Structure of mammalian DNA methyltransferase as deduced from the inferred amino acid sequence and direct studies of the protein

1988 ◽  
Vol 16 (6) ◽  
pp. 944-947 ◽  
Author(s):  
TIMOTHY BESTOR
Keyword(s):  
Amino Acid ◽  
Metal Binding ◽  
Dna Methyltransferase ◽  
Active Species ◽  
Metal Binding Sites ◽  
Terminal Domain ◽  
Catalytically Active ◽  
Methylation Patterns

Abstract DNA (cytosine-5)-methyltransferase (DNA MeTase) establishes and maintains methylation patterns in the genome of higher eukaryotes. This enzyme has been purified, and the cDNA which encodes it has been cloned and sequenced. DNA MeTase appears to contain a large (1000 amino acid) N-terminal domain that contains potential metal-binding sites. This domain appears to contain a series of five to seven structural units of Mr about 20 000, since post-translational processing in vivo or partial proteolysis of the purified protein in vitro leads to the production of a series of catalytically active species differing in Mr by units of 20 000. The N-terminal domain is fused to a smaller (570 amino acid) C-terminal domain that is related to bacterial type II cytosine methyltransferases. The relevance of these findings for the biological function of DNA MeTase is discussed.

The C-terminal domain of Saccharomyces cerevisiae DNA topoisomerase II

1994 ◽  
Vol 14 (5) ◽  
pp. 3197-3207
Author(s):  
P R Caron ◽  
P Watt ◽  
J C Wang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Localization ◽  
Topoisomerase Ii ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Mutant Proteins ◽  
Terminal Domain ◽  
Catalytically Active

A set of carboxy-terminal deletion mutants of Saccharomyces cerevisiae DNA topoisomerase II were constructed for studying the functions of the carboxyl domain in vitro and in vivo. The wild-type yeast enzyme is a homodimer with 1,429 amino acid residues in each of the two polypeptides; truncation of the C terminus to Ile-1220 has little effect on the function of the enzyme in vitro or in vivo, whereas truncations extending beyond Gln-1138 yield completely inactive proteins. Several mutant enzymes with C termini in between these two residues were found to be catalytically active but unable to complement a top2-4 temperature-sensitive mutation. Immunomicroscopy results suggest that the removal of a nuclear localization signal in the C-terminal domain is likely to contribute to the physiological dysfunction of these proteins; the ability of these mutant proteins to relax supercoiled DNA in vivo shows, however, that at least some of the mutant proteins are present in the nuclei in a catalytically active form. In contrast to the ability of the catalytically active mutant proteins to relax supercoiled intracellular DNA, all mutants that do not complement the temperature-dependent lethality and high frequency of chromosomal nondisjunction of top2-4 were found to lack decatenation activity in vivo. The plausible roles of the DNA topoisomerase II C-terminal domain, in addition to providing a signal for nuclear localization, are discussed in the light of these results.

scholarly journals Distinct interactions of eIF4A and eIF4E with RNA helicase Ded1 stimulate translation in vivo

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Suna Gulay ◽  
Neha Gupta ◽  
Jon R Lorsch ◽  
Alan G Hinnebusch
Keyword(s):  
Amino Acid ◽  
Cell Growth ◽  
Translation Initiation ◽  
Rna Helicase ◽  
Preinitiation Complex ◽  
Terminal Domain ◽  
Dead Box ◽  
Stimulation Of

Yeast DEAD-box helicase Ded1 stimulates translation initiation, particularly of mRNAs with structured 5'UTRs. Interactions of the Ded1 N-terminal domain (NTD) with eIF4A, and Ded1-CTD with eIF4G, subunits of eIF4F, enhance Ded1 unwinding activity and stimulation of preinitiation complex (PIC) assembly in vitro. However, the importance of these interactions, and of Ded1-eIF4E association, in vivo were poorly understood. We identified separate amino acid clusters in the Ded1-NTD required for binding to eIF4A or eIF4E in vitro. Disrupting each cluster selectively impairs native Ded1 association with eIF4A or eIF4E, and reduces cell growth, polysome assembly, and translation of reporter mRNAs with structured 5'UTRs. It also impairs Ded1 stimulation of PIC assembly on a structured mRNA in vitro. Ablating Ded1 interactions with eIF4A/eIF4E unveiled a requirement for the Ded1-CTD for robust initiation. Thus, Ded1 function in vivo is stimulated by independent interactions of its NTD with eIF4E and eIF4A, and its CTD with eIF4G.

scholarly journals DNMT1 but not its interaction with the replication machinery is required for maintenance of DNA methylation in human cells

2007 ◽  
Vol 176 (5) ◽  
pp. 565-571 ◽  
Author(s):  
Fabio Spada ◽  
Andrea Haemmer ◽  
David Kuch ◽  
Ulrich Rothbauer ◽  
Lothar Schermelleh ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Regulation Of Gene Expression ◽  
Human Cells ◽  
Nuclear Antigen ◽  
Replication Machinery ◽  
Catalytically Active ◽  
Methylation Patterns ◽  
Proliferating Cell

DNA methylation plays a central role in the epigenetic regulation of gene expression in vertebrates. Genetic and biochemical data indicated that DNA methyltransferase 1 (Dnmt1) is indispensable for the maintenance of DNA methylation patterns in mice, but targeting of the DNMT1 locus in human HCT116 tumor cells had only minor effects on genomic methylation and cell viability. In this study, we identified an alternative splicing in these cells that bypasses the disrupting selective marker and results in a catalytically active DNMT1 protein lacking the proliferating cell nuclear antigen–binding domain required for association with the replication machinery. Using a mechanism-based trapping assay, we show that this truncated DNMT1 protein displays only twofold reduced postreplicative DNA methylation maintenance activity in vivo. RNA interference–mediated knockdown of this truncated DNMT1 results in global genomic hypomethylation and cell death. These results indicate that DNMT1 is essential in mouse and human cells, but direct coupling of the replication of genetic and epigenetic information is not strictly required.

scholarly journals Murine De Novo Methyltransferase Dnmt3a Demonstrates Strand Asymmetry and Site Preference in the Methylation of DNA In Vitro

2002 ◽  
Vol 22 (3) ◽  
pp. 704-723 ◽  
Author(s):  
Iping G. Lin ◽  
Li Han ◽  
Alexander Taghva ◽  
Laura E. O’Brien ◽  
Chih-Lin Hsieh
Keyword(s):  
Dna Methyltransferase ◽  
De Novo ◽  
Dna Methyltransferases ◽  
Site Preference ◽  
Cpg Sites ◽  
Methylation Of Dna ◽  
Wide Range ◽  
Methylation Patterns

ABSTRACT CpG methylation is involved in a wide range of biological processes in vertebrates as well as in plants and fungi. To date, three enzymes, Dnmt1, Dnmt3a, and Dnmt3b, are known to have DNA methyltransferase activity in mouse and human. It has been proposed that de novo methylation observed in early embryos is predominantly carried out by the Dnmt3a and Dnmt3b methyltransferases, while Dntm1 is believed to be responsible for maintaining the established methylation patterns upon replication. Analysis of the sites methylated in vivo using the bisulfite genomic sequencing method confirms the previous finding that some regions of the plasmid are much more methylated by Dnmt3a than other regions on the same plasmid. However, the preferred targets of the enzyme cannot be determined due to the presence of other methylases, DNA binding proteins, and chromatin structure. To discern the DNA targets of Dnmt3a without these compounding factors, sites methylated by Dnmt3a in vitro were analyzed. These analyses revealed that the two cDNA strands have distinctly different methylation patterns. Dnmt3a prefers CpG sites on a strand in which it is flanked by pyrimidines over CpG sites flanked by purines in vitro. These findings indicate that, unlike Dnmt1, Dnmt3a most likely methylates one strand of DNA without concurrent methylation of the CpG site on the complementary strand. These findings also indicate that Dnmt3a may methylate some CpG sites more frequently than others, depending on the sequence context. Methylation of each DNA strand independently and with possible sequence preference is a novel feature among the known DNA methyltransferases.

scholarly journals The C-terminal domain of Saccharomyces cerevisiae DNA topoisomerase II.

1994 ◽  
Vol 14 (5) ◽  
pp. 3197-3207 ◽  
Author(s):  
P R Caron ◽  
P Watt ◽  
J C Wang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Localization ◽  
Topoisomerase Ii ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Mutant Proteins ◽  
Terminal Domain ◽  
Catalytically Active

A set of carboxy-terminal deletion mutants of Saccharomyces cerevisiae DNA topoisomerase II were constructed for studying the functions of the carboxyl domain in vitro and in vivo. The wild-type yeast enzyme is a homodimer with 1,429 amino acid residues in each of the two polypeptides; truncation of the C terminus to Ile-1220 has little effect on the function of the enzyme in vitro or in vivo, whereas truncations extending beyond Gln-1138 yield completely inactive proteins. Several mutant enzymes with C termini in between these two residues were found to be catalytically active but unable to complement a top2-4 temperature-sensitive mutation. Immunomicroscopy results suggest that the removal of a nuclear localization signal in the C-terminal domain is likely to contribute to the physiological dysfunction of these proteins; the ability of these mutant proteins to relax supercoiled DNA in vivo shows, however, that at least some of the mutant proteins are present in the nuclei in a catalytically active form. In contrast to the ability of the catalytically active mutant proteins to relax supercoiled intracellular DNA, all mutants that do not complement the temperature-dependent lethality and high frequency of chromosomal nondisjunction of top2-4 were found to lack decatenation activity in vivo. The plausible roles of the DNA topoisomerase II C-terminal domain, in addition to providing a signal for nuclear localization, are discussed in the light of these results.

SUMOylation enhances DNA methyltransferase 1 activity

2009 ◽  
Vol 421 (3) ◽  
pp. 449-461 ◽  
Author(s):  
Bongyong Lee ◽  
Mark T. Muller
Keyword(s):  
Dna Methyltransferase ◽  
Gene Control ◽  
Dna Methyltransferase 1 ◽  
Dna Interactions ◽  
Protein Dna Interactions ◽  
Catalytically Active ◽  
Endogenous Activity ◽  
Methyltransferase 1

DNA methylation regulates gene expression through a complex network of protein–protein and protein–DNA interactions in chromatin. The maintenance methylase, DNMT1 (DNA methyltransferase 1), is a prominent enzyme in the process that is linked to DNA replication and drives the heritable nature of epigenetic modifications. The mechanistic details that explain how DNMT1 catalytic action is directed and regulated in chromatin are important in our overall understanding of gene control. In this work, we show that DNMT1 is modified by SUMOylation and we have mapped these SUMOylation sites by defined mutations. SUMOylated DNMT1 is catalytically active on genomic DNA in vivo and we find that SUMOylation significantly enhances the methylase activity of DNMT1 both in vitro and in chromatin. These data suggest that SUMOylation modulates the endogenous activity of a prominent epigenetic maintenance pathway in somatic cells.

scholarly journals Functional Analysis of AtlA, the Major N-Acetylglucosaminidase of Enterococcus faecalis

2006 ◽  
Vol 188 (24) ◽  
pp. 8513-8519 ◽  
Author(s):  
Catherine Eckert ◽  
Maxime Lecerf ◽  
Lionel Dubost ◽  
Michel Arthur ◽  
Stéphane Mesnage
Keyword(s):  
Enzyme Activity ◽  
Enterococcus Faecalis ◽  
Proteinase K ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Terminal Domain ◽  
Catalytically Active ◽  
Limited Digestion

ABSTRACT The major peptidoglycan hydrolase of Enterococcus faecalis, AtlA, has been identified, but its enzyme activity remains unknown. We have used tandem mass spectrometry analysis of peptidoglycan hydrolysis products obtained using the purified protein to show that AtlA is an N-acetylglucosaminidase. To gain insight into the regulation of its enzyme activity, the three domains of AtlA were purified alone or in combination following expression of truncated forms of the atlA gene in Escherichia coli or partial digestion of AtlA by proteinase K. The central domain of AtlA was catalytically active, but its activity was more than two orders of magnitude lower than that of the complete protein. Partial proteolysis of AtlA was detected in vivo: zymograms of E. faecalis extracts revealed two catalytically active protein bands of 62 and 72 kDa that were both absent in extracts from an atlA null mutant. Limited digestion of AtlA by proteinase K in vitro suggested that the proteolytic cleavage of AtlA in E. faecalis extracts corresponds to the truncation of the N-terminal domain, which is rich in threonine and glutamic acid residues. We show that the truncation of the N-terminal domain from recombinant AtlA has no impact on enzyme activity. The C-terminal domain of the protein, which contains six LysM modules bound to highly purified peptidoglycan, was required for optimal enzyme activity. These data indicate that AtlA is not produced as a proenzyme and that control of the AtlA glucosaminidase activity is likely to occur at the level of LysM-mediated binding to peptidoglycan.

scholarly journals Evolutionary persistence of DNA methylation for millions of years after ancient loss of a de novo methyltransferase

2017 ◽  
Author(s):  
Sandra Catania ◽  
Phillip A. Dumesic ◽  
Harold Pimentel ◽  
Ammar Nasif ◽  
Caitlin I. Stoddard ◽  
...  
Keyword(s):  
Dna Methyltransferase ◽  
Cytosine Methylation ◽  
De Novo ◽  
Darwinian Evolution ◽  
Methylation Of Dna ◽  
Efficient Replication ◽  
Methylation Patterns ◽  
Evolutionary Persistence

SUMMARYCytosine methylation of DNA is a widespread modification of DNA that plays numerous critical roles, yet has been lost many times in diverse eukaryotic lineages. In the yeast Cryptococcus neoformans, CG methylation occurs in transposon-rich repeats and requires the DNA methyltransferase, Dnmt5. We show that Dnmt5 displays exquisite maintenance-type specificity in vitro and in vivo and utilizes similar in vivo cofactors as the metazoan maintenance methylase Dnmt1. Remarkably, phylogenetic and functional analysis revealed that the ancestral species lost the gene for a de novo methylase, DnmtX, between 50-150 MYA. We examined how methylation has persisted since the ancient loss of DnmtX. Experimental and comparative studies reveal efficient replication of methylation patterns in C. neoformans, rare stochastic methylation loss and gain events, and the action of natural selection. We propose that an epigenome has been propagated for >50 MY through a process analogous to Darwinian evolution of the genome.

Oxytocin analogues with non-coded amino acid residues in position 8: [8-Neopentylglycine]oxytocin and [8-cycloleucine]oxytocin

1987 ◽  
Vol 52 (9) ◽  
pp. 2317-2325 ◽  
Author(s):  
Jan Hlaváček ◽  
Jan Pospíšek ◽  
Jiřina Slaninová ◽  
Walter Y. Chan ◽  
Victor J. Hruby
Keyword(s):  
Amino Acid ◽  
Solid Phase Synthesis ◽  
Solid Phase ◽  
The Other ◽  
Amino Acid Residues ◽  
Phase Synthesis ◽  
Other Hand ◽  
Fragment Condensation

[8-Neopentylglycine]oxytocin (II) and [8-cycloleucine]oxytocin (III) were prepared by a combination of solid-phase synthesis and fragment condensation. Both analogues exhibited decreased uterotonic potency in vitro, each being about 15-30% that of oxytocin. Analogue II also displayed similarly decreased uterotonic potency in vivo and galactogogic potency. On the other hand, analogue III exhibited almost the same potency as oxytocin in the uterotonic assay in vivo and in the galactogogic assay.

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