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Query: UNIPROT:P06889 (Mol)
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Observations made with Escherichia coli have suggested that a lag between replication and methylation regulates initiation of replication. To address the question of whether a similar mechanism operates in mammalian cells, we have determined the temporal relationship between initiation of replication and methylation in mammalian cells both at a comprehensive level and at specific sites. First, newly synthesized DNA containing origins of replication was isolated from primate-transformed and primary cell lines (HeLa cells, primary human fibroblasts, African green monkey kidney fibroblasts [CV-1], and primary African green monkey kidney cells) by the nascent-strand extrusion method followed by sucrose gradient sedimentation. By a modified nearest-neighbor analysis, the levels of cytosine methylation residing in all four possible dinucleotide sequences of both nascent and genomic DNAs were determined. The levels of cytosine methylation observed in the nascent and genomic DNAs were equivalent, suggesting that DNA replication and methylation are concomitant events. Okazaki fragments were also demonstrated to be methylated, suggesting that the rapid kinetics of methylation is a feature of both the leading and the lagging strands of nascent DNA. However, in contrast to previous observations, neither nascent nor genomic DNA contained detectable levels of methylated cytosines at dinucleotide contexts other than CpG (i.e., CpA, CpC, and CpT are not methylated). The nearest-neighbor analysis also shows that cancer cell lines are hypermethylated in both nascent and genomic DNAs relative to the primary cell lines. The extent of methylation in nascent and genomic DNAs at specific sites was determined as well by bisulfite mapping of CpG sites at the lamin B2, c-myc, and beta-globin origins of replication. The methylation patterns of genomic and nascent clones are the same, confirming the hypothesis that methylation occurs concurrently with replication. Interestingly, the c-myc origin was found to be unmethylated in all clones tested. These results show that, like genes, different origins of replication exhibit different patterns of methylation. In summary, our results demonstrate tight coordination of DNA methylation and replication, which is consistent with recent observations showing that DNA methyltransferase is associated with proliferating cell nuclear antigen in the replication fork.
Mol Cell Biol 1998 Jun
PMID:Concurrent replication and methylation at mammalian origins of replication. 958 87

Mammalian DNA cytosine-C5 methyltransferase modifies the CpG dinucleotide in the context of many different genomic sequences. A rigorous DNA binding assay was developed for the murine enzyme and used to define how sequences flanking the CpG dinucleotide affect the stability of the enzyme:DNA complex. Oligonucleotides containing a single CpG site form reversible 1:1 complexes with the enzyme that are sequence-specific. A guanine/cytosine-rich 30 base-pair sequence, a mimic of the GC-box cis-element, bound threefold more tightly than an adenine/thymine-rich sequence, a mimic of the cyclic AMP responsive element. However, the binding discrimination between hemi- and unmethylated forms of these DNA substrates was small, as we previously observed at the K(m)DNA level (Biochemistry, 35, 7308-7315 (1996)). Single-stranded substrates are bound much more weakly than double-stranded DNA forms. An in vitro screening method was used to select for CpG flanking sequence preferences of the DNA methyltransferase from a large, divergent population of DNA substrates. After five iterative rounds of increasing selective pressure, guanosine/cytosine-rich sequences were abundant and contributed to binding stabilization for at least 12 base-pairs on either side of a central CpG. Our results suggest a read-out of sequence-dependent conformational features, such as helical flexibility, minor groove dimensions and critical phosphate orientation and mobility, rather than interactions with specific bases over the course of two complete helical turns. Thus, both studies reveal a preference for guanosine/cytosine deoxynucleotides flanking the cognate CpG. The enzyme specificity for similar sequences in the genome may contribute to the in vivo functions of this vital enzyme.
J Mol Biol 1998 May 29
PMID:DNA binding discrimination of the murine DNA cytosine-C5 methyltransferase. 963 3

The immune response to pathogens is regulated by a delicate balance of cytokines. The dysregulation of cytokine gene expression, including interleukin-12, tumor necrosis factor alpha, and gamma interferon (IFN-gamma), following human retrovirus infection is well documented. One process by which such gene expression may be modulated is altered DNA methylation. In subsets of T-helper cells, the expression of IFN-gamma, a cytokine important to the immune response to viral infection, is regulated in part by DNA methylation such that mRNA expression inversely correlates with the methylation status of the promoter. Of the many possible genes whose methylation status could be affected by viral infection, we examined the IFN-gamma gene as a candidate. We show here that acute infection of cells with human immunodeficiency virus type 1 (HIV-1) results in (i) increased DNA methyltransferase expression and activity, (ii) an overall increase in methylation of DNA in infected cells, and (iii) the de novo methylation of a CpG dinucleotide in the IFN-gamma gene promoter, resulting in the subsequent downregulation of expression of this cytokine. The introduction of an antisense methyltransferase construct into lymphoid cells resulted in markedly decreased methyltransferase expression, hypomethylation throughout the IFN-gamma gene, and increased IFN-gamma production, demonstrating a direct link between methyltransferase and IFN-gamma gene expression. The ability of increased DNA methyltransferase activity to downregulate the expression of genes like the IFN-gamma gene may be one of the mechanisms for dysfunction of T cells in HIV-1-infected individuals.
Mol Cell Biol 1998 Sep
PMID:Infection with human immunodeficiency virus type 1 upregulates DNA methyltransferase, resulting in de novo methylation of the gamma interferon (IFN-gamma) promoter and subsequent downregulation of IFN-gamma production. 971 Jun 1

Mutants that show reduced DNA methylation were identified in a mutant screen based on the assumptions that (i) the nucleoside analog 5-azacytidine (5-azaC) promotes the formation of potentially lethal DNA-methyltransferase adducts; (ii) reduction in DNA methyltransferase will decrease the sensitivity of cells to 5-azaC; and (iii) this potential selective advantage will be enhanced in mutants that are deficient in the repair of 5-azaC-induced DNA damage. Of fifteen potential repair mutants screened for sensitivity to 5-azaC, five (mus-9, mus-10, mus-11, mus-18, and uvs-3) showed moderately increased sensitivity and two (mus-20, mei-3) showed highly increased sensitivity. A mus-20 mutation was used to isolate three non-complementing methylation mutants. The mutations, named dim-1 (defective in methylation), reduced female fertility, reduced methylation by 40-50%, and altered patterns of methylation. In wild-type strains hypomethylation per se fails to alter methylation specificity. We demonstrate a growth-phase-dependent change in methylation patterns, detectable only in hypomethylated DNA from dim+ cultures. This may represent a growth-phase-dependent change in the relative amounts of distinct species of methyltransferase, one of which may be encoded by the dim-1 gene.
Mol Gen Genet 1998 Jul
PMID:Mutations in the dim-1 gene of Neurospora crassa reduce the level of DNA methylation. 973 81

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.
J Mol Biol 1998 Dec 11
PMID:The DNA recognition subunit of the type IB restriction-modification enzyme EcoAI tolerates circular permutions of its polypeptide chain. 983 17

DNA methyltransferases flip their target base out of the DNA helix. Here, we have investigated base flipping by wild-type EcoRV DNA methyltransferase (M.EcoRV) and five M.EcoRV variants (D193A, Y196A, S229A, W231R and Y258A). These variants bind to DNA and S-adenosylmethionine but have a severely reduced catalytic efficiency or are catalytically inactive. To measure base flipping three different assays were used, viz. analysis of the yields of photocrosslinking reactions between the enzymes and a substrate in which the target base is replaced by 5-iodouracil, analysis of the binding constants to substrates containing a mismatch base-pair at the target position and analysis of the salt dependence of specific complex formation. Our data show that the Y196A, W231R and Y258A variants are not able to stabilize a flipped target base, suggesting that the aromatic amino acid residues (Tyr196, Trp231 and Tyr258) are involved in hydrophobic interactions with the flipped base. The D193A variant behaves like wild-type M.EcoRV with respect to base flipping. The fact that this variant is catalytically inactive indicates that Asp193 has a function in chemical catalysis. The S229A variant can better flip modified bases but does not tightly lock the flipped base into the adenine-binding pocket, suggesting that Ser229 could form a contact to the flipped adenine.
J Mol Biol 1999 Jan 22
PMID:Mutational analysis of target base flipping by the EcoRV adenine-N6 DNA methyltransferase. 991 20

CpG island hypermethylation is known to be associated with gene silencing in cancer. This epigenetic event is generally accepted as a stochastic process in tumor cells resulting from aberrant DNA methyltransferase (DNA-MTase) activities. Specific patterns of CpG island methylation could result from clonal selection of cells having growth advantages due to silencing of associated tumor suppressor genes. Alternatively, methylation patterns may be determined by other, as yet unidentified factors. To explore further the underlying mechanisms, we developed a novel array-based method, called differential methylation hybridization (DMH), which allows a genome-wide screening of hypermethylated CpG islands in tumor cells. DMH was used to determine the methylation status of >276 CpG island loci in a group of breast cancer cell lines. Between 5 and 14% of these loci were hypermethylated extensively in these cells relative to a normal control. Pattern analysis of 30 positive loci by Southern hybridization indicated that CpG islands might differ in their susceptibility to hypermethylation. Loci exhibiting pre-existing methylation in normal controls were more susceptible to de novo methylation in these cancer cells than loci without this condition. In addition, these cell lines exhibited different intrinsic abilities to methylate CpG islands not directly associated with methyltransferase activities. Our study provides evidence that, aside from random DNA-MTase action, additional cellular factors exist that govern aberrant methylation in breast cancer cells.
Hum Mol Genet 1999 Mar
PMID:Methylation profiling of CpG islands in human breast cancer cells. 994 5

Type I DNA restriction enzymes are large, molecular machines possessing DNA methyltransferase, ATPase, DNA translocase and endonuclease activities. The ATPase, DNA translocase and endonuclease activities are specified by the restriction (R) subunit of the enzyme. We demonstrate that the R subunit of the Eco KI type I restriction enzyme comprises several different functional domains. An N-terminal domain contains an amino acid motif identical with that forming the catalytic site in simple restriction endonucleases, and changes within this motif lead to a loss of nuclease activity and abolish the restriction reaction. The central part of the R subunit contains amino acid sequences characteristic of DNA helicases. We demonstrate, using limited proteolysis of this subunit, that the helicase motifs are contained in two domains. Secondary structure prediction of these domains suggests a structure that is the same as the catalytic domains of DNA helicases of known structure. The C-terminal region of the R subunit can be removed by elastase treatment leaving a large fragment, stable in the presence of ATP, which can no longer bind to the other subunits of Eco KI suggesting that this domain is required for protein assembly. Considering these results and previous models of the methyltransferase part of these enzymes, a structural and operational model of a type I DNA restriction enzyme is presented.
J Mol Biol 1999 Jul 09
PMID:On the structure and operation of type I DNA restriction enzymes. 1039 Mar 54

DNA methylation patterns are a critical component of the epigenetic machinery that controls the expression of genetic programs in vertebrates. DNA methyltransferase gene (dnmt1) encodes the enzyme catalyzing the methylation of DNA during replication. We tested the hypothesis that the expression of dnmt1 is regulated with the developmental state of neuronal cells. We show that DNA methyltransferase (Dnmt1) activity is sharply reduced 4 days after induction of differentiation of PC12 cells with NGF. Similarly, the adult brain expresses reduced levels of Dnmt1 activity. We propose that the level of Dnmt1 is downregulated to adjust the activity of the DNA methyltransferase to a different role in mature post-mitotic neurons. Both the abundance of dnmt1 mRNA as well as the Dnmt1 polypeptide are downregulated. Downregulation of dnmt1 parallels other indicators of withdrawal from the cell cycle such as induction of p21, and downregulation of the S phase maker PCNA (proliferating cell nuclear antigen). The temporal pattern of downregulation of dnmt1 in nerve growth factor (NGF)-induced PC12 cells is different from myotube differentiation where downregulation of DNA methyltransferase and demethylation is an early event and was proposed to play a causal role in differentiation. We propose that NGF differentiation of PC12 cells represents a different paradigm of involvement of DNA methylation in terminal differentiation.
Brain Res Mol Brain Res 1999 Jul 23
PMID:Downregulation of DNA (cytosine-5-)methyltransferase is a late event in NGF-induced PC12 cell differentiation. 1040 83

A molecular dynamics study was performed on the DNA methyltransferase M. Hha I in a ternary complex with DNA and AdoMet in solution. Methylation involves addition of the Cys81 sulfhydryl anion to the 6-position of Cyt18, followed by a nucleophilic attack of the resultant carbanion at C5 on the AdoMet methyl group. It was found in this simulation that the distances between the sulfhydryl group (SG) of Cys81 to the C6 of Cyt18 (SG-C6) and methyl carbon (CH3) of AdoMet to the C5 of cytosine (CH3-C5) are dependent on the dihedral angle chi (O4'-C1'-N1-C2) of the nucleotide. When the chi angle of Cyt18 is low (< -80 degrees), the SG-C6 and CH3-C5 distances are large. A high chi angle (> -80 degrees) for the target cytosine residue reduces the distances for both SG-C6 and CH3-C5, and the angles formed between the cytosine ring and AdoMet correspond well to values for the transition state structures formed during methylation of cytosine from ab initio calculations. Two possible proton sources for protonation of N3 of the cytosine residue upon formation of the covalent intermediate were found in the simulation. The protonated amine group of AdoMet could provide a proton via a water bridge, or Arg163 could also be the source of the proton for N3 via a water bridge. The simulation provides insights into how the H5 of cytosine could go from the active site into solvent. Conserved residues Asn304 and Gln82 stabilize a water network within the active site of M. Hha I which provides a route for H5 to diffuse into bulk solvent. An initially distant water molecule was able to diffuse into the active site of the enzyme and replace a position of a crystallographic water molecule in close proximity to the C5 of cytosine. The movement of this water molecule showed that a channel exists between Gln82 and the AdoMet in M. Hha I which allows both water and protons to easily gain access to the active site of the enzyme.
J Mol Biol 1999 Oct 15
PMID:Active site dynamics of the HhaI methyltransferase: insights from computer simulation. 1051 11


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