 677TT genotype and folate interact to lower global leukocyte DNA methylation patterns in young Mexican American women.
DNA methyltransferases might be used as powerful tools for studies in molecular and cell biology due to their ability to recognize and modify nitrogen bases in specific sequences of the genome. Methylation of the eukaryotic genome using exogenous DNA methyltransferases appears to be a promising approach for studies on chromatin structure. Currently, the development of new methods for targeted methylation of specific genetic loci using DNA methyltransferases fused with DNA-binding proteins is especially interesting. In the present review, expression of exogenous DNA methyltransferase for purposes of in vivo analysis of the functional chromatin structure along with investigation of the functional role of DNA methylation in cell processes are discussed, as well as future prospects for application of DNA methyltransferases in epigenetic therapy and in plant selection.
DNA methylation is an integral part of the mechanism of a remodeling and modification of the chromatin structure. The global
complex net of chromatin modification and remodeling reactions is still to be determined, and studies of the mechanisms controlling
the epigenetic processes of histone modification and DNA methylation are in their infancy. Cytosine methylation occurs predominantly
in CpG sequences of the eukaryotic genome, and it also takes place at symmetric CpHpG and nonsymmetric CpHpH sites (where
H is A, T, or C). The modification efficiency of the three types of DNA methylation sites depends on their genomic localization.
Different regions of the eukaryotic genome are remarkable for their methylation features: CpG-islands, CpG-island shores,
differentially methylated regions of imprinted genes, and regions of nonalternative site-specific modification. The three
canonical sites (CpG, CpHpG, and CpHpH) differ in DNA methylation efficiency depending on their nucleotide context. An epigenetic
code of DNA methylation can be assumed with context differences playing a specific functional role. The review summarizes
the main up-to-date data on the structural and functional features of site-specific cytosine methylation in eukaryotic genomes.
Pathogenesis-related alterations in the methylation pattern of the eukaryotic genome are considered.
Key wordsDNA methylation-repetitive sequences-CpG-CpHpG-CpHpH methylation-sequence contexts-chromatin-carcinogenesis-genomic imprinting
Current data suggest that angiogenesis, smooth muscle cell migration, differentiation and proliferation may be epigenetically regulated. Prokaryotic DNA methyltransferases have been proposed as tools to modify mammalian DNA methylation. In order to assess the impact of DNA hypermethylation on smooth muscle pathophysiology, we expressed an HpaII site-specific methyltransferase transgene in smooth muscle cells in mice. The enzyme is expected to target only a subset (CCGG) of unmethylated CpG dinucleotides, thus avoiding possible deleterious effects of widespread hypermethylation. Transgenics of two independent lines were born at expected frequencies, showed no obvious abnormalities and were fertile. Nevertheless, ~30% of > 1 year-old transgenics developed organomegaly and ~20% showed a range of tumors. Global DNA methylation was unchanged in transgenic tissue whether hyperplastic or normal, but tumor DNA showed a pronounced global hypermethylation. DNA hypermethylation was not indiscriminate, as five tested tumor suppressor genes showed promoter CpG and non-CpG hypermethylation and transcriptional down-regulation, whereas the methylation status of one intergenic CpG islands, repeated elements (n=2) and non-tumor suppressor gene promoters (n=3) was unchanged. Our work is the first report on the effects of HpaII methyltransferase on endogenous chromatin and in a whole animal. Furthermore, our data expand previous findings that imply that global DNA hypomethylation is not an obligate oncogenic pathway at least in the tumor types examined here.
Non-CG methylation is well characterized in plants, where it appears to play a role in gene silencing and genomic imprinting. Although strong evidence for the presence of non-CG methylation in animals has been available for some time, both its origin and function remain elusive. In this review we discuss available evidence on non-CG methylation in animals in light of evidence suggesting that the human stem cell methylome contains significant levels of methylation outside the CG site.
The VNTR at c-Ha-ras resides in a hotspot for DNA methylation on chromosome 11 in human tumors, where it is flanked by two MspI restriction sites. We have investigated the nature of the MspI site polymorphism at the c-Ha-ras VNTR observed in variety of tumors including breast cancer.We find that the MspI site 5' to the VNTR is present in a Non-B DNA structure with single-strand character that renders it accessible to bisulfite modification under native conditions, while the MspI site 3' to the VNTR appears to reside in a normal B-form structure that is inaccessible to bisulfite. The non-B DNA structure accounts for the observed polymorphism since MspI cannot cleave single-stranded DNA and control experiments show that the MspI sites were neither mutated nor abnormally methylated. Southern blotting showed that structural polymorphism was present in tumor DNA and tumor adjacent normal tissue DNA but absent from lymphocyte DNA from the same patients. We conclude that the non-B DNA structural polymorphism detected in human tumors near the c-Ha-ras VNTR is a self-perpetuating epigenetic mark that manifests itself spontaneously during breast carcinogenesis in a methylation hot spot.
